Immunohistochemistry - In Situ Hybridization

5-Hydroxytryptamine-2B receptor (5HT2BR) stimulation is known to cause fibroblast mitogenesis, and the mitogenic effect has been proposed to trigger valvular heart disease in humans. In this study, we used real-time polymerase chain reaction (TaqMan) to quantify transcript levels of 5HT2B, 5HT2C, and 5HT1B receptors and immunohistochemistry (IHC) to detect the tissue localization of these receptors in the normal heart valves of cynomolgus (CM) monkeys and Sprague-Dawley (S-D) rats. In both species, positive immunostaining was noted for 5HT1B and 5HT2B receptors in mitral, tricuspid, aortic, and pulmonary valves, and the cell types showing positive staining were interstitial cells and endothelial cells lining the valve leaflet. In CM monkeys, 5HT2CR was expressed only in the endothelial cells lining the leaflet, whereas S-D valves were negative for this receptor. IHC results were correlated with 5HT2B and 5HT1B receptor transcripts for all four valves. However, 5HT2C receptor transcripts were lower than 5HT2B or 5HT1B in all CM monkey valves, whereas 5HT2C transcripts were below the level of detection in any of the S-D rat valves. Our data showed the expression of 5HT2B, 5HT1B, and 5HT2C receptors in the normal heart valves of CM monkeys and S-D rats, and IHC and TaqMan techniques may be used to study the potential mechanism of compounds with 5HT2BR agonist activity.

(J Histochem Cytochem 53:671–677, 2005)

5-Hydroxytryptamine-2B receptor (5HT2BR) stimulation is known to cause fibroblast mitogenesis, and the mitogenic effect has been proposed to trigger valvular heart disease in humans. In this study, we used real-time polymerase chain reaction (TaqMan) to quantify transcript levels of 5HT2B, 5HT2C, and 5HT1B receptors and immunohistochemistry (IHC) to detect the tissue localization of these receptors in the normal heart valves of cynomolgus (CM) monkeys and Sprague-Dawley (S-D) rats. In both species, positive immunostaining was noted for 5HT1B and 5HT2B receptors in mitral, tricuspid, aortic, and pulmonary valves, and the cell types showing positive staining were interstitial cells and endothelial cells lining the valve leaflet. In CM monkeys, 5HT2CR was expressed only in the endothelial cells lining the leaflet, whereas S-D valves were negative for this receptor. IHC results were correlated with 5HT2B and 5HT1B receptor transcripts for all four valves. However, 5HT2C receptor transcripts were lower than 5HT2B or 5HT1B in all CM monkey valves, whereas 5HT2C transcripts were below the level of detection in any of the S-D rat valves. Our data showed the expression of 5HT2B, 5HT1B, and 5HT2C receptors in the normal heart valves of CM monkeys and S-D rats, and IHC and TaqMan techniques may be used to study the potential mechanism of compounds with 5HT2BR agonist activity.

(J Histochem Cytochem 53:671–677, 2005)

5-Hydroxytryptamine-2B receptor (5HT2BR) stimulation is known to cause fibroblast mitogenesis, and the mitogenic effect has been proposed to trigger valvular heart disease in humans. In this study, we used real-time polymerase chain reaction (TaqMan) to quantify transcript levels of 5HT2B, 5HT2C, and 5HT1B receptors and immunohistochemistry (IHC) to detect the tissue localization of these receptors in the normal heart valves of cynomolgus (CM) monkeys and Sprague-Dawley (S-D) rats. In both species, positive immunostaining was noted for 5HT1B and 5HT2B receptors in mitral, tricuspid, aortic, and pulmonary valves, and the cell types showing positive staining were interstitial cells and endothelial cells lining the valve leaflet. In CM monkeys, 5HT2CR was expressed only in the endothelial cells lining the leaflet, whereas S-D valves were negative for this receptor. IHC results were correlated with 5HT2B and 5HT1B receptor transcripts for all four valves. However, 5HT2C receptor transcripts were lower than 5HT2B or 5HT1B in all CM monkey valves, whereas 5HT2C transcripts were below the level of detection in any of the S-D rat valves. Our data showed the expression of 5HT2B, 5HT1B, and 5HT2C receptors in the normal heart valves of CM monkeys and S-D rats, and IHC and TaqMan techniques may be used to study the potential mechanism of compounds with 5HT2BR agonist activity.

(J Histochem Cytochem 53:671–677, 2005)

We report a case of a de novo complex chromosomal rearrangement among five chromosomes found in a clinically healthy woman. The only indication for chromosome analysis was a planned intracytoplasmatic sperm injection. Physical examination, including internal and external genitals, and ovaries and hormone status were normal. Banding cytogenetics showed a rearrangement among chromosomes #3, #4, #7, #9, and #17. Twenty-four-color fluorescence in situ hybridization and multicolor banding were applied to characterize the translocations and breakpoints more precisely. This confirmed the involved chromosomes and revealed two breakpoints in chromosome #4. This six-breakpoint rearrangement [der(3)t(3;4), der(4)t(17;4;7), der(7)t(3;7), der(9)t(4;9), and der(17)t(9;17)] seemed to be balanced on a molecular cytogenetic level, although submicroscopic deletions or duplications close to the breakpoints cannot be excluded. (J Histochem Cytochem 53:355–357, 2005)

Spinal cord injury (SCI) initiates a cascade of biochemical changes that results in necrotic and apoptotic cell death. There is evidence that caspase-3 activation and apoptotic cell death occur within hours after SCI. However, the time course and cellular localization of activated caspase-3 has not been examined. Such information is essential because caspase-3–independent apoptotic pathways do exist. In this experiment, we describe the distribution of and cell types containing activated caspase-3 at 4 hr, 1 day, 2 days, 4 days, and 8 days following SCI in rats. Numerous caspase-3–positive cells were observed at 4 hr and 1 day postinjury and colocalized most often with CC1, a marker for oligodendroglia. Both markers disappeared near the injury epicenter over the next several days. Activated caspase-3 was again present in the injured spinal cord on postoperative day 8, which coincided with a reemergence of CC1-positive cells. Many of these CC1-positive cells again colocalized activated caspase-3. NeuN-positive neurons of the dorsal horn were occasionally immunopositive for activated caspase-3 at early time points. OX42-positive microglia/macrophages rarely contained activated caspase-3. The results indicate a biphasic pattern of caspase-3 activation during the first 8 days postinjury, suggesting that at least two mechanisms activate caspase-3 following SCI. This time-course study provides a framework for investigating and understanding the different signaling events contributing to this biphasic pattern of caspase-3 activation. (J Histochem Cytochem 53:809–819, 2005)

Spinal cord injury (SCI) initiates a cascade of biochemical changes that results in necrotic and apoptotic cell death. There is evidence that caspase-3 activation and apoptotic cell death occur within hours after SCI. However, the time course and cellular localization of activated caspase-3 has not been examined. Such information is essential because caspase-3–independent apoptotic pathways do exist. In this experiment, we describe the distribution of and cell types containing activated caspase-3 at 4 hr, 1 day, 2 days, 4 days, and 8 days following SCI in rats. Numerous caspase-3–positive cells were observed at 4 hr and 1 day postinjury and colocalized most often with CC1, a marker for oligodendroglia. Both markers disappeared near the injury epicenter over the next several days. Activated caspase-3 was again present in the injured spinal cord on postoperative day 8, which coincided with a reemergence of CC1-positive cells. Many of these CC1-positive cells again colocalized activated caspase-3. NeuN-positive neurons of the dorsal horn were occasionally immunopositive for activated caspase-3 at early time points. OX42-positive microglia/macrophages rarely contained activated caspase-3. The results indicate a biphasic pattern of caspase-3 activation during the first 8 days postinjury, suggesting that at least two mechanisms activate caspase-3 following SCI. This time-course study provides a framework for investigating and understanding the different signaling events contributing to this biphasic pattern of caspase-3 activation. (J Histochem Cytochem 53:809–819, 2005)

A molecular mechanism of formalin fixation and antigen retrieval.

Am J Clin Pathol. 2004 Jul;122(1):154; author reply 154-5

Authors: O'Leary TJ, Mason JT

PMID: 15272544 [PubMed - indexed for MEDLINE]

The development of a non-invasive method of prenatal diagnosis in maternal blood has been the goal of our investigations during the last years. We have developed several anti-CD71 monoclonal antibodies and optimized a protocol for the isolation of nucleated red blood cells (NRBC) from peripheral maternal blood. The enhanced traffic of fetal erythroblasts into the maternal circulation in preeclampsia has been investigated by several groups. In this study, we compared one of our antibodies, 2F6.3, with a commercial anti-CD71 antibody in blood samples from pregnant women suffering pregnancy-induced hypertension (PIH) and in a control group of pregnant women without clinical features suggestive of an increased risk of developing preeclampsia. The mAb 2F6.3, developed by our group, has succeeded in isolating a significantly higher number of erythroblasts with less maternal cell contamination than the commercial antibody in both women with PIH and in the control group (p<0.01; Wilcoxon Signed Ranks Test). Florescence in situ hybridization analysis also demonstrated that 2F6.3 is a better antibody for the isolation of fetal NRBC in maternal blood than the commercial anti-CD71 antibody.

(J Histochem Cytochem 53:345–350, 2005)

In the search for immunohistochemical markers of the developing human brain, a monoclonal antibody, HFB-16, was raised against homogenates from the cerebrum of a 15-gestational-week-old (GW) human fetus and screened on paraffin-embedded human embryonic brain specimens. This antibody was particularly useful as a marker for Purkinje cells in the developing human cerebellum. Positive immunoreactivities with HFB-16 first appeared in the Purkinje cell layer at 17 GW. From 20 to 24 GW, positive immunoreactivities were found above the lamina dissecans. After 25 GW, dendrites of Purkinje cells were found with the HFB-16 antibody, and the nerve fibers of the Purkinje cells became positive after 35 GW. Neurons in the dentate nucleus and external and internal granular layers reacted negatively to this antibody. After 1 year, when the external granular layer faded out, the dendrites of the Purkinje cells reached the pial surface of the cerebellum, and nerve fibers began to develop in the white matter. This antibody was also useful for characterization of components in heterotopic neurons found in various anomaly syndromes such as trisomy 13. Expressional cloning indicated the antigen against HFB-16 to be human KIAA0864 protein, which is supposed to be an alternative splicing product of p116Rip, whose function has not yet been elucidated. The antigenicity of the KIAA0864 protein was confirmed using human cDNA of the KIAA0864 protein, a protein expression vector, and an HFB-16 antibody. (J Histochem Cytochem 53:423–430, 2005)

In the search for immunohistochemical markers of the developing human brain, a monoclonal antibody, HFB-16, was raised against homogenates from the cerebrum of a 15-gestational-week-old (GW) human fetus and screened on paraffin-embedded human embryonic brain specimens. This antibody was particularly useful as a marker for Purkinje cells in the developing human cerebellum. Positive immunoreactivities with HFB-16 first appeared in the Purkinje cell layer at 17 GW. From 20 to 24 GW, positive immunoreactivities were found above the lamina dissecans. After 25 GW, dendrites of Purkinje cells were found with the HFB-16 antibody, and the nerve fibers of the Purkinje cells became positive after 35 GW. Neurons in the dentate nucleus and external and internal granular layers reacted negatively to this antibody. After 1 year, when the external granular layer faded out, the dendrites of the Purkinje cells reached the pial surface of the cerebellum, and nerve fibers began to develop in the white matter. This antibody was also useful for characterization of components in heterotopic neurons found in various anomaly syndromes such as trisomy 13. Expressional cloning indicated the antigen against HFB-16 to be human KIAA0864 protein, which is supposed to be an alternative splicing product of p116Rip, whose function has not yet been elucidated. The antigenicity of the KIAA0864 protein was confirmed using human cDNA of the KIAA0864 protein, a protein expression vector, and an HFB-16 antibody. (J Histochem Cytochem 53:423–430, 2005)

In the search for immunohistochemical markers of the developing human brain, a monoclonal antibody, HFB-16, was raised against homogenates from the cerebrum of a 15-gestational-week-old (GW) human fetus and screened on paraffin-embedded human embryonic brain specimens. This antibody was particularly useful as a marker for Purkinje cells in the developing human cerebellum. Positive immunoreactivities with HFB-16 first appeared in the Purkinje cell layer at 17 GW. From 20 to 24 GW, positive immunoreactivities were found above the lamina dissecans. After 25 GW, dendrites of Purkinje cells were found with the HFB-16 antibody, and the nerve fibers of the Purkinje cells became positive after 35 GW. Neurons in the dentate nucleus and external and internal granular layers reacted negatively to this antibody. After 1 year, when the external granular layer faded out, the dendrites of the Purkinje cells reached the pial surface of the cerebellum, and nerve fibers began to develop in the white matter. This antibody was also useful for characterization of components in heterotopic neurons found in various anomaly syndromes such as trisomy 13. Expressional cloning indicated the antigen against HFB-16 to be human KIAA0864 protein, which is supposed to be an alternative splicing product of p116Rip, whose function has not yet been elucidated. The antigenicity of the KIAA0864 protein was confirmed using human cDNA of the KIAA0864 protein, a protein expression vector, and an HFB-16 antibody. (J Histochem Cytochem 53:423–430, 2005)

The composition of extracellular matrix during growth and regression of the neointima was analyzed during healing in a baboon aorto-iliac polytetrafluoroethylene graft. Graft neointimal thickening can be modulated by altering blood flow by construction of downstream arteriovenous fistulas. Normal flow with normal shear stress induces neointimal thickening, whereas high flow with high shear stress upstream of a fistula induces regression of established neointima. The neointima formed under normal shear stress is enriched in hyaluronan and proteoglycans, particularly versican. On the other hand, the neointima near the graft material is enriched in collagen and biglycan. Neointimal regression in response to high shear stress is associated with a loss of proteoglycans as detected by histochemical staining. Immunostaining with an antibody against an ADAMTS cleavage neoepitope of versican increases after switching to high flow, although immunostaining for versican core protein is not appreciably changed by high flow. The present data demonstrate that the graft neointima is enriched with proteoglycans, particularly versican and hyaluronan, as well as collagen, and there is a differential distribution of each. Neointimal atrophy occurs with an apparent loss of proteoglycans and evidence of versican degradation. (J Histochem Cytochem 53:131–140, 2005)

Protease-activated receptors (PARs) are multifunctional G protein–coupled receptors. Among the four existing PARs, PAR₄ is preferentially expressed in the human lung tissue. However, the function of PAR₄ has not been defined in the lung endothelial cells. Because PAR₁-mediated cellular effects are deeply related to the morphological changes, we focused on the actin fiber and p38 mitogen-activated protein kinase (MAPK) signaling involved in actin polymerization to elucidate the role of PAR₄. RT-PCR and Western blot analyses identified PAR₄ expression in human pulmonary artery endothelial cells and in human microvascular endothelial cells from lung. We then examined the changes in actin fibers in endothelial cells treated with PAR₄-activating peptide. PAR₁-activating peptide was used for comparison. Activation of PAR₄ and PAR₁ by their corresponding peptides induced actin fiber formation; however, the actin filaments were broadly bundled in PAR₄ as compared with the ringlike actin filaments in PAR₁ activation. Correspondingly, the magnitude of p38 MAPK phosphorylation was different between cells treated with PAR₄ and PAR₁, with PAR₄-activating peptide showing a significantly higher sensitivity to p38 MAPK inhibitor, SB203580. Taken together, these results demonstrate that activation of PAR₄ results in the formation of actin fiber distinct from that by PAR₁ activation, suggesting PAR₄ may play specific roles in the lung endothelial cells. (J Histochem Cytochem 53:1121–1129, 2005)

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Acupuncture prevents cognitive deficits and oxidative stress in cerebral multi-infarction rats.

Neurosci Lett. 2005 Oct 14;

Authors: Liu CZ, Yu JC, Zhang XZ, Fu WW, Wang T, Han JX

The aim of this study is to investigate the effects of acupuncture on cognitive deficits and oxidative stress in cerebral multi-infarction rats. The results showed that acupunctural treatment attenuated memory impairment induced by cerebral multi-infarction, as evaluated by shortened escape latency and increased swimming time of rats with memory impairment in the target quadrant. The data additionally suggested that acupunctural treatment ameliorated oxidative injuries induced by cerebral multi-infarction by increasing the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in the hippocampus. Further investigation by in situ hybridization and immunohistochemistry revealed that acupunctural treatment significantly increased the expression of CuZnSOD mRNA and protein in the hippocampus of the impaired rats. The findings demonstrate that acupuncture can exert beneficial effects on spatial memory and antioxidant status of cerebral multi-infarction rats.

PMID: 16236447 [PubMed - as supplied by publisher]

Several proteins implicated in hormonogenesis of the adrenal cortex have alternatively spliced isoforms, which respond differently to adrenocorticotropic hormone (ACTH). Heterogeneous nuclear ribonucleoproteins A2 and B1 are among the abundant pre-mRNA-binding proteins involved in alternative splicing. We examined the expression of A2 and B1 in normal adrenal cortex and tumors. B1 was variably expressed in the zona fasciculata-reticularis, although A2 was diffusely expressed in the three zones. B1 was more abundant in compact cells than clear cells, and B1 expression was frequent in the zona reticularis, which consists mainly of compact cells. In three kinds of cortical adenomas autonomously producing hormones, B1 was generally overexpressed and there were no significant differences among them. In cortisol-producing tumors, non-tumor parts of the cortex, which were generally atrophic due to low ACTH, had less B1 protein than normal adrenals. These results suggested a correlation between B1 expression and the hormonal activity responding to ACTH. In vitro ACTH stimulation induced a biphasic expression of B1 in an H295R cortical carcinoma cell line, and it paralleled hormonogenesis. Conclusively, B1 expression varied in relation to the hormonal activity responding to the ACTH, and it may provide a key to elucidating the splicing mechanisms involved in hormonogenesis.

(J Histochem Cytochem 53:487–495, 2005)

Several proteins implicated in hormonogenesis of the adrenal cortex have alternatively spliced isoforms, which respond differently to adrenocorticotropic hormone (ACTH). Heterogeneous nuclear ribonucleoproteins A2 and B1 are among the abundant pre-mRNA-binding proteins involved in alternative splicing. We examined the expression of A2 and B1 in normal adrenal cortex and tumors. B1 was variably expressed in the zona fasciculata-reticularis, although A2 was diffusely expressed in the three zones. B1 was more abundant in compact cells than clear cells, and B1 expression was frequent in the zona reticularis, which consists mainly of compact cells. In three kinds of cortical adenomas autonomously producing hormones, B1 was generally overexpressed and there were no significant differences among them. In cortisol-producing tumors, non-tumor parts of the cortex, which were generally atrophic due to low ACTH, had less B1 protein than normal adrenals. These results suggested a correlation between B1 expression and the hormonal activity responding to ACTH. In vitro ACTH stimulation induced a biphasic expression of B1 in an H295R cortical carcinoma cell line, and it paralleled hormonogenesis. Conclusively, B1 expression varied in relation to the hormonal activity responding to the ACTH, and it may provide a key to elucidating the splicing mechanisms involved in hormonogenesis.

(J Histochem Cytochem 53:487–495, 2005)

Several proteins implicated in hormonogenesis of the adrenal cortex have alternatively spliced isoforms, which respond differently to adrenocorticotropic hormone (ACTH). Heterogeneous nuclear ribonucleoproteins A2 and B1 are among the abundant pre-mRNA-binding proteins involved in alternative splicing. We examined the expression of A2 and B1 in normal adrenal cortex and tumors. B1 was variably expressed in the zona fasciculata-reticularis, although A2 was diffusely expressed in the three zones. B1 was more abundant in compact cells than clear cells, and B1 expression was frequent in the zona reticularis, which consists mainly of compact cells. In three kinds of cortical adenomas autonomously producing hormones, B1 was generally overexpressed and there were no significant differences among them. In cortisol-producing tumors, non-tumor parts of the cortex, which were generally atrophic due to low ACTH, had less B1 protein than normal adrenals. These results suggested a correlation between B1 expression and the hormonal activity responding to ACTH. In vitro ACTH stimulation induced a biphasic expression of B1 in an H295R cortical carcinoma cell line, and it paralleled hormonogenesis. Conclusively, B1 expression varied in relation to the hormonal activity responding to the ACTH, and it may provide a key to elucidating the splicing mechanisms involved in hormonogenesis.

(J Histochem Cytochem 53:487–495, 2005)

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Amplification of EMSY, a novel oncogene on 11q13, in high grade ovarian surface epithelial carcinomas.

Gynecol Oncol. 2005 Oct 14;

Authors: Brown LA, Irving J, Parker R, Kim H, Press JZ, Longacre TA, Chia S, Magliocco A, Makretsov N, Gilks B, Pollack J, Huntsman D

OBJECTIVES.: Amplification of the 11q13 locus is commonly observed in a number of human cancers including both breast and ovarian cancer. Cyclin D1 and EMS1 have been implicated as candidate oncogenes involved in the emergence of amplification at this locus. Detailed analysis of the 11q13 amplicon in breast cancer led to the discovery of four regions of amplification suggesting the involvement of other genes. Here, we investigate the role of EMSY, a recently described BRCA2 interacting protein, as a key element of the 11q13 amplicon in ovarian cancer. EMSY maps to 11q13.5 and is amplified in 13% of breast and 17% of ovarian carcinomas. METHODS.: EMSY amplification was assessed by fluorescent in-situ hybridization (FISH) in 674 ovarian cancers in a tissue microarray and correlated with histopathological subtype and tumor grade. A detailed map of the 11q13 amplicon in 51 cases of ovarian cancer was obtained using cDNA-array-based comparative genomic hybridization (aCGH). To further characterize the role of EMSY within this amplicon, we evaluated both the amplification profiles and RNA expression levels of EMSY and two other genes from the 11q13 amplicon in an additional series of 22 ovarian carcinomas. RESULTS.: EMSY amplification was seen in 52/285 (18%) high grade papillary serous carcinomas, 4/27 (15%) high grade endometrioid carcinomas, 3/38 (8%) clear cell carcinomas, and 3/10 (30%) undifferentiated carcinomas. aCGH mapping of 11q13 in ovarian cancer showed that EMSY localized to the region with the highest frequency of copy number gain. Cyclin D1 and EMS1 showed a lower frequency of copy number gain. A highly significant correlation between EMSY gene amplification and RNA expression was also observed (P = 0.0001). This was a stronger correlation than for other genes at 11q13 including Cyclin D1 and PAK1. CONCLUSIONS.: These findings support the role of EMSY as a key oncogene within the 11q13 amplicon in ovarian cancer.

PMID: 16236351 [PubMed - as supplied by publisher]

Because of its sensitivity, immunohistochemistry (IHC) of abnormal prion protein (PrPsc) is used more often in the diagnosis of transmissible spongiform encephalopathies (TSEs), such as scrapie and bovine spongiform encephalopathy (BSE). PrPsc IHC requires a combination of pretreatments (chemical, heating, and enzymatic). The method of application may depend on the anti-prion antibody considered. If these pretreatments are efficient for diagnostic purpose, it may, however, be interesting to use an alternative method to efficiently detect PrPsc IHC immunohistochemically using chemical pretreatments solely. Here we describe such pretreatments reporting the difficulty (section adhesion) but also the potential advantages of such methods (easy, quick, inexpensive, and amplifying effect). (J Histochem Cytochem 53:1199–1202, 2005)

Because of its sensitivity, immunohistochemistry (IHC) of abnormal prion protein (PrPsc) is used more often in the diagnosis of transmissible spongiform encephalopathies (TSEs), such as scrapie and bovine spongiform encephalopathy (BSE). PrPsc IHC requires a combination of pretreatments (chemical, heating, and enzymatic). The method of application may depend on the anti-prion antibody considered. If these pretreatments are efficient for diagnostic purpose, it may, however, be interesting to use an alternative method to efficiently detect PrPsc IHC immunohistochemically using chemical pretreatments solely. Here we describe such pretreatments reporting the difficulty (section adhesion) but also the potential advantages of such methods (easy, quick, inexpensive, and amplifying effect). (J Histochem Cytochem 53:1199–1202, 2005)

A number of applied molecular cytogenetic studies require the quantitative assessment of fluorescence in situ hybridization (FISH) signals (for example, interphase FISH analysis of aneuploidy by chromosome enumeration DNA probes; analysis of somatic pairing of homologous chromosomes in interphase nuclei; identification of chromosomal heteromorphism after FISH with satellite DNA probes for differentiation of parental origin of homologous chromosome, etc.). We have performed a pilot study to develop a simple technique for quantitative assessment of FISH signals by means of the digital capturing of microscopic images and the intensity measuring of hybridization signals using Scion Image software, commonly used for quantification of electrophoresis gels. We have tested this approach by quantitative analysis of FISH signals after application of chromosome-specific DNA probes for aneuploidy scoring in interphase nuclei in cells of different human tissues. This approach allowed us to exclude or confirm a low-level mosaic form of aneuploidy by quantification of FISH signals (for example, discrimination of pseudo-monosomy and artifact signals due to over-position of hybridization signals). Quantification of FISH signals was also used for analysis of somatic pairing of homologous chromosomes in nuclei of postmortem brain tissues after FISH with "classical" satellite DNA probes for chromosomes 1, 9, and 16. This approach has shown a relatively high efficiency for the quantitative registration of chromosomal heteromorphism due to variations of centromeric alphoid DNA in homologous parental chromosomes. We propose this approach to be efficient and to be considered as a useful tool in addition to visual FISH signal analysis for applied molecular cytogenetic studies. (J Histochem Cytochem 53:401–408, 2005)

Sixty blood samples from pregnant women during gestational weeks 9–28 were investigated. Cell-free fetal DNA was extracted from maternal plasma or serum to be detected by nested PCR for determination of fetal gender. The SRY gene as a marker for fetal Y chromosome was detected in 34/36 women carrying a male fetus. In 3/24 women carrying female fetuses, the SRY sequence was also detected. Overall, fetal sex was correctly predicted in 91.7% of the cases. Therefore, the new, non-invasive method of prenatal diagnosis of fetal gender for women at risk of producing children with X-linked disorders is reliable, secure, and can substantially reduce invasive prenatal tests.

(J Histochem Cytochem 53:297–299, 2005)

Green fluorescent protein (GFP) expression was evaluated in tissues of different transgenic rodents—Sprague-Dawley (SD) rat strain [SD-Tg(GFP)Bal], W rat strain [Wistar-TgN(CAG-GFP)184ys], and M mouse strain [Tg(GFPU)5Nagy/J]—by direct fluorescence of native GFP expression and by immunohistochemistry. The constitutively expressing GFP transgenic strains showed tissue-specific differences in GFP expression, and GFP immunohistochemistry amplified the fluorescent signal. The fluorescence of stem/progenitor cells cultured as neurospheres from the ependymal region of the adult spinal cord from the GFP SD and W rat strains was assessed in vitro. After transplantation of the cells into wild-type spinal cord, the ability to track the grafted cells was evaluated in vivo. Cultured stem/progenitor cells from the SD strain required GFP immunostaining to be visualized. Likewise, after transplantation of SD cells into the spinal cord, immunohistochemical amplification of the GFP signal was required for detection. In contrast, GFP expression of stem/progenitor cells generated from the W strain was readily detected by direct fluorescence both in vitro and in vivo without the need for immunohistochemical amplification. The cultured stem/progenitor cells transplanted into the spinal cord survived for at least 49 days after transplantation, and continued to express GFP, demonstrating stable expression of the GFP transgene in vivo. (J Histochem Cytochem 53:1215–1226, 2005)

Green fluorescent protein (GFP) expression was evaluated in tissues of different transgenic rodents—Sprague-Dawley (SD) rat strain [SD-Tg(GFP)Bal], W rat strain [Wistar-TgN(CAG-GFP)184ys], and M mouse strain [Tg(GFPU)5Nagy/J]—by direct fluorescence of native GFP expression and by immunohistochemistry. The constitutively expressing GFP transgenic strains showed tissue-specific differences in GFP expression, and GFP immunohistochemistry amplified the fluorescent signal. The fluorescence of stem/progenitor cells cultured as neurospheres from the ependymal region of the adult spinal cord from the GFP SD and W rat strains was assessed in vitro. After transplantation of the cells into wild-type spinal cord, the ability to track the grafted cells was evaluated in vivo. Cultured stem/progenitor cells from the SD strain required GFP immunostaining to be visualized. Likewise, after transplantation of SD cells into the spinal cord, immunohistochemical amplification of the GFP signal was required for detection. In contrast, GFP expression of stem/progenitor cells generated from the W strain was readily detected by direct fluorescence both in vitro and in vivo without the need for immunohistochemical amplification. The cultured stem/progenitor cells transplanted into the spinal cord survived for at least 49 days after transplantation, and continued to express GFP, demonstrating stable expression of the GFP transgene in vivo. (J Histochem Cytochem 53:1215–1226, 2005)

Here we report a prenatally detected small supernumerary marker chromosome (sSMC) derived from chromosome 2 as demonstrated by cenM-FISH (centromere-specific multicolor fluorescence in situ hybridization). By application of a recently described subcentromere-specific probe set (subcenM-FISH) for chromosome 2, the presence of a small partial trisomy due to a karyotype 47,XX,+r(2)(::p11.1->q11.2::) was demonstrated. Including this case, a total of 11 patients with sSMC(2) are described throughout the literature. Based on that data, a first genotype/phenotype correlation according to the size and structure of the marker is suggested. (J Histochem Cytochem 53:367–370, 2005)

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Antibodies that label paraffin-embedded mouse tissues: a collaborative endeavor.

Toxicol Pathol. 2004 Mar-Apr;32(2):181-91

Authors: Mikaelian I, Nanney LB, Parman KS, Kusewitt DF, Ward JM, Näf D, Krupke DM, Eppig JT, Bult CJ, Seymour R, Ichiki T, Sundberg JP

Histology and immunohistochemistry are important tools in the study of human diseases and their respective animal models. The study of mouse models has been hampered by the absence of a large set of mouse-specific antibodies adapted to paraffin-embedded tissues. A total of 196 antibodies were tested on paraffin-embedded mouse tissues preserved in five different fixatives (Fekete's acid-alcohol-formalin, 10% neutral buffered formalin, 4% paraformaldehyde, IHC Zinc Fixative, and Bouin's fixative). The antibodies were targeted to proteins of the cytoplasm (n = 100), plasma membrane (n = 48), nucleus (n = 36), extracellular compartment (n = 5), cytoplasm/cell membrane (n = 4), and viral proteins (n = 3). A total of 83 antibodies provided an adequate signal to noise ratio. Of these, adequate labeling required heat-mediated epitope retrieval or enzymatic digestion for 32 and 8 antibodies, respectively. Epitope recognition was best for tissues fixed with Fekete's acid-alcohol-formalin. However, some proteins could be detected only in IHC Zinc Fixative, confirming that there is no single fixative suitable for the preservation of all epitopes. Four of 13 antibodies that failed to label their cellular targets on tissue sections successfully labeled whole-mount tissues, indicating that tissue processing plays an important role in epitope degradation. Regularly updated information on immunohistochemistry of normal and neoplastic mouse tissues is accessible online at (http://tumor.informatics.jax.org); links to antibody suppliers' web sites are provided.

PMID: 15200156 [PubMed - indexed for MEDLINE]

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Antibody selection in immunohistochemical detection of cyclin d1 in mantle cell lymphoma.

Am J Clin Pathol. 2005 Nov;124(5):1-8

Authors: Torlakovic E, Nielsen S, Vyberg M

An assessment in Nordic immunohistochemical Quality Control (NordiQC) revealed that only 23% participant laboratories performed optimal staining for detection of cyclin D1 (CyD1) in mantle cell lymphoma (MCL). False-negative results were secondary to suboptimal protocols. We compared the 5 anti-CyD1 antibodies (monoclonal SP4, P2D11F11, and DCS-6 and polyclonal CP236 and 06-137) used in the Scandinavian laboratories. Evaluated were 31 MCLs, 16 other malignant lymphomas, and 19 samples of normal tissues. Sensitivity was as follows: CP236, 100%; SP4, 95%; P2D11F11, 90%; DCS-6, 84%; and 06-137, 53%. SP4 produced the strongest staining. Correlation of CyD1 with the proliferative index was best with polyclonal antibodies CP236 and 06-137. The use of heat-induced epitope retrieval in an alkaline buffer such as 1/10 mmol/L of Tris-EDTA buffer, pH 9, seemed mandatory. For the optimal detection of CyD1 expression, both SP4 and CP236 antibodies should be available in the laboratory.

PMID: 16203276 [PubMed - in process]

Apolipoprotein D (apoD) expression has been shown to correlate both with cell cycle arrest and with prognosis in several types of malignancy, including central nervous system astrocytomas and medulloblastomas. ApoD expression was investigated by real-time quantitative RT-PCR using RNA extracted from 68 formalin-fixed, paraffin-embedded brain specimens. Glyceraldehyde phosphate dehydrogenase was used as an internal control. Quantitation was achieved on all specimens. Sixteen poorly infiltrating WHO grade I glial neoplasms (i.e., pilocytic astrocytomas and gangliogliomas) showed an average 20-fold higher apoD expression level compared with the 20 diffusely infiltrating glial neoplasms (i.e., glioblastoma, anaplastic astrocytoma, oligodendrogliomas; p=0.00004). A small number of exceptions (i.e., two high-expressing glioblastomas and three low-expressing gangliogliomas) were identified. Analyzed as individual tumor groups, poorly infiltrating grade I pilocytic astrocytomas and gangliogliomas differed significantly from each tumor type within the diffusely infiltrating higher-grade category (p<0.05 for each comparison) but not from each other (p>0.05). Conversely, each individual tumor type within the diffusely infiltrating category differed significantly from both pilocytic astrocytomas and gangliogliomas (p<0.05) but did not vary from other infiltrating tumors (p>0.05). Ependymomas, non-infiltrating grade II neoplasms, expressed levels of apoD similar to or lower than levels expressed by the diffusely infiltrating gliomas. Ten medulloblastomas with survival longer than 3 years averaged slightly higher apoD expression than four fatal medulloblastomas; however, this result was not statistically significant and individual exceptions were notable. In 17 of the medulloblastomas, MIB-1 proliferation rates quantitated by image cytometry did not correlate with apoD expression. In addition, apoD expression was 5-fold higher in the slowly proliferating grade I glial neoplasms compared with non-proliferating normal brain tissue (p=0.01), suggesting that apoD expression is not simply an inverse measure of proliferation. ApoD expression measured by quantitative RT-PCR may be useful in the differential diagnosis of primary brain tumors, particularly pilocytic astrocytomas and gangliogliomas. (J Histochem Cytochem 53:963–969, 2005)

Apolipoprotein D (apoD) expression has been shown to correlate both with cell cycle arrest and with prognosis in several types of malignancy, including central nervous system astrocytomas and medulloblastomas. ApoD expression was investigated by real-time quantitative RT-PCR using RNA extracted from 68 formalin-fixed, paraffin-embedded brain specimens. Glyceraldehyde phosphate dehydrogenase was used as an internal control. Quantitation was achieved on all specimens. Sixteen poorly infiltrating WHO grade I glial neoplasms (i.e., pilocytic astrocytomas and gangliogliomas) showed an average 20-fold higher apoD expression level compared with the 20 diffusely infiltrating glial neoplasms (i.e., glioblastoma, anaplastic astrocytoma, oligodendrogliomas; p=0.00004). A small number of exceptions (i.e., two high-expressing glioblastomas and three low-expressing gangliogliomas) were identified. Analyzed as individual tumor groups, poorly infiltrating grade I pilocytic astrocytomas and gangliogliomas differed significantly from each tumor type within the diffusely infiltrating higher-grade category (p<0.05 for each comparison) but not from each other (p>0.05). Conversely, each individual tumor type within the diffusely infiltrating category differed significantly from both pilocytic astrocytomas and gangliogliomas (p<0.05) but did not vary from other infiltrating tumors (p>0.05). Ependymomas, non-infiltrating grade II neoplasms, expressed levels of apoD similar to or lower than levels expressed by the diffusely infiltrating gliomas. Ten medulloblastomas with survival longer than 3 years averaged slightly higher apoD expression than four fatal medulloblastomas; however, this result was not statistically significant and individual exceptions were notable. In 17 of the medulloblastomas, MIB-1 proliferation rates quantitated by image cytometry did not correlate with apoD expression. In addition, apoD expression was 5-fold higher in the slowly proliferating grade I glial neoplasms compared with non-proliferating normal brain tissue (p=0.01), suggesting that apoD expression is not simply an inverse measure of proliferation. ApoD expression measured by quantitative RT-PCR may be useful in the differential diagnosis of primary brain tumors, particularly pilocytic astrocytomas and gangliogliomas. (J Histochem Cytochem 53:963–969, 2005)

Intranuclear localization of signal molecules and chromosome territories has become more attractive in relation to postgenomic analyses of cellular functions. Cryotechniques and freeze-substitution (CrT-FS) have been generally used for electron microscopic observation to obtain better ultrastructure and immunoreactivity. To investigate benefits of applying the CrT-FS method to immunostaining of intranuclear signal molecules and FISH for chromosome territories, we performed an immunohistochemical study of phosphorylated cAMP-responsive element binding protein (pCREB) in mouse cerebellar tissues and a FISH study of chromosome 18 territory in human thyroid tissues using various cryotechniques. The immunoreactivity of pCREB was more clearly detected without antigen retrieval treatment on sections prepared by the CrT-FS method than those prepared by the conventional dehydration method. In the FISH study, more definite probe labeling of the chromosome territory could be obtained on paraffin sections by the CrT-FS method without microwave treatment, although such labeling was not clear even with microwave treatment on sections prepared by the routine dehydration method. The CrT-FS preserved relatively native morphology by preventing shrinkage of nuclei, and produced better immunoreactivity. Because the reduction of routine pretreatments in the present study might reveal more native morphology, the CrT-FS method would be a useful technique for intranuclear immunostaining and FISH. (J Histochem Cytochem 53:55–62, 2005)

Non-invasive prenatal diagnosis tests based on the analysis of fetal DNA in maternal plasma have potential to be a safer alternative to invasive methods. So far, different studies have shown mainly fetal sex, fetal RhD, and quantitative variations of fetal DNA during gestation with fetal chromosomal anomalies or gestations at risk for preeclampsia. The objective of our research was to evaluate the use of fetal DNA in maternal plasma for clinical application. In our study, we have established the methodology needed for the analysis of fetal DNA. Different methods were used, according to the requirements of the assay. We have used quantitative fluorescent polymerase chain reaction (QF-PCR) to perform fetal sex detection with 90% sensitivity. The same technique permitted the detection of fetal DNA from the 10th week of gestation to hours after delivery. We have successfully carried out the diagnosis of two inherited disorders, cystic fibrosis (conventional PCR and restriction analysis) and Huntington disease (QF-PCR). Ninety percent of the cases studied for fetal RhD by real-time PCR were correctly diagnosed. The detection of fetal DNA sequences is a reality and could reduce the risk of invasive techniques for certain fetal disorders in the near future. (J Histochem Cytochem 53:307–314, 2005)

Murine arylamine N-acetyltransferase 2 (NAT2) is expressed in the developing heart and in the neural tube at the time of closure. Classically described as a xenobiotic metabolizing enzyme, there is increasing evidence for a distinct biological role for murine NAT2. We have characterized the expression of arylamine N-acetyltransferase 2 during cardiogenesis, mapping its expression in vivo, using a lacZ insertion deletion, and also in vitro, by measuring NAT2 enzyme activity. These findings show that cardiac Nat2 expression is both temporally and spatially regulated during development. In neonatal mice, cardiac Nat2 expression is most extensive in the central fibrous body and is evident in the atrioventricular valves and the valves of the great vessels. Whereas Nat2 expression is not detected in ventricular myocardial cells, Nat2 is strongly expressed in scattered cells in the region of the sinus node, the epicardium of the right atrial appendage, and in the pulmonary artery. Expression of active NAT2 protein is maximal when the developing heart attains the adult circulation pattern and moves from metabolizing glucose to fatty acids. NAT2 acetylating activity in cardiac tissue from Nat2^–/– and Nat2^+/– mice indicates a lack of compensating acetylating activity either from other acetylating enzymes or by NAT2 encoded by the wild-type Nat2 allele in Nat2^+/– heterozygotes. The temporal and spatial control of murine Nat2 expression points to an endogenous role distinct from xenobiotic metabolism and indicates that Nat2 expression may be useful as a marker in cardiac development.

(J Histochem Cytochem 53:583–592, 2005)

Murine arylamine N-acetyltransferase 2 (NAT2) is expressed in the developing heart and in the neural tube at the time of closure. Classically described as a xenobiotic metabolizing enzyme, there is increasing evidence for a distinct biological role for murine NAT2. We have characterized the expression of arylamine N-acetyltransferase 2 during cardiogenesis, mapping its expression in vivo, using a lacZ insertion deletion, and also in vitro, by measuring NAT2 enzyme activity. These findings show that cardiac Nat2 expression is both temporally and spatially regulated during development. In neonatal mice, cardiac Nat2 expression is most extensive in the central fibrous body and is evident in the atrioventricular valves and the valves of the great vessels. Whereas Nat2 expression is not detected in ventricular myocardial cells, Nat2 is strongly expressed in scattered cells in the region of the sinus node, the epicardium of the right atrial appendage, and in the pulmonary artery. Expression of active NAT2 protein is maximal when the developing heart attains the adult circulation pattern and moves from metabolizing glucose to fatty acids. NAT2 acetylating activity in cardiac tissue from Nat2^–/– and Nat2^+/– mice indicates a lack of compensating acetylating activity either from other acetylating enzymes or by NAT2 encoded by the wild-type Nat2 allele in Nat2^+/– heterozygotes. The temporal and spatial control of murine Nat2 expression points to an endogenous role distinct from xenobiotic metabolism and indicates that Nat2 expression may be useful as a marker in cardiac development.

(J Histochem Cytochem 53:583–592, 2005)

Murine arylamine N-acetyltransferase 2 (NAT2) is expressed in the developing heart and in the neural tube at the time of closure. Classically described as a xenobiotic metabolizing enzyme, there is increasing evidence for a distinct biological role for murine NAT2. We have characterized the expression of arylamine N-acetyltransferase 2 during cardiogenesis, mapping its expression in vivo, using a lacZ insertion deletion, and also in vitro, by measuring NAT2 enzyme activity. These findings show that cardiac Nat2 expression is both temporally and spatially regulated during development. In neonatal mice, cardiac Nat2 expression is most extensive in the central fibrous body and is evident in the atrioventricular valves and the valves of the great vessels. Whereas Nat2 expression is not detected in ventricular myocardial cells, Nat2 is strongly expressed in scattered cells in the region of the sinus node, the epicardium of the right atrial appendage, and in the pulmonary artery. Expression of active NAT2 protein is maximal when the developing heart attains the adult circulation pattern and moves from metabolizing glucose to fatty acids. NAT2 acetylating activity in cardiac tissue from Nat2^–/– and Nat2^+/– mice indicates a lack of compensating acetylating activity either from other acetylating enzymes or by NAT2 encoded by the wild-type Nat2 allele in Nat2^+/– heterozygotes. The temporal and spatial control of murine Nat2 expression points to an endogenous role distinct from xenobiotic metabolism and indicates that Nat2 expression may be useful as a marker in cardiac development.

(J Histochem Cytochem 53:583–592, 2005)

Apoptosis competence is central to the prevention of cancer. Frequency of apoptotic cells, after a sample of colonic tissue is stressed, can be used to gauge apoptosis competence and, thus, possible susceptibility to colon cancer. The gold standard for assessment of apoptosis is morphological evaluation, but this requires an experienced microscopist. Easier-to-use immunohistochemical markers of apoptosis, applicable in archived paraffin-embedded tissue, have been commercially developed. Potentially useful apoptosis markers include cleaved cytokeratin-18 (c-CK18), cleaved caspase-3 (c-cas-3), cleaved lamin A (c-lam-A), phosphorylated histone H2AX (H2AX), cleaved poly(ADP ribose) polymerase (c-PARP), and translocation of apoptosis-inducing factor (AIF). When tissue samples from freshly resected colon segments were challenged ex vivo with the bile acid deoxycholate, ~50% of goblet cells became apoptotic by morphologic criteria. This high level of morphologic apoptosis allowed quantitative comparison with the usefulness and specificity of immunohistochemical markers of apoptosis. The antibody to c-CK18 was almost as useful and about as specific as morphology for identifying apoptotic colonic epithelial cells. Antibodies to c-cas-3, c-lam-A, and H2AX, though specific for apoptotic cells, were less useful. The antibody to c-PARP, though specific for apoptotic cells, had low usefulness, and the antibody to AIF was relatively nonspecific, under our conditions.

(J Histochem Cytochem 53:229–235, 2005)

The recent success of pancreatic islet transplantation has generated considerable enthusiasm. To better understand the quality and characteristics of human islets used for transplantation, we performed detailed analysis of islet architecture and composition using confocal laser scanning microscopy. Human islets from six separate isolations provided by three different islet isolation centers were compared with isolated mouse and non-human primate islets. As expected from histological sections of murine pancreas, in isolated murine islets and cells resided at the periphery of the ßbeta;-cell core. However, human islets were markedly different in that , ßbeta;, and cells were dispersed throughout the islet. This pattern of cell distribution was present in all human islet preparations and islets of various sizes and was also seen in histological sections of human pancreas. The architecture of isolated non-human primate islets was very similar to that of human islets. Using an image analysis program, we calculated the volume of , ßbeta;, and cells. In contrast to murine islets, we found that populations of islet cell types varied considerably in human islets. The results indicate that human islets not only are quite heterogeneous in terms of cell composition but also have a substantially different architecture from widely studied murine islets.

(J Histochem Cytochem 53:1087–1097, 2005)

Traditional histological descriptions of the pancreas distinguish between the exocrine and the endocrine pancreas, as if they were two functionally distinct glands. This view has been proven incorrect and can be considered obsolete. Interactions between acinar and islet tissues have been well established through numerous studies that reveal the existence of anatomical and functional relationships between these compartments of the gland. Less attention, however, has traditionally been paid to the relationships occurring between the endocrine pancreas and the ductal system. Associations between islet tissue and ducts are considered by most researchers as only a transient epiphenomenon of endocrine development. This article reviews the evidence that has emerged in the last 10 years demonstrating the existence of stable, close, and systematic relationships between these two pancreatic compartments. Functional and pathophysiological implications are considered, and the existence of an "acinar–duct–islet" axis is put forward. The pancreas appears at present to be an integrated organ composed of three functionally related components of well-orchestrated endocrine and exocrine physiological responses. (J Histochem Cytochem 53:1071–1086, 2005)

Rupture of vulnerable atherosclerotic plaques that are characterized by extensive neointimal accumulation of lipid is a cause of acute coronary syndromes. To identify whether insulin resistance alters atherogenesis, we characterized the composition of atherosclerotic lesions in the proximal aortas in mice deficient in apolipoprotein E (ApoE^–/–) and in ApoE^–/– mice in which insulin resistance was intensified by a concomitant heterozygous deficiency in insulin receptor substrate type 2 (IRS2^+/– ApoE^–/– mice). In addition, we characterized the effect of an insulin sensitizer, pioglitazone, on the atherogenesis in IRS2^+/– ApoE^–/– mice. The extent of the aortic intima occupied by lesion was increased in the IRS2^+/– ApoE^–/– compared with ApoE^–/– mice (79 ± 3% compared with 68 ± 8%, p<0.05). Treatment with pioglitazone decreased the neointimal content of lipid in 20-week-old mice from 50 ± 6% to 30 ± 7%, p=0.005 and decreased the cellularity reflected by the multisection cross-sectional areas of lesions comprising cells in atheroma from 24 ± 1% to 19 ± 3%, p=0.018. Accordingly, genetically induced intensification of insulin resistance increases atheroma formation. Furthermore, attenuation of insulin resistance by treatment with pioglitazone decreases accumulation of lipid in the neointima.

(J Histochem Cytochem 53:603–610, 2005)

Rupture of vulnerable atherosclerotic plaques that are characterized by extensive neointimal accumulation of lipid is a cause of acute coronary syndromes. To identify whether insulin resistance alters atherogenesis, we characterized the composition of atherosclerotic lesions in the proximal aortas in mice deficient in apolipoprotein E (ApoE^–/–) and in ApoE^–/– mice in which insulin resistance was intensified by a concomitant heterozygous deficiency in insulin receptor substrate type 2 (IRS2^+/– ApoE^–/– mice). In addition, we characterized the effect of an insulin sensitizer, pioglitazone, on the atherogenesis in IRS2^+/– ApoE^–/– mice. The extent of the aortic intima occupied by lesion was increased in the IRS2^+/– ApoE^–/– compared with ApoE^–/– mice (79 ± 3% compared with 68 ± 8%, p<0.05). Treatment with pioglitazone decreased the neointimal content of lipid in 20-week-old mice from 50 ± 6% to 30 ± 7%, p=0.005 and decreased the cellularity reflected by the multisection cross-sectional areas of lesions comprising cells in atheroma from 24 ± 1% to 19 ± 3%, p=0.018. Accordingly, genetically induced intensification of insulin resistance increases atheroma formation. Furthermore, attenuation of insulin resistance by treatment with pioglitazone decreases accumulation of lipid in the neointima.

(J Histochem Cytochem 53:603–610, 2005)

Rupture of vulnerable atherosclerotic plaques that are characterized by extensive neointimal accumulation of lipid is a cause of acute coronary syndromes. To identify whether insulin resistance alters atherogenesis, we characterized the composition of atherosclerotic lesions in the proximal aortas in mice deficient in apolipoprotein E (ApoE^–/–) and in ApoE^–/– mice in which insulin resistance was intensified by a concomitant heterozygous deficiency in insulin receptor substrate type 2 (IRS2^+/– ApoE^–/– mice). In addition, we characterized the effect of an insulin sensitizer, pioglitazone, on the atherogenesis in IRS2^+/– ApoE^–/– mice. The extent of the aortic intima occupied by lesion was increased in the IRS2^+/– ApoE^–/– compared with ApoE^–/– mice (79 ± 3% compared with 68 ± 8%, p<0.05). Treatment with pioglitazone decreased the neointimal content of lipid in 20-week-old mice from 50 ± 6% to 30 ± 7%, p=0.005 and decreased the cellularity reflected by the multisection cross-sectional areas of lesions comprising cells in atheroma from 24 ± 1% to 19 ± 3%, p=0.018. Accordingly, genetically induced intensification of insulin resistance increases atheroma formation. Furthermore, attenuation of insulin resistance by treatment with pioglitazone decreases accumulation of lipid in the neointima.

(J Histochem Cytochem 53:603–610, 2005)

Related Articles

B(7) molecule mRNA expression in colorectal carcinoma.

World J Gastroenterol. 2005 Sep 28;11(36):5655-8

Authors: Xiao JX, Bai PS, Lai BC, Li L, Zhu J, Wang YL

AIM: To observe the status of tumor-associated B(7) molecule mRNA expression in human colorectal cancer tissue by in situ hybridization. METHODS: The mRNA expression patterns of cancer-associated B(7-1),B(7)H(1),B(7)H(2),ICOS in 22 specimens of human colorectal cancer tissue were monitored by in situ hybridization (ISH) with digoxin-labeled oligonucleotide probes. RESULTS: B(7-1),B(7)H(1),B(7)H(2),ICOS mRNA were detected in both cancer cells and tumor infiltrating lymphocytes (TIL). The mRNA expression level of these molecules in tumor cells was higher than that in TIL (0.76+/-0.54-1.62+/-0.82 vs 0.38+/-0.19-0.65+/-0.33, P<0.001). There was no relationship between expression level of tested B(7) family molecules and patients' sex, age, differentiation status of cancer and regional lymph node metastasis. CONCLUSION: Th2 cytokine predominant in tumor microenvironment might be related to the expression of B(7)H(1),B(7)H(2) co-signal molecules in tumor cells and TIL.

PMID: 16237760 [PubMed - in process]

To elucidate the behavior of autologously transplanted mesenchymal cells in osteochondral defects, we followed transplanted cells using green fluorescent protein (GFP) transgenic rats, in which all cells express GFP signals in their cytoplasm and nuclei as transplantation donors. Bone marrow–derived mesenchymal cells, which contain mesenchymal stem cells (MSCs), were obtained from transgenic rats. Then, dense mesenchymal cell masses created by hanging-drop culture were transplanted and fixed with fibrin glue into osteochondral defects of wild-type rats. At 24 weeks after surgery, the defects were repaired with hyaline-like cartilage and subchondral bone. GFP positive cells, indicating transplanted mesenchymal-derived cells, were observed in the regenerated tissues for 24 weeks although GFP positive cells decreased in number with time. Because GFP causes no immunological rejection and requires no chemicals for visualization, transplantation between transgenic and wild-type rats can be regarded as a simulation of autologous transplantation, and the survivability of transplanted cells are able to be followed easily and reliably. Thus, the behavior of transplanted mesenchymal cells was able to be elucidated in vivo by this strategy, and the results could be essential in future tissue engineering for the regeneration of osteochondral defects with original hyaline cartilage and subchondral bone. (J Histochem Cytochem 53:207–216, 2005)

Biological dosimetry in a group of radiologists by the analysis of dicentrics and translocations.

Radiat Res. 2005 Nov;164(5):612-7

Authors: Montoro A, Rodríguez P, Almonacid M, Villaescusa JI, Verdú G, Caballín MR, Barrios L, Barquinero JF

Montoro, A., Rodríguez, P., Almonacid, M., Villaescusa, J. I., Verdú, G., Caballín, M. R., Barrios, L. and Barquinero, J. F. Biological Dosimetry in a Group of Radiologists by the Analysis of Dicentrics and Translocations. Radiat. Res. 164, 612-617 (2005). The results of a cytogenetic study carried out in a group of nine radiologists are presented. Chromosome aberrations were detected by fluorescence plus Giemsa staining and fluorescence in situ hybridization. Dose estimates were obtained by extrapolating the yield of dicentrics and translocations to their respective dose-effect curves. In seven individuals, the 95% confidence limits of the doses estimated by dicentrics did not include 0 Gy. The 99 dicentrics observed in 17,626 cells gave a collective estimated dose of 115 mGy (95% confidence limits 73-171). For translocations, five individuals had estimated doses that were clearly higher than the total accumulated recorded dose. The 82 total apparently simple translocations observed in 9722 cells gave a collective estimated dose of 275 mGy (132-496). The mean genomic frequencies (x100 +/- SE) of complete and total apparently simple translocations observed in the group of radiologists (1.91 +/- 0.30 and 2.67 +/- 0.34, respectively) were significantly higher than those observed in a matched control group (0.53 +/- 0.10 and 0.87 +/- 0.13, P < 0.01 in both cases) and in another occupationally exposed matched group (0.79 +/- 0.12 and 1.14 +/-0.14, P < 0.03 and P < 0.01, respectively). The discrepancies observed between the physically recorded doses and the biologically estimated doses indicate that the radiologists did not always wear their dosimeters or that the dosimeters were not always in the radiation field.

PMID: 16238438 [PubMed - in process]

Breast cancer is a major cause of cancer-related mortality in women. There are major discrepancies concerning the usefulness of various antibodies in detecting breast cancer susceptibility gene 1 (BRCA1) protein and its subcellular localization. The aim of the present study was to determine the specificity and sensitivity of immunohistochemistry (IHC) as a screening method for demonstrating BRCA1 expression. BRCA1 gene expression in archival paraffin-embedded breast cancer tissues was studied simultaneously at the protein and mRNA levels, and the two findings were compared. Forty-eight archival paraffin-embedded breast cancer tissues were studied for BRCA1 gene expression at protein level by IHC using four different antibodies against different BRCA1 epitopes and at mRNA level using real-time RT-PCR. BRCA1 mRNA expression was reduced or absent in 79% of the samples, and this finding correlated significantly with loss of BRCA1 protein expression in 83% of breast cancer tissues using one BRCA1 antibody studied (AB-1, against N-terminus epitope). The specificity of this antibody was 91.3%, and its sensitivity was 66.6%. There was no significant correlation between BRCA1 mRNA and protein expression as demonstrated by the remaining three antibodies. Antibody 8F7 had the highest sensitivity of 100%, but its specificity was 30.4% if mRNA levels were considered as the reference standard. (J Histochem Cytochem 53:621–629, 2005)

Breast cancer is a major cause of cancer-related mortality in women. There are major discrepancies concerning the usefulness of various antibodies in detecting breast cancer susceptibility gene 1 (BRCA1) protein and its subcellular localization. The aim of the present study was to determine the specificity and sensitivity of immunohistochemistry (IHC) as a screening method for demonstrating BRCA1 expression. BRCA1 gene expression in archival paraffin-embedded breast cancer tissues was studied simultaneously at the protein and mRNA levels, and the two findings were compared. Forty-eight archival paraffin-embedded breast cancer tissues were studied for BRCA1 gene expression at protein level by IHC using four different antibodies against different BRCA1 epitopes and at mRNA level using real-time RT-PCR. BRCA1 mRNA expression was reduced or absent in 79% of the samples, and this finding correlated significantly with loss of BRCA1 protein expression in 83% of breast cancer tissues using one BRCA1 antibody studied (AB-1, against N-terminus epitope). The specificity of this antibody was 91.3%, and its sensitivity was 66.6%. There was no significant correlation between BRCA1 mRNA and protein expression as demonstrated by the remaining three antibodies. Antibody 8F7 had the highest sensitivity of 100%, but its specificity was 30.4% if mRNA levels were considered as the reference standard. (J Histochem Cytochem 53:621–629, 2005)

Breast cancer is a major cause of cancer-related mortality in women. There are major discrepancies concerning the usefulness of various antibodies in detecting breast cancer susceptibility gene 1 (BRCA1) protein and its subcellular localization. The aim of the present study was to determine the specificity and sensitivity of immunohistochemistry (IHC) as a screening method for demonstrating BRCA1 expression. BRCA1 gene expression in archival paraffin-embedded breast cancer tissues was studied simultaneously at the protein and mRNA levels, and the two findings were compared. Forty-eight archival paraffin-embedded breast cancer tissues were studied for BRCA1 gene expression at protein level by IHC using four different antibodies against different BRCA1 epitopes and at mRNA level using real-time RT-PCR. BRCA1 mRNA expression was reduced or absent in 79% of the samples, and this finding correlated significantly with loss of BRCA1 protein expression in 83% of breast cancer tissues using one BRCA1 antibody studied (AB-1, against N-terminus epitope). The specificity of this antibody was 91.3%, and its sensitivity was 66.6%. There was no significant correlation between BRCA1 mRNA and protein expression as demonstrated by the remaining three antibodies. Antibody 8F7 had the highest sensitivity of 100%, but its specificity was 30.4% if mRNA levels were considered as the reference standard. (J Histochem Cytochem 53:621–629, 2005)

Cardiac apoptosis in severe relapsing Fever borreliosis.

Infect Immun. 2005 Nov;73(11):7669-76

Authors: Londoño D, Bai Y, Zückert WR, Gelderblom H, Cadavid D

Previous studies revealed that the heart suffers significant injury during experimental Lyme and relapsing fever borreliosis when the immune response is impaired (D. Cadavid, Y. Bai, E. Hodzic, K. Narayan, S. W. Barthold, and A. R. Pachner, Lab. Investig. 84:1439-1450, 2004; D. Cadavid, T. O'Neill, H. Schaefer, and A. R. Pachner, Lab. Investig. 80:1043-1054, 2000; and D. Cadavid, D. D. Thomas, R. Crawley, and A. G. Barbour, J. Exp. Med. 179:631-642, 1994). To investigate cardiac injury in borrelia carditis, we used antibody-deficient mice persistently infected with isogenic serotypes of the relapsing fever agent Borrelia turicatae. We studied infection in hearts 1 to 2 months after inoculation by TaqMan reverse transcription-PCR and immunohistochemistry (IHC) and inflammation by hematoxylin and eosin and trichrome staining, IHC, and in situ hybridization (ISH). We studied apoptosis by terminal transferase-mediated DNA nick end labeling assay and measured expression of apoptotic molecules by RNase protection assay, immunofluorescence, and immunoblot. All antibody-deficient mice, but none of the immunocompetent controls, developed persistent infection of the heart. Antibody-deficient mice infected with serotype 2 had more severe cardiac infection and injury than serotype 1-infected mice. The injury was more severe around the base of the heart and pericardium, corresponding to sites of marked infiltration by activated macrophages and upregulation of interleukin-6 (IL-6). Infected hearts showed evidence of apoptosis of macrophages and cardiomyocytes as well as significant upregulation of caspases, most notably caspase-1. We conclude that persistent infection with relapsing fever borrelias causes significant loss of cardiomyocytes associated with prominent infiltration by activated macrophages, upregulation of IL-6, induction of caspase-1, and apoptosis.

PMID: 16239571 [PubMed - in process]

Cardiac apoptosis in severe relapsing Fever borreliosis.

Infect Immun. 2005 Nov;73(11):7669-76

Authors: Londoño D, Bai Y, Zückert WR, Gelderblom H, Cadavid D

Previous studies revealed that the heart suffers significant injury during experimental Lyme and relapsing fever borreliosis when the immune response is impaired (D. Cadavid, Y. Bai, E. Hodzic, K. Narayan, S. W. Barthold, and A. R. Pachner, Lab. Investig. 84:1439-1450, 2004; D. Cadavid, T. O'Neill, H. Schaefer, and A. R. Pachner, Lab. Investig. 80:1043-1054, 2000; and D. Cadavid, D. D. Thomas, R. Crawley, and A. G. Barbour, J. Exp. Med. 179:631-642, 1994). To investigate cardiac injury in borrelia carditis, we used antibody-deficient mice persistently infected with isogenic serotypes of the relapsing fever agent Borrelia turicatae. We studied infection in hearts 1 to 2 months after inoculation by TaqMan reverse transcription-PCR and immunohistochemistry (IHC) and inflammation by hematoxylin and eosin and trichrome staining, IHC, and in situ hybridization (ISH). We studied apoptosis by terminal transferase-mediated DNA nick end labeling assay and measured expression of apoptotic molecules by RNase protection assay, immunofluorescence, and immunoblot. All antibody-deficient mice, but none of the immunocompetent controls, developed persistent infection of the heart. Antibody-deficient mice infected with serotype 2 had more severe cardiac infection and injury than serotype 1-infected mice. The injury was more severe around the base of the heart and pericardium, corresponding to sites of marked infiltration by activated macrophages and upregulation of interleukin-6 (IL-6). Infected hearts showed evidence of apoptosis of macrophages and cardiomyocytes as well as significant upregulation of caspases, most notably caspase-1. We conclude that persistent infection with relapsing fever borrelias causes significant loss of cardiomyocytes associated with prominent infiltration by activated macrophages, upregulation of IL-6, induction of caspase-1, and apoptosis.

PMID: 16239571 [PubMed - in process]

Individual cardiomyocytes are lengthened in dilated cardiomyopathy. However, it is not known how the new sarcomeres are added to preexisting myofibrils. Using a three-dimensional microtextured culturing system, a 10% mechanical static strain was applied to aligned, well-attached cardiomyocytes from neonatal rat. The morphology of the myofibrils and the ends of the myocytes were examined. Disruptions of the sarcomeric pattern for actin showed a progression from weak to intense staining over 4 hr. The lightly stained sarcomeres were common at 1 hr after being strained, peaked at 2 hr, and then subsided. In contrast, the numbers of intensely stained sarcomeres were initially low, peaked at 3 hr, and then began to decline when compared with control values. The myocyte ends showed elongations and convolutions after 3 hr and 4 hr of mechanical strain when observed with -actinin and N-cadherin staining. We suggest that myocytes from neonatal rat hearts remodel by insertion of new sarcomeres throughout the cell length and also by enhancement at the intercalated discs. (J Histochem Cytochem 53:839–844, 2005)

Individual cardiomyocytes are lengthened in dilated cardiomyopathy. However, it is not known how the new sarcomeres are added to preexisting myofibrils. Using a three-dimensional microtextured culturing system, a 10% mechanical static strain was applied to aligned, well-attached cardiomyocytes from neonatal rat. The morphology of the myofibrils and the ends of the myocytes were examined. Disruptions of the sarcomeric pattern for actin showed a progression from weak to intense staining over 4 hr. The lightly stained sarcomeres were common at 1 hr after being strained, peaked at 2 hr, and then subsided. In contrast, the numbers of intensely stained sarcomeres were initially low, peaked at 3 hr, and then began to decline when compared with control values. The myocyte ends showed elongations and convolutions after 3 hr and 4 hr of mechanical strain when observed with -actinin and N-cadherin staining. We suggest that myocytes from neonatal rat hearts remodel by insertion of new sarcomeres throughout the cell length and also by enhancement at the intercalated discs. (J Histochem Cytochem 53:839–844, 2005)

The multidrug-resistant (MDR)-1 gene-encoded P-glycoprotein (Pgp-170) is not normally present in the cardiomyocyte. Given that in other tissues Pgp-170 is not found under normoxic conditions but is expressed during hypoxia, we searched for Pgp-170 in chronically ischemic porcine cardiomyocytes. Pgp-170 was detected and localized via immunohistochemistry in ischemic and nonischemic cardiomyocytes of eight adult pigs 8 weeks after placement of an Ameroid constrictor at the origin of the left circumflex artery (Cx). Regional myocardial ischemia in the Cx bed was documented with nuclear perfusion scans. Pgp-170 mass was quantified using Western blot analysis. In all pigs, Pgp-170 was consistently present in the sarcolemma and T invaginations of the cardiomyocytes of the ischemic zone. Pgp-170 expression decreased toward the border of the ischemic zone and was negative in nonischemic regions as well as in the myocardium of sham-operated animals. Western blot analysis yielded significantly higher Pgp-170 mass in ischemic than in nonischemic areas. We conclude that Pgp-170 is consistently expressed in the cardiomyocytes of chronically ischemic porcine myocardium. Its role in the ischemic heart as well as in conditions such as myocardial hibernation, stunning, and preconditioning may have potentially relevant clinical implications and merits further investigation.

(J Histochem Cytochem 53:845–850, 2005)

The multidrug-resistant (MDR)-1 gene-encoded P-glycoprotein (Pgp-170) is not normally present in the cardiomyocyte. Given that in other tissues Pgp-170 is not found under normoxic conditions but is expressed during hypoxia, we searched for Pgp-170 in chronically ischemic porcine cardiomyocytes. Pgp-170 was detected and localized via immunohistochemistry in ischemic and nonischemic cardiomyocytes of eight adult pigs 8 weeks after placement of an Ameroid constrictor at the origin of the left circumflex artery (Cx). Regional myocardial ischemia in the Cx bed was documented with nuclear perfusion scans. Pgp-170 mass was quantified using Western blot analysis. In all pigs, Pgp-170 was consistently present in the sarcolemma and T invaginations of the cardiomyocytes of the ischemic zone. Pgp-170 expression decreased toward the border of the ischemic zone and was negative in nonischemic regions as well as in the myocardium of sham-operated animals. Western blot analysis yielded significantly higher Pgp-170 mass in ischemic than in nonischemic areas. We conclude that Pgp-170 is consistently expressed in the cardiomyocytes of chronically ischemic porcine myocardium. Its role in the ischemic heart as well as in conditions such as myocardial hibernation, stunning, and preconditioning may have potentially relevant clinical implications and merits further investigation.

(J Histochem Cytochem 53:845–850, 2005)

CD117/c-kit positive interstitial (Cajal-like) cells in human pancreas.

J Cell Mol Med. 2005 Jul-Sep;9(3):738-9

Authors: Popescu LM, Hinescu ME, Radu E, Ciontea SM, Cretoiu D, Leabu M, Ardeleanu C

We provide evidence that interstitial Cajal-like cells, previously described in human pancreas - pICC (J Cel Mol Med, 9: 169, 2005), are positive for c-kit irrespective of immunohistochemical procedures used. Various sample types (fresh cryosections or formalin-fixed, paraffin-embedded specimens), various slide pretreatments (with or without heat-induced epitope retrieval) or different antibodies used (Dako polyclonal or Santa Cruz monoclonal), all showed CD117-positive pICC.

PMID: 16202222 [PubMed - in process]

Cyclosporine A (CsA) is the first immunosuppressant used in allotransplantation. Its use is associated with side effects that include nephrotoxicity. This study explored the anatomic structures involved in CsA nephrotoxicity and the effect of heme oxygenase (HO) in preventing CsA injury. Rats were divided into four groups, which were treated with olive oil, CsA (15 mg/kg/day), CsA plus the HO inhibitor (SnMP; 30 µM/kg/day), and with the HO inducer (CoPP; 5 mg/100 g bw). Renal tissue was treated for morphological, biochemical, and immunohistochemical studies. CsA-treated rats showed degenerative changes with renal fibrosis localized mainly around proximal tubules. Collapsed vessels were sometimes seen in glomeruli. No HO-1 expression and increased expression of endothelin-1 (ET-1) were observed in CsA-treated rats compared with controls. In CsA plus SnMP-treated rats, HO-1 expression was further reduced and the morphology was not changed compared to the CsA group, whereas CsA plus CoPP-treated animals again showed normal morphology and with restoration and an increase in HO-1 levels. HO activity and immunohistochemical data showed similar alterations as HO expression. No changes were observed for HO-2 analysis. The observations indicate that HO-1 downregulation and ET-1 upregulation by CsA might be one mechanism underlying CsA-induced nephrotoxicity. Therefore, attempts to preserve HO levels attenuate CsA nephrotoxicity. (J Histochem Cytochem 53:105–112, 2005)

Osteocrin (Ostn), a bone-active molecule, has been shown in animals to be highly expressed in cells of the osteoblast lineage. We have characterized this protein in human cultured primary human osteoblasts, in developing human neonatal bone, and in iliac crest bone biopsies from adult women. In vivo, Ostn expression was localized in developing human neonatal rib bone, with intense immunoreactivity in osteoblasts on bone-forming surfaces, in newly incorporated osteocytes, and in some late hypertrophic chondrocytes. In adult bone, Ostn expression was specifically localized to osteoblasts and young osteocytes at bone-forming sites. In vitro, Ostn expression decreased time dependently (p<0.02) in osteoblasts cultured for 2, 3, and 6 days. Expression was further decreased in cultures containing 200 nM hydrocortisone by 1.5-, 2.3-, and 3.1-fold (p<0.05) at the same time points. In contrast, alkaline phosphatase expression increased with osteoblast differentiation (p<0.05). Low-dose estradiol decreased Ostn expression time dependently (p<0.05), whereas Ostn expression in cultures treated with high-dose estradiol was not significantly changed. These results demonstrate that Ostn is expressed in human skeletal tissue, particularly in osteoblasts in developing bone and at sites of bone remodeling, suggesting a role in bone formation. Thus, Ostn provides a marker of osteoblast lineage cells and appears to correlate with osteoblast activity. (J Histochem Cytochem 53:1181–1187, 2005)

Osteocrin (Ostn), a bone-active molecule, has been shown in animals to be highly expressed in cells of the osteoblast lineage. We have characterized this protein in human cultured primary human osteoblasts, in developing human neonatal bone, and in iliac crest bone biopsies from adult women. In vivo, Ostn expression was localized in developing human neonatal rib bone, with intense immunoreactivity in osteoblasts on bone-forming surfaces, in newly incorporated osteocytes, and in some late hypertrophic chondrocytes. In adult bone, Ostn expression was specifically localized to osteoblasts and young osteocytes at bone-forming sites. In vitro, Ostn expression decreased time dependently (p<0.02) in osteoblasts cultured for 2, 3, and 6 days. Expression was further decreased in cultures containing 200 nM hydrocortisone by 1.5-, 2.3-, and 3.1-fold (p<0.05) at the same time points. In contrast, alkaline phosphatase expression increased with osteoblast differentiation (p<0.05). Low-dose estradiol decreased Ostn expression time dependently (p<0.05), whereas Ostn expression in cultures treated with high-dose estradiol was not significantly changed. These results demonstrate that Ostn is expressed in human skeletal tissue, particularly in osteoblasts in developing bone and at sites of bone remodeling, suggesting a role in bone formation. Thus, Ostn provides a marker of osteoblast lineage cells and appears to correlate with osteoblast activity. (J Histochem Cytochem 53:1181–1187, 2005)

Facial and calvarial bones form intramembranously without a cartilagenous model; however, cultured chick calvarial mesenchyme cells may differentiate into both osteoblasts and chondroblasts and, in rodents, small cartilages occasionally form at the sutures in vivo. Therefore, we wanted to investigate what factors regulate normal differentiation of calvarial mesenchymal cells directly into osteoblasts. In embryonic mouse heads and in cultured tissue explants, we analyzed the expression of selected transcription factors and extracellular matrix molecules associated with bone and cartilage development. Cartilage markers Sox9 and type II collagen were expressed in all craniofacial cartilages. In addition, Msx2 and type I collagen were expressed in sense capsule cartilages. We also observed that the undifferentiated calvarial mesenchyme and the osteogenic fronts in the jaw expressed Col2A1. Moreover, we found that cultured mouse calvarial mesenchyme could develop into cartilage. Of the 49 explants that contained mesenchyme, intramembranous ossification occurred in 35%. Only cartilage formed in 4%, and both cartilage and bone formed in 4%. Our study confirms that calvarial mesenchyme, which normally gives rise to intramembranous bone, also has chondrogenic potential.

(J Histochem Cytochem 53:653–663, 2005)

Facial and calvarial bones form intramembranously without a cartilagenous model; however, cultured chick calvarial mesenchyme cells may differentiate into both osteoblasts and chondroblasts and, in rodents, small cartilages occasionally form at the sutures in vivo. Therefore, we wanted to investigate what factors regulate normal differentiation of calvarial mesenchymal cells directly into osteoblasts. In embryonic mouse heads and in cultured tissue explants, we analyzed the expression of selected transcription factors and extracellular matrix molecules associated with bone and cartilage development. Cartilage markers Sox9 and type II collagen were expressed in all craniofacial cartilages. In addition, Msx2 and type I collagen were expressed in sense capsule cartilages. We also observed that the undifferentiated calvarial mesenchyme and the osteogenic fronts in the jaw expressed Col2A1. Moreover, we found that cultured mouse calvarial mesenchyme could develop into cartilage. Of the 49 explants that contained mesenchyme, intramembranous ossification occurred in 35%. Only cartilage formed in 4%, and both cartilage and bone formed in 4%. Our study confirms that calvarial mesenchyme, which normally gives rise to intramembranous bone, also has chondrogenic potential.

(J Histochem Cytochem 53:653–663, 2005)

Facial and calvarial bones form intramembranously without a cartilagenous model; however, cultured chick calvarial mesenchyme cells may differentiate into both osteoblasts and chondroblasts and, in rodents, small cartilages occasionally form at the sutures in vivo. Therefore, we wanted to investigate what factors regulate normal differentiation of calvarial mesenchymal cells directly into osteoblasts. In embryonic mouse heads and in cultured tissue explants, we analyzed the expression of selected transcription factors and extracellular matrix molecules associated with bone and cartilage development. Cartilage markers Sox9 and type II collagen were expressed in all craniofacial cartilages. In addition, Msx2 and type I collagen were expressed in sense capsule cartilages. We also observed that the undifferentiated calvarial mesenchyme and the osteogenic fronts in the jaw expressed Col2A1. Moreover, we found that cultured mouse calvarial mesenchyme could develop into cartilage. Of the 49 explants that contained mesenchyme, intramembranous ossification occurred in 35%. Only cartilage formed in 4%, and both cartilage and bone formed in 4%. Our study confirms that calvarial mesenchyme, which normally gives rise to intramembranous bone, also has chondrogenic potential.

(J Histochem Cytochem 53:653–663, 2005)

We investigated the localization of several markers for lysosomes and aggresomes in the chromatoid bodies (CBs) by immunoelectron microscopy. We found so-called aggresomal markers such as Hsp70 and ubiquitin in the core of the CBs and vimentin and proteasome subunit around the CBs. Ubiquitin-conjugating enzyme (E2) was also found in the CBs. In tubulovesicular structures surrounding the CBs, lysosomal markers were detected but an endoplasmic reticulum retention signal (KDEL) was not. Moreover, proteins located in each subcellular compartment, including the cytosol, mitochondria, and nucleus, were detected in the CBs. Signals for cytochrome oxidase I (COXI) coded on mitochondrial DNA were also found in the CBs. Quantitative analysis of labeling density showed that all proteins examined were concentrated in the CBs to some extent. These results show that the CBs have some aggresomal features, suggesting that they are not a synthetic site as proposed previously but a degradation site where unnecessary DNA, RNA, and proteins are digested. (J Histochem Cytochem 53:455–465, 2005)

We investigated the localization of several markers for lysosomes and aggresomes in the chromatoid bodies (CBs) by immunoelectron microscopy. We found so-called aggresomal markers such as Hsp70 and ubiquitin in the core of the CBs and vimentin and proteasome subunit around the CBs. Ubiquitin-conjugating enzyme (E2) was also found in the CBs. In tubulovesicular structures surrounding the CBs, lysosomal markers were detected but an endoplasmic reticulum retention signal (KDEL) was not. Moreover, proteins located in each subcellular compartment, including the cytosol, mitochondria, and nucleus, were detected in the CBs. Signals for cytochrome oxidase I (COXI) coded on mitochondrial DNA were also found in the CBs. Quantitative analysis of labeling density showed that all proteins examined were concentrated in the CBs to some extent. These results show that the CBs have some aggresomal features, suggesting that they are not a synthetic site as proposed previously but a degradation site where unnecessary DNA, RNA, and proteins are digested. (J Histochem Cytochem 53:455–465, 2005)

We investigated the localization of several markers for lysosomes and aggresomes in the chromatoid bodies (CBs) by immunoelectron microscopy. We found so-called aggresomal markers such as Hsp70 and ubiquitin in the core of the CBs and vimentin and proteasome subunit around the CBs. Ubiquitin-conjugating enzyme (E2) was also found in the CBs. In tubulovesicular structures surrounding the CBs, lysosomal markers were detected but an endoplasmic reticulum retention signal (KDEL) was not. Moreover, proteins located in each subcellular compartment, including the cytosol, mitochondria, and nucleus, were detected in the CBs. Signals for cytochrome oxidase I (COXI) coded on mitochondrial DNA were also found in the CBs. Quantitative analysis of labeling density showed that all proteins examined were concentrated in the CBs to some extent. These results show that the CBs have some aggresomal features, suggesting that they are not a synthetic site as proposed previously but a degradation site where unnecessary DNA, RNA, and proteins are digested. (J Histochem Cytochem 53:455–465, 2005)

Related Articles

Cloning and transcription analysis of an AGAMOUS- and SEEDSTICK ortholog in the orchid Dendrobium thyrsiflorum (Reichb. f.).

Gene. 2005 Oct 15;

Authors: Skipper M, Johansen LB, Pedersen KB, Frederiksen S, Johansen BB

Studies have shown that several plant species posses AGAMOUS (AG) and SEEDSTICK (STK) orthologs. These genes are part of the so-called C- and D MADS-box gene lineages and play key roles in ovule development in Arabidopsis thaliana. We have cloned an AG- and STK ortholog in the orchid Dendrobium thyrsiflorum, named DthyrAG1 and DthyrAG2, respectively, and analyzed their expression patterns. Quantification by real-time RT-PCR analysis shows that both are transcribed in the mature flowers and during ovule development. Localization of the transcripts by in situ hybridization analysis in flowers reveals that both genes are transcribed in the rostellum, stigma, and stylar canal. Expression analysis during ovule development shows that DthyrAG1 is expressed only in the initial periods of placenta- and ovule development, whereas the DthyrAG2 is transcribed throughout ovule development. These results suggest that both C- and D lineage orthologs are involved in various aspects of flower development and that DthyrAG2 have a more prominent role than DthyrAG1 in late ovule development in D. thyrsiflorum.

PMID: 16236468 [PubMed - as supplied by publisher]

We previously reported that CD1d, a molecule responsible for the presentation of lipid antigens, is expressed in atherosclerotic lesions and that its expression is restricted to dendritic cells. Recent studies demonstrating that CD1d-restricted natural killer T (NKT) cells are involved in atherogenesis prompted the present study investigating whether NKT cells are present in human atherosclerotic lesions and, if so, whether there is an association between NKT cells and dendritic cells. We found that NKT cells do accumulate in rupture-prone shoulders of atherosclerotic plaques and observed direct contacts of dendritic cells with NKT cells in rupture-prone regions of plaque.

(J Histochem Cytochem 53:781–785, 2005)

We previously reported that CD1d, a molecule responsible for the presentation of lipid antigens, is expressed in atherosclerotic lesions and that its expression is restricted to dendritic cells. Recent studies demonstrating that CD1d-restricted natural killer T (NKT) cells are involved in atherogenesis prompted the present study investigating whether NKT cells are present in human atherosclerotic lesions and, if so, whether there is an association between NKT cells and dendritic cells. We found that NKT cells do accumulate in rupture-prone shoulders of atherosclerotic plaques and observed direct contacts of dendritic cells with NKT cells in rupture-prone regions of plaque.

(J Histochem Cytochem 53:781–785, 2005)

We previously reported that CD1d, a molecule responsible for the presentation of lipid antigens, is expressed in atherosclerotic lesions and that its expression is restricted to dendritic cells. Recent studies demonstrating that CD1d-restricted natural killer T (NKT) cells are involved in atherogenesis prompted the present study investigating whether NKT cells are present in human atherosclerotic lesions and, if so, whether there is an association between NKT cells and dendritic cells. We found that NKT cells do accumulate in rupture-prone shoulders of atherosclerotic plaques and observed direct contacts of dendritic cells with NKT cells in rupture-prone regions of plaque.

(J Histochem Cytochem 53:781–785, 2005)

Apolipoprotein AI (apoAI), a major component of high-density lipoproteins, is one of the major amyloid fibril proteins and a minor constituent of the senile plaques observed in Alzheimer's disease. We examined colocalization of apoAI in various kinds of systemic amyloidosis in this study. Forty-three of 48 formalin-fixed paraffin-embedded heart specimens with various forms of systemic amyloidosis reacted immunohistochemically with anti-human apoAI antibody. ApoAI was also detected in water-extracted amyloid material by immunoblotting. In addition, we observed colocalization of apoAI and murine amyloid A (AA) amyloidosis in human apoAI transgenic mice. This is the first report of colocalization of apoAI with amyloid deposits in various forms of human systemic amyloidosis and murine AA amyloidosis in human apoAI transgenic mice. ApoAI may not always be a major component of amyloid fibrils, even when it is present in systemic amyloid deposits.

(J Histochem Cytochem 53:237–242, 2005)

Complex de novo cryptic subtelomeric rearrangements in a fetus with multiple ultrasonographic abnormalities and a normal karyotype at amniocentesis.

Prenat Diagn. 2005 Oct 21;

Authors: Iqbal MA, Ramadan S, Ali FA, Kurdi W

OBJECTIVE: Prenatal diagnosis is usually offered to the majority of pregnancies with fetal structural abnormalities detected by prenatal ultrasound; however, only a small proportion show an abnormal karyotype. We wanted to detect cryptic subtelomeric rearrangements (CSTR) in a fetus with multiple abnormal ultrasonographic findings that revealed a normal karyotype at amniocentesis. METHODS: Fetal chromosome analysis was performed from amniotic fluid cells. Parental chromosome analysis was done on PHA stimulated lymphocyte cultures. For fluorescence in situ hybridization (FISH) analysis, ToTelVysion multicolor DNA probe mixture was used to hybridize the p and q telomeres of each chromosome. RESULTS: The amniotic fluid chromosome analysis revealed an apparently normal 46,XY karyotype. A follow-up FISH analysis showed three apparently balanced complex translocations involving (1) the chromosome 4p and 22q telomeres (2) 4q and 11q telomeres and (3) 8p, 20p and 20q telomeres. Parental chromosome and subtelomere FISH analysis was found to be normal. CONCLUSION: To our knowledge, this is the first report of complex de novo cryptic translocations in an abnormal fetus. These CSTR identified by FISH with subtelomere-specific probes are not detected by other cytogenetic and/or molecular cytogenetic approaches. However, to confirm the balanced nature of CSTR, array-CGH can be helpful. Further studies are in progress to determine the frequency of CSTR and its significance in the etiology of fetal abnormalities. Copyright (c) 2005 John Wiley & Sons, Ltd.

PMID: 16240463 [PubMed - as supplied by publisher]

The temporal and spatial expression of transforming growth factor (TGF)-ßbeta;₁ and connective tissue growth factor (CTGF) was assessed in the left ventricle of a myocardial infarction (MI) model of injury with and without angiotensin-converting enzyme (ACE) inhibition. Coronary artery ligated rats were killed 1, 3, 7, 28, and 180 days after MI. TGF-ßbeta;₁, CTGF, and procollagen 1(I) mRNA were localized by in situ hybridization, and TGF-ßbeta;₁ and CTGF protein levels by immunohistochemistry. Collagen protein was measured using picrosirius red staining. In a separate group, rats were treated for 6 months with an ACE inhibitor. There were temporal and regional differences in the expression of TGF-ßbeta;₁, CTGF, and collagen after MI. Procollagen 1(I) mRNA expression increased in the border zone and scar peaking 1 week after MI, whereas collagen protein increased in all areas of the heart over the 180 days. Expression of TGF-ßbeta;₁ mRNA and protein showed major increases in the border zone and scar peaking 1 week after MI. The major increases in CTGF mRNA and protein occurred in the viable myocardium at 180 days after MI. Long-term ACE inhibition reduced left ventricular mass and decreased fibrosis in the viable myocardium, but had no effect on cardiac TGF-ßbeta;₁ or CTGF. TGF-ßbeta;₁ is involved in the initial, acute phase of inflammation and repair after MI, whereas CTGF is involved in the ongoing fibrosis of the heart. The antifibrotic benefits of captopril are not mediated through a reduction in CTGF.

(J Histochem Cytochem 53:1245–1256, 2005)

The temporal and spatial expression of transforming growth factor (TGF)-ßbeta;₁ and connective tissue growth factor (CTGF) was assessed in the left ventricle of a myocardial infarction (MI) model of injury with and without angiotensin-converting enzyme (ACE) inhibition. Coronary artery ligated rats were killed 1, 3, 7, 28, and 180 days after MI. TGF-ßbeta;₁, CTGF, and procollagen 1(I) mRNA were localized by in situ hybridization, and TGF-ßbeta;₁ and CTGF protein levels by immunohistochemistry. Collagen protein was measured using picrosirius red staining. In a separate group, rats were treated for 6 months with an ACE inhibitor. There were temporal and regional differences in the expression of TGF-ßbeta;₁, CTGF, and collagen after MI. Procollagen 1(I) mRNA expression increased in the border zone and scar peaking 1 week after MI, whereas collagen protein increased in all areas of the heart over the 180 days. Expression of TGF-ßbeta;₁ mRNA and protein showed major increases in the border zone and scar peaking 1 week after MI. The major increases in CTGF mRNA and protein occurred in the viable myocardium at 180 days after MI. Long-term ACE inhibition reduced left ventricular mass and decreased fibrosis in the viable myocardium, but had no effect on cardiac TGF-ßbeta;₁ or CTGF. TGF-ßbeta;₁ is involved in the initial, acute phase of inflammation and repair after MI, whereas CTGF is involved in the ongoing fibrosis of the heart. The antifibrotic benefits of captopril are not mediated through a reduction in CTGF.

(J Histochem Cytochem 53:1245–1256, 2005)

Cyclical up-regulated iNOS and long-term down-regulated nNOS are the basis for relapse and quiescent phases in a rat model of IBD.

Am J Physiol Gastrointest Liver Physiol. 2005 Oct 20;

Authors: Porras M, Martin MT, Torres R, Vergara P

Background: We previously reported that indomethacin induces a chronic intestinal inflammation in the rat where the cyclical characteristic phases of Crohn's disease are manifested with a few days' interval and lasting for several months: active phase (high inflammation, hypomotility, bacterial traslocation) and reactive phase (low inflammation, hypermotility, no bacterial traslocation). Aims: In this study we investigated the possible role of both constitutive and inducible isoforms of nitric oxide synthase (NOS) and cyclooxygenase (COX) in the cyclicity of active and reactive phases in rats with chronic intestinal inflammation. Methods: Rats selected at either active or reactive phases and from 2 to 60 days after indomethacin treatment were used. mRNA expression of both constitutive and inducible NOS and COX isoforms in each phase was evaluated by RT-PCR and cellular enzyme localization by immunohistochemistry. The effects of different COX and NOS inhibitors on the intestinal motor activity were tested. Results: mRNA expression of COX-1 was not modified by inflammation whereas mRNA expression of nNOS was reduced in all indomethacin treated rats. In contrast, NOS and COX inducible forms showed a cyclical oscillation. mRNA expression and protein of both iNOS and COX-2 increased only during active phases. The intestinal hypomotility associated with active phases was turned into hypermotility after the administration of selective iNOS inhibitors. Conclusions: Sustained down-regulation of constitutive NOS caused hypermotility, possibly as a defense mechanism. However, this reaction was masked during the active phases due to the inhibitory effects of nitric oxide resulting from the increased levels of inducible NOS isoform.

PMID: 16239401 [PubMed - as supplied by publisher]

In DNA histograms of some human solid tumors stained with nuclear isolation medium–4,6-diamidino-2-phenylindole dihydrochloride (NIM-DAPI), the coefficient of variation (CV) of the G₀/G₁ peak was broad, and in nuclear volume vs DNA scattergrams, a prominent slope was seen. To determine the cause for this, nuclei from frozen breast tumors were stained with NIM-DAPI and analyzed after dilution or resuspension in PBS. In two-color (blue vs red) analysis, most of the slope and broad CV was due to red fluorescence of nuclei stained with NIM-DAPI, which was reduced on dilution or resuspension in PBS, resulting in elimination of the slope and tightening of the CV.

(J Histochem Cytochem 53:1033–1036, 2005)

In DNA histograms of some human solid tumors stained with nuclear isolation medium–4,6-diamidino-2-phenylindole dihydrochloride (NIM-DAPI), the coefficient of variation (CV) of the G₀/G₁ peak was broad, and in nuclear volume vs DNA scattergrams, a prominent slope was seen. To determine the cause for this, nuclei from frozen breast tumors were stained with NIM-DAPI and analyzed after dilution or resuspension in PBS. In two-color (blue vs red) analysis, most of the slope and broad CV was due to red fluorescence of nuclei stained with NIM-DAPI, which was reduced on dilution or resuspension in PBS, resulting in elimination of the slope and tightening of the CV.

(J Histochem Cytochem 53:1033–1036, 2005)

Articular cartilage is a heterogeneous tissue, with cell density and organization varying with depth from the surface. The objectives of the present study were to establish a method for localizing individual cells in three-dimensional (3D) images of cartilage and quantifying depth-associated variation in cellularity and cell organization at different stages of growth. Accuracy of nucleus localization was high, with 99% sensitivity relative to manual localization. Cellularity (million cells per cm³) decreased from 290, 310, and 150 near the articular surface in fetal, calf, and adult samples, respectively, to 120, 110, and 50 at a depth of 1.0 mm. The distance/angle to the nearest neighboring cell was 7.9 µm/31°, 7.1 µm/31°, and 9.1 µm/31° for cells at the articular surface of fetal, calf, and adult samples, respectively, and increased/decreased to 11.6 µm/31°, 12.0 µm/30°, and 19.2 µm/25° at a depth of 0.7 mm. The methodologies described here may be useful for analyzing the 3D cellular organization of cartilage during growth, maturation, aging, degeneration, and regeneration. (J Histochem Cytochem 53:1109–1119, 2005)

Design and assessment of a tissue-engineered model of human phalanges and a small joint.

Orthod Craniofac Res. 2005 Nov;8(4):303-12

Authors: Landis W, Jacquet R, Hillyer J, Lowder E, Yanke A, Siperko L, Asamura S, Kusuhara H, Enjo M, Chubinskaya S, Potter K, Isogai N

Objectives - To develop models of human phalanges and small joints by suturing different cell-polymer constructs that are then implanted in athymic (nude) mice. Design - Models consisted of bovine periosteum, cartilage, and/or tendon cells seeded onto biodegradable polymer scaffolds of either polyglycolic acid (PGA) or copolymers of PGA and poly-L-lactic acid (PLLA) or poly-epsilon-caprolactone (PCL) and PLLA. Constructs were fabricated to produce a distal phalanx, middle phalanx, or distal interphalangeal joint. Setting and Sample Population - Studies of more than 250 harvested implants were conducted at the Northeastern Ohio Universities College of Medicine. Experimental Variable - Polymer scaffold, cell type, and implantation time were examined. Outcome Measure - Tissue-engineered specimens were characterized by histology, transmission electron microscopy, in situ hybridization, laser capture microdissection and qualitative and quantitative polymerase chain reaction analysis, magnetic resonance microscopy, and X-ray microtomography. Results - Over periods to 60 weeks of implantation, constructs developed through vascularity from host mice; formed new cartilage, bone, and/or tendon; expressed characteristic genes of bovine origin, including type I, II and X collagen, osteopontin, aggrecan, biglycan, and bone sialoprotein; secreted corresponding proteins; responded to applied mechanical stimuli; and maintained shapes of human phalanges with small joints. Conclusion - Results give insight into construct processes of tissue regeneration and development and suggest more complete tissue-engineered cartilage, bone, and tendon models. These should have significant future scientific and clinical applications in medicine, including their use in plastic surgery, orthopaedics, craniofacial reconstruction, and teratology.

PMID: 16238611 [PubMed - in process]

Matrix metalloproteinase-7 (MMP-7) is upregulated during carcinogenesis and its expression correlates with metastasis of human endometrial and gastrointestinal carcinomas. In the present study, we have developed a new method to localize the activity of MMP-7 within tissues. Polyethylene terephthalate films were uniformly coated with crosslinked carboxymethylated transferrin (CCm-Tf) as a substrate and incubated with frozen tissue sections mounted on the films. CCm-Tf on the films was degraded selectively by MMP-7, but showed little or no susceptibility to MMP-1, -2, -3, -9, or -13; MT1-MMP; MT3-MMP; or ADAMTS4. Although some serine proteinases such as elastase also digested CCm-Tf, CCm-Tf films impregnated with serine proteinase inhibitors prevented the digestion. When frozen sections of human endometrial carcinoma and lung carcinoma tissues were incubated on CCm-Tf films or those treated with proteinase inhibitors, the activity was detected in the carcinoma cell nests, where MMP-7 was immunolocalized. The present in situ zymography using CCm-Tf may be a useful method to analyze the functions of MMP-7 in pathophysiological conditions. (J Histochem Cytochem 53:1227–1234, 2005)

Matrix metalloproteinase-7 (MMP-7) is upregulated during carcinogenesis and its expression correlates with metastasis of human endometrial and gastrointestinal carcinomas. In the present study, we have developed a new method to localize the activity of MMP-7 within tissues. Polyethylene terephthalate films were uniformly coated with crosslinked carboxymethylated transferrin (CCm-Tf) as a substrate and incubated with frozen tissue sections mounted on the films. CCm-Tf on the films was degraded selectively by MMP-7, but showed little or no susceptibility to MMP-1, -2, -3, -9, or -13; MT1-MMP; MT3-MMP; or ADAMTS4. Although some serine proteinases such as elastase also digested CCm-Tf, CCm-Tf films impregnated with serine proteinase inhibitors prevented the digestion. When frozen sections of human endometrial carcinoma and lung carcinoma tissues were incubated on CCm-Tf films or those treated with proteinase inhibitors, the activity was detected in the carcinoma cell nests, where MMP-7 was immunolocalized. The present in situ zymography using CCm-Tf may be a useful method to analyze the functions of MMP-7 in pathophysiological conditions. (J Histochem Cytochem 53:1227–1234, 2005)

In this study, we describe the development of fluorescent oligonucleotide probes to variable regions in the small subunit of 16S rRNA in three distinct Giardia species. Sense and antisense probes (17–22 mer) to variable regions 1, 3, and 8 were labeled with digoxygenin or selected fluorochomes (FluorX, Cy3, or Cy5). Optimal results were obtained with fluorochome-labeled oligonucleotides for detection of rRNA in Giardia cysts. Specificity of fluorescent in situ hybridization (FISH) was shown using RNase digestion and high stringency to diminish the hybridization signal, and oligonucleotide probes for rRNA in Giardia lamblia, Giardia muris, and Giardia ardeae were shown to specifically stain rRNA only within cysts or trophozoites of those species. The fluorescent oligonucleotide specific for rRNA in human isolates of Giardia was positive for ten different strains. A method for simultaneous FISH detection of cysts using fluorescent antibody (genotype marker) and two oligonucleotide probes (species marker) permitted visualization of G. lamblia and G. muris cysts in the same preparation. Testing of an environmental water sample revealed the presence of FISH-positive G. lamblia cysts with a specific rDNA probe for rRNA, while negative cysts were presumed to be of animal or bird origin. (J Histochem Cytochem 53:917–927, 2005)

In this study, we describe the development of fluorescent oligonucleotide probes to variable regions in the small subunit of 16S rRNA in three distinct Giardia species. Sense and antisense probes (17–22 mer) to variable regions 1, 3, and 8 were labeled with digoxygenin or selected fluorochomes (FluorX, Cy3, or Cy5). Optimal results were obtained with fluorochome-labeled oligonucleotides for detection of rRNA in Giardia cysts. Specificity of fluorescent in situ hybridization (FISH) was shown using RNase digestion and high stringency to diminish the hybridization signal, and oligonucleotide probes for rRNA in Giardia lamblia, Giardia muris, and Giardia ardeae were shown to specifically stain rRNA only within cysts or trophozoites of those species. The fluorescent oligonucleotide specific for rRNA in human isolates of Giardia was positive for ten different strains. A method for simultaneous FISH detection of cysts using fluorescent antibody (genotype marker) and two oligonucleotide probes (species marker) permitted visualization of G. lamblia and G. muris cysts in the same preparation. Testing of an environmental water sample revealed the presence of FISH-positive G. lamblia cysts with a specific rDNA probe for rRNA, while negative cysts were presumed to be of animal or bird origin. (J Histochem Cytochem 53:917–927, 2005)

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Diagnostic utility of CDX-2 expression in separating metastatic gastrointestinal adenocarcinoma from other metastatic adenocarcinoma in fine-needle aspiration cytology using cell blocks.

Cancer. 2004 Jun 25;102(3):168-73

Authors: Saad RS, Essig DL, Silverman JF, Liu Y

BACKGROUND: CDX-2 gene is a transcription factor that is involved in the proliferation and differentiation of intestinal epithelial cells. Recent studies have shown that CDX-2 could be used as an immunohistochemical marker to differentiate metastatic gastrointestinal adenocarcinoma from other metastatic adenocarcinomas in surgical pathology. The objective of the current study was to investigate the diagnostic value of CDX-2 to separate metastatic gastrointestinal adenocarcinoma from other metastatic adenocarcinomas in fine-needle aspiration cytology (FNAC). METHODS: Sixty-two FNAC specimens of metastatic adenocarcinomas with corresponding cell blocks were retrieved from the hospital computer system. There were 22 specimens of metastatic gastrointestinal adenocarcinoma, 20 specimens of metastatic pulmonary adenocarcinoma, and 20 specimens of metastatic adenocarcinomas from other sites, including 10 from the breast, 3 from the ovaries, 4 from the pancreas, and 3 from the prostate. Radiology and histologic evaluation confirmed all cases. Sections were immunostained for CDX-2 and thyroid transcription factor-1 (TTF-1) using a heat-induced epitope retrieval technique. RESULTS: In metastatic gastrointestinal adenocarcinoma, CDX-2 demonstrated positive nuclear staining in 19 of 22 specimens (86%). Other specimens of metastatic adenocarcinoma were negative for CDX-2. TTF-1 expression was detected in 0%, 80%, and 0% of metastatic gastrointestinal adenocarcinoma, pulmonary adenocarcinoma, and other adenocarcinoma specimens, respectively. CONCLUSIONS: The results of the current study demonstrated that CDX-2 is a sensitive and a specific marker to separate metastatic gastrointestinal adenocarcinoma from other metastatic adenocarcinomas in FNAC specimens.

PMID: 15211476 [PubMed - indexed for MEDLINE]

Immunohistochemistry was used to study the distribution of laminin (Ln) chains, collagen types IV ( 1/2), VII, and XVIII and Lutheran antigen (Lu) in 36 frozen ovarian carcinoma samples. Surface epithelial basement membrane (BM) of the normal ovary showed immunoreactivity for Ln 1, 3-5, ßbeta;1-3, 1, and 2 chains and type IV and XVIII collagens. Chains of Ln-5 (3ßbeta;32) and Ln-10 (5ßbeta;11) as well as type IV and XVIII collagens were found in most tumor BMs, but Ln 2 chain and type VII collagen were detected only in few tumors. Contrary to serous tumors, BMs of mucinous carcinomas showed Ln 4 chain, but not Ln 1 and ßbeta;2 chains. Ln 1 chain was found in most endometrioid carcinomas, whereas chains of Ln-5 were only moderately detectable in comparison with serous and mucinous carcinomas. In the normal ovary, Lu immunoreactivity was confined to basal aspect in the ovarian epithelial cells, but in tumor specimens Lu immunostainings showed variable polarized and nonpolarized patterns. The results suggest that the three types of ovarian carcinoma have distinct differences in their Ln distribution and can be grouped based on their expression pattern. This suggests that they may have histogenetically different precursors and may help to distinguish these tumors from each other.

(J Histochem Cytochem 53:1293–1300, 2005)

Immunohistochemistry was used to study the distribution of laminin (Ln) chains, collagen types IV ( 1/2), VII, and XVIII and Lutheran antigen (Lu) in 36 frozen ovarian carcinoma samples. Surface epithelial basement membrane (BM) of the normal ovary showed immunoreactivity for Ln 1, 3-5, ßbeta;1-3, 1, and 2 chains and type IV and XVIII collagens. Chains of Ln-5 (3ßbeta;32) and Ln-10 (5ßbeta;11) as well as type IV and XVIII collagens were found in most tumor BMs, but Ln 2 chain and type VII collagen were detected only in few tumors. Contrary to serous tumors, BMs of mucinous carcinomas showed Ln 4 chain, but not Ln 1 and ßbeta;2 chains. Ln 1 chain was found in most endometrioid carcinomas, whereas chains of Ln-5 were only moderately detectable in comparison with serous and mucinous carcinomas. In the normal ovary, Lu immunoreactivity was confined to basal aspect in the ovarian epithelial cells, but in tumor specimens Lu immunostainings showed variable polarized and nonpolarized patterns. The results suggest that the three types of ovarian carcinoma have distinct differences in their Ln distribution and can be grouped based on their expression pattern. This suggests that they may have histogenetically different precursors and may help to distinguish these tumors from each other.

(J Histochem Cytochem 53:1293–1300, 2005)

To better understand the mechanism(s) of leukocyte Ca²⁺ signaling, we have studied the intracellular locations of two Ca²⁺-mobilizing receptors, the inositol 1,4,5-trisphosphate receptor and ryanodine receptor, by immunofluorescence microscopy. Our results show that localization differs not only between receptor classes within a cell, but among leukocyte types as well. We also illustrate the importance of preserving labile cellular filaments in maintaining cell integrity by fixation with the Safiejko-Mroczka and Bell protocol, because conventional fixation methods distort receptor patterns. We suggest that the observed differences influence intracellular Ca²⁺ signaling.

(J Histochem Cytochem 53:913–916, 2005)

To better understand the mechanism(s) of leukocyte Ca²⁺ signaling, we have studied the intracellular locations of two Ca²⁺-mobilizing receptors, the inositol 1,4,5-trisphosphate receptor and ryanodine receptor, by immunofluorescence microscopy. Our results show that localization differs not only between receptor classes within a cell, but among leukocyte types as well. We also illustrate the importance of preserving labile cellular filaments in maintaining cell integrity by fixation with the Safiejko-Mroczka and Bell protocol, because conventional fixation methods distort receptor patterns. We suggest that the observed differences influence intracellular Ca²⁺ signaling.

(J Histochem Cytochem 53:913–916, 2005)

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Distinct but overlapping T helper epitopes in the 37-58 region of SSX-2.

Clin Immunol. 2005 Jan;114(1):70-8

Authors: Ayyoub M, Merlo A, Hesdorffer CS, Speiser D, Rimoldi D, Cerottini JC, Ritter G, Chen YT, Old LJ, Stevanovic S, Valmori D

Because of their specific expression in tumors of different histological types, the products of the SSX genes are important candidate targets for development of cancer vaccines. We have previously identified two immunodominant SSX-2-derived T cell epitopes recognized by HLA-A2-restricted CD8+ T cells (SSX-2 41-49) and HLA-DR11-restricted CD4+ T cells (SSX-2 45-59), respectively. In this study, we report the identification of an HLA-DR3-restricted epitope mapping to the 37-51 region of SSX-2, overlapping both previously identified epitopes. As about one fifth of individuals from several major ethnic groups express HLA-DR3, the identification of this epitope significantly increases the percent of patients that are expected to mount specific CD4+ T cell responses following vaccination with peptides in this region of SSX-2. Retrieval of multiple overlapping epitopes in a defined region of SSX-2 protein suggests the presence of a "hot spot" for T cell recognition that may prove sufficient for the induction of immune responses.

PMID: 15596411 [PubMed - indexed for MEDLINE]

Vasculogenic mimicry (VM) has been observed in melanoma and in some nonmelanoma tumor types. It is unknown whether a similar VM phenomenon exists in astrocytoma. The present study was to examine 45 astrocytomas (including World Health Organization grade II 15 cases, grade III 15 cases, and grade IV 15 cases) by CD34 endothelial marker periodic acid–Schiff (PAS) dual staining to see if VM existing in these tumors. The results demonstrated that endothelium-lined vessels dominated the tumor microvasculature and stained positively for PAS, laminin, and endothelial marker. PAS-positive pattern of VM was found in two grade IV astrocytomas. Channels stained positively for PAS, laminin, and negatively for CD34 of the VM entrapped in the tumor tissue. Erythrocytes could be observed in some of these channels. In these networks of PAS-positive pattern, spots of weak reaction for CD34 were observed, suggesting the incorporation of VM channel and normal vessel. Furthermore, in astrocytoma, especially glioblastoma, focus of anaplastic tumor cells appeared with CD34 expression, whereas some tumor cells lost glial fibrillary acid protein expression. It is assumed that genetically deregulated tumor cells in astrocytoma could lose the astrocyte-specific protein and express inappropriate markers not expected in cells of astrocyte lineage. The present results suggest that VM phenomenon exists in some malignant astrocytoma. (J Histochem Cytochem 53:997–1002, 2005)

Vasculogenic mimicry (VM) has been observed in melanoma and in some nonmelanoma tumor types. It is unknown whether a similar VM phenomenon exists in astrocytoma. The present study was to examine 45 astrocytomas (including World Health Organization grade II 15 cases, grade III 15 cases, and grade IV 15 cases) by CD34 endothelial marker periodic acid–Schiff (PAS) dual staining to see if VM existing in these tumors. The results demonstrated that endothelium-lined vessels dominated the tumor microvasculature and stained positively for PAS, laminin, and endothelial marker. PAS-positive pattern of VM was found in two grade IV astrocytomas. Channels stained positively for PAS, laminin, and negatively for CD34 of the VM entrapped in the tumor tissue. Erythrocytes could be observed in some of these channels. In these networks of PAS-positive pattern, spots of weak reaction for CD34 were observed, suggesting the incorporation of VM channel and normal vessel. Furthermore, in astrocytoma, especially glioblastoma, focus of anaplastic tumor cells appeared with CD34 expression, whereas some tumor cells lost glial fibrillary acid protein expression. It is assumed that genetically deregulated tumor cells in astrocytoma could lose the astrocyte-specific protein and express inappropriate markers not expected in cells of astrocyte lineage. The present results suggest that VM phenomenon exists in some malignant astrocytoma. (J Histochem Cytochem 53:997–1002, 2005)

We report on the case of a pregnant woman with hyposomia who was previously suspected of having Turner syndrome. Prenatal cytogenetic diagnostics showed a fetal karyotype of 46,XX,dup(13)(q14.2q21.1) ish.13q14(RB1 x 3). Parental and grandparental chromosome analyses were performed and the dup(13) was found to be of maternal origin (de novo). The pregnancy was continued and a healthy female child was born with normal development apart from growth retardation. The reported chromosomal aberration is, together with two other cases reported in the literature, the first hint of a short stature–like phenotype due to dup(13)(q14.2q14.3). (J Histochem Cytochem 53:365–366, 2005)

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Effective antigen-retrieval method for immunohistochemical detection of abnormal isoform of prion proteins in animals.

Acta Neuropathol (Berl). 2005 Mar;109(3):263-71

Authors: Furuoka H, Yabuzoe A, Horiuchi M, Tagawa Y, Yokoyama T, Yamakawa Y, Shinagawa M, Sata T

For immunohistochemistry of the prion diseases, several pretreatment methods to enhance the immunoreactivity of human and animal abnormal proteinase-resistant prion protein (PrP(Sc)) on the tissue sections have been employed. The method of 121 degree C hydrated autoclaving pretreatment or the combination method of 121 degree C hydrated autoclaving with a certain chemical reagent (formic acid or proteinase K, etc) are now widely used. We found that an improved hydrated autoclaving method at 135 degrees C, more effectively enhanced PrP(Sc) immunoreactivity for the antibodies recognizing the linear epitope. In addition, this method was more effective for the long-term fixation samples as compared with other previous methods. However, this modified method could not retrieve PrP(Sc) antigenic epitopes composed of conformational structures or several discontinuous epitopes. We describe the comparative studies between our improved method and other antigen-retrieval procedures reported previously. Based on the differences of reaction among the antibodies, we also discuss the mechanisms of the hydrated autoclaving methods to retrieve PrP(Sc) immunoreactivity.

PMID: 15616789 [PubMed - indexed for MEDLINE]

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Electron microscopic localisation of P2X4 receptor subunit immunoreactivity to pre- and post-synaptic neuronal elements and glial processes in the dorsal vagal complex of the rat.

Brain Res. 2004 Nov 5;1026(1):44-55

Authors: Ashour F, Deuchars J

P2X receptors are ligand gated ion channels activated by extracellular ATP. There are seven P2X subunits, P2X(1-7), and all are expressed in the CNS. The P2X(4) receptor subunit (P2X(4)R) is likely to be important in the CNS as it has been reported to be expressed throughout the brain and spinal cord. However, P2X(4)Rs have been identified as restricted to neurones, only in glia or expressed in both neurones and glia with no discernible relationship to CNS region or epitope target of antibodies used for staining. In addition, although there are particularly high levels of mRNA encoding P2X(4)R in the brainstem, previous immunohistochemical studies have revealed only indistinct staining. We therefore examined the distribution of P2X(4)R in the dorsal vagal complex (DVC) of the brainstem using immunohistochemistry in sections obtained from adult Wistar rats transcardially perfused with aldehyde fixatives. When this revealed staining identifiable only as small puncta at the light microscope level, we examined the area with electron microscopy. This ultrastructural study revealed that P2X(4)R immunoreactivity (IR) was present in neurones at both pre- and post-synaptic sites as well as in glial cell processes and somata. This P2X(4)R-IR was localised adjacent to plasma membranes, as well as internally in membrane bound structures resembling endosomes. Immunoreactivity in endosomes was more prominent following antigen retrieval protocols. Localisation of P2X(4)R-IR in astrocytes, identified by the presence of glial fibrillary acidic protein (GFAP), was confirmed using immunofluorescence. The presence of P2X(4)Rs in the dorsal vagal complex is consistent with expression studies, but some reasons for a lack of correlation with pharmacological studies are discussed. The P2X(4)R is therefore expressed by neurones and glia in the dorsal vagal complex and may play a role in mediating extracellular signalling by ATP in this region.

PMID: 15476696 [PubMed - indexed for MEDLINE]

RNA interference has become a valuable tool to identify and investigate proteins involved in the formation of peroxisomes. We demonstrate that the elongation of peroxisomes serves as an excellent indicator for efficient knock down of dynamin-like protein 1 (DLP1) in mammalian cells. We took advantage of the silencing-dependent morphological changes of peroxisomes to compare different transfection methods and show that a single transfection of DLP1 siRNA by electroporation is sufficient to effectively silence DLP1. We present a fast, easy, and convenient protocol for efficient gene silencing in a large number of cells, which can be used for quantitative and biochemical studies.

(J Histochem Cytochem 53:1037–1040, 2005)

RNA interference has become a valuable tool to identify and investigate proteins involved in the formation of peroxisomes. We demonstrate that the elongation of peroxisomes serves as an excellent indicator for efficient knock down of dynamin-like protein 1 (DLP1) in mammalian cells. We took advantage of the silencing-dependent morphological changes of peroxisomes to compare different transfection methods and show that a single transfection of DLP1 siRNA by electroporation is sufficient to effectively silence DLP1. We present a fast, easy, and convenient protocol for efficient gene silencing in a large number of cells, which can be used for quantitative and biochemical studies.

(J Histochem Cytochem 53:1037–1040, 2005)

Leptin is a hormone that plays important roles in nutritional status and in obesity. By means of immunocytochemistry, two populations of leptin-secreting cells were found in the lower half of the gastric mucosa. One consists of numerous large cells located around the gastric pits, the Chief epithelial cells, whereas the second refers to much smaller cells, strongly stained, few in number, and scattered between the gastric pits, the endocrine cells. By double immunostaining, leptin and pepsinogen were colocalized in the Chief cells, whereas the endocrine cells were positive only for leptin. Immunoelectron microscopy showed that leptin is present along the rough endoplasmic reticulum–Golgi-granules secretory pathways of the Chief and endocrine cells. On the other hand, leptin-receptor (long and short forms) immunolabelings, although absent in the gastric epithelial cell plasma membranes, were present in enterocytes at the level of their apical and basolateral membranes. Duodenal, jejunal, and ileal enterocytes displayed similar labelings for the leptin receptor. Thus, exocrine and endocrine secretions of leptin together with the presence of leptin receptors on enterocyte plasma membranes constitute a gastroenteric axis that coordinates the role played by leptin in the digestive tract.

(J Histochem Cytochem 53:851–860, 2005)

Leptin is a hormone that plays important roles in nutritional status and in obesity. By means of immunocytochemistry, two populations of leptin-secreting cells were found in the lower half of the gastric mucosa. One consists of numerous large cells located around the gastric pits, the Chief epithelial cells, whereas the second refers to much smaller cells, strongly stained, few in number, and scattered between the gastric pits, the endocrine cells. By double immunostaining, leptin and pepsinogen were colocalized in the Chief cells, whereas the endocrine cells were positive only for leptin. Immunoelectron microscopy showed that leptin is present along the rough endoplasmic reticulum–Golgi-granules secretory pathways of the Chief and endocrine cells. On the other hand, leptin-receptor (long and short forms) immunolabelings, although absent in the gastric epithelial cell plasma membranes, were present in enterocytes at the level of their apical and basolateral membranes. Duodenal, jejunal, and ileal enterocytes displayed similar labelings for the leptin receptor. Thus, exocrine and endocrine secretions of leptin together with the presence of leptin receptors on enterocyte plasma membranes constitute a gastroenteric axis that coordinates the role played by leptin in the digestive tract.

(J Histochem Cytochem 53:851–860, 2005)

EPIMHC: a curated database of MHC-binding peptides for customized computational vaccinology.

Bioinformatics. 2005 May 1;21(9):2140-1

Authors: Reche PA, Zhang H, Glutting JP, Reinherz EL

SUMMARY: EPIMHC is a relational database of MHC-binding peptides and T cell epitopes that are observed in real proteins. Currently, the database contains 4867 distinct peptide sequences from various sources, including 84 tumor-associated antigens. The EPIMHC database is accessible through a web server that has been designed to facilitate research in computational vaccinology. Importantly, peptides resulting from a query can be selected to derive specific motif-matrices. Subsequently, these motif-matrices can be used in combination with a dynamic algorithm for predicting MHC-binding peptides from user-provided protein queries. AVAILABILITY: The EPIMHC database server is hosted by the Dana-Farber Cancer Institute at the site http://immunax.dfci.harvard.edu/bioinformatics/epimhc/

PMID: 15657103 [PubMed - indexed for MEDLINE]

To investigate, in real time, the transport and secretion of pituitary hormone, we have developed an experimental pituitary cell line, GH3 cell, which has secretory granules of growth hormone (GH) linked to enhanced yellow fluorescein protein (EYFP). This stable GH3 cell secretes secretory granules of GH linked to EYFP on stimulation by Ca²⁺ influx or Ca² release from storage. This GH3 cell will be useful for the real-time visualization of the intracellular transport and secretion of GH. (J Histochem Cytochem 53:1177–1180, 2005)

We performed a comparative study of the enrichment of erythroblasts by a soybean agglutinin galactose-specific lectin method and a standardized magnetic cell-sorting (MACS) protocol. Blood samples, obtained from 11 pregnant women at between 11 and 40 weeks of gestation, were split and examined by each method in parallel. The number of erythroblasts recovered by the lectin method was approximately eightfold higher than the number obtained by MACS. Our data suggest that the lectin-based method may provide a better approach for the enrichment of rare fetal erythroblasts from maternal blood.

(J Histochem Cytochem 53:329–330, 2005)

The primary function of the exocrine pancreas consists of the synthesis and secretion of several digestive enzymes. It is well established that amylase secretion by rat pancreatic tissue or by isolated acinar cells in culture can be stimulated by the cholinergic agonist carbachol. However, the effect of this secretagogue on enzyme synthesis remains unclear. Some studies demonstrated increases in rates of synthesis, whereas others reported increases in secretion with or without decreases in synthesis. We have evaluated changes in pancreatic amylase mRNA and total RNA after a single injection of carbachol and under fasting conditions. Two approaches in molecular morphology were applied on rat pancreatic tissue: in situ hybridization and RNase A–gold. Both revealed decreases in RNA labeling at the level of the rough endoplasmic reticulum (RER) 5 min after stimulation of secretion and after fasting. Gradual recovery was registered 15 and 30 min after stimulation of secretion. Northern blotting confirmed drastic decreases in amylase mRNA 5 min after stimulation and after fasting. The combination of such different approaches has demonstrated drastic decreases in RNA at the RER level, reflecting declines in rates of synthesis at the translational level under all conditions tested. (J Histochem Cytochem 53:93–103, 2005)

Numerical chromosomal imbalances are a common feature of spontaneous abortions. However, the incidence of mosaic forms of chromosomal abnormalities has not been evaluated. We have applied interphase multicolor fluorescence in situ hybridization using original DNA probes for chromosomes 1, 9, 13, 14, 15, 16, 18, 21, 22, X, and Y to study chromosomal abnormalities in 148 specimens of spontaneous abortions. We have detected chromosomal abnormalities in 89/148 (60.1%) of specimens. Among them, aneuploidy was detected in 74 samples (83.1%). In the remaining samples, polyploidy was detected. The mosaic forms of chromosome abnormality, including autosomal and sex chromosomal aneuploidies and polyploidy (31 and 12 cases, respectively), were observed in 43/89 (48.3%) of specimens. The most frequent mosaic form of aneuploidy was related to chromosome X (19 cases). The frequency of mosaic forms of chromosomal abnormalities in samples with male chromosomal complement was 50% (16/32 chromosomally abnormal), and in samples with female chromosomal complement, it was 47.4% (27/57 chromosomally abnormal). The present study demonstrates that the postzygotic or mitotic errors leading to chromosomal mosaicism in spontaneous abortions are more frequent than previously suspected. Chromosomal mosaicsm may contribute significantly to both pregnancy complications and spontaneous fetal loss. (J Histochem Cytochem 53:375–380, 2005)

The majority of newly acquired HIV infections are believed to occur following transmission of virus infectivity across mucosal surfaces, although many mechanistic details still remain unresolved. We have used human ex vivo organ cultures and primary cell populations to analyze the cellular and molecular basis for mucosal HIV transmission. By using human palatine tonsil from routine tonsillectomies and semen from HIV-positive donors, we have created an experimental equivalent to oral HIV transmission. HIV infection was readily transferred into tonsillar lymphocytes, but this transmission into lymphocytes was dramatically reduced when the exposed lymphocyte populations were protected by intact mucosal surfaces. In this study, we consider the impact that leukocyte activation and morphological aberrations in surface structure may have on susceptibility to primary HIV infection and introduce novel time-lapse confocal microscopy procedures that begin to reveal the dynamic complexity associated with cell-mediated HIV transmission. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials. (J Histochem Cytochem 53:631–642, 2005)

The majority of newly acquired HIV infections are believed to occur following transmission of virus infectivity across mucosal surfaces, although many mechanistic details still remain unresolved. We have used human ex vivo organ cultures and primary cell populations to analyze the cellular and molecular basis for mucosal HIV transmission. By using human palatine tonsil from routine tonsillectomies and semen from HIV-positive donors, we have created an experimental equivalent to oral HIV transmission. HIV infection was readily transferred into tonsillar lymphocytes, but this transmission into lymphocytes was dramatically reduced when the exposed lymphocyte populations were protected by intact mucosal surfaces. In this study, we consider the impact that leukocyte activation and morphological aberrations in surface structure may have on susceptibility to primary HIV infection and introduce novel time-lapse confocal microscopy procedures that begin to reveal the dynamic complexity associated with cell-mediated HIV transmission. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials. (J Histochem Cytochem 53:631–642, 2005)

The majority of newly acquired HIV infections are believed to occur following transmission of virus infectivity across mucosal surfaces, although many mechanistic details still remain unresolved. We have used human ex vivo organ cultures and primary cell populations to analyze the cellular and molecular basis for mucosal HIV transmission. By using human palatine tonsil from routine tonsillectomies and semen from HIV-positive donors, we have created an experimental equivalent to oral HIV transmission. HIV infection was readily transferred into tonsillar lymphocytes, but this transmission into lymphocytes was dramatically reduced when the exposed lymphocyte populations were protected by intact mucosal surfaces. In this study, we consider the impact that leukocyte activation and morphological aberrations in surface structure may have on susceptibility to primary HIV infection and introduce novel time-lapse confocal microscopy procedures that begin to reveal the dynamic complexity associated with cell-mediated HIV transmission. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials. (J Histochem Cytochem 53:631–642, 2005)

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Exposure of nuclear antigens in formalin-fixed, paraffin-embedded necropsy human spinal cord tissue: Detection of NeuN.

J Neurosci Methods. 2005 Oct 15;148(1):26-35

Authors: Gill SK, Ishak M, Rylett RJ

Immunohistochemical and immunofluorescence staining approaches are powerful tools for characterization of the endogenous protein expression and subcellular compartmentalization. However, several technical problems hamper identification of low-abundance nuclear proteins in archival formalin-fixed, paraffin-embedded human neural tissue. These include loss of protein antigenicity during tissue fixation and processing, and intrinsic auto-fluorescence associated with the tissue related to its fixation and the presence of lipofuscin. We evaluated several antigen retrieval methods to establish a strategy for detection of neuronal nuclear proteins in human spinal cord formalin-fixed, paraffin-embedded tissue. Thus, using immunostaining of the neuron-specific nuclear protein NeuN as the outcome measure, we found that heating tissue sections in an alkaline pH buffer unmasked protein epitopes most effectively. Moreover, staining by immunohistochemistry with diaminobenzidine tetrahydrochloride chromagen was superior to immunofluorescence labeling, likely due to the signal amplification steps included in the former approach. Auto-fluorescence in the tissue sections can be effectively reduced, but a sufficient fluorescence signal associated with specific antibody labeling could not be detected above this background for NeuN in the nucleus.

PMID: 16176837 [PubMed - in process]

PMP22 is a structural protein of Schwann cells, but it also influences cell proliferation. In the present study, quantitative RT-PCR (QRT-PCR) and immunohistochemistry were used to determine PMP22 mRNA levels and to localize PMP22 in the normal pancreas (n=20), chronic pancreatitis (CP) (n=22), pancreatic ductal adenocarcinoma (PDAC) (n=31), intraductal papillary mucinous neoplasms (IPMN) (n=9), mucinous cystic tumors (MCN) (n=4), and in a panel of PanIN lesions (n=29). PMP22 mRNA levels were significantly higher in CP (3-fold) and PDAC (2.5-fold), compared to normal pancreatic tissues. PMP22 expression was restricted to nerves in the normal pancreas, while in CP and PDAC PMP22 was also expressed in PanIN lesions and in a small percentage of pancreatic cancer cells. PMP22 was weak to absent in the tumor cells of IPMNs and MCNs. PMP22 mRNA was present at different levels in cultured pancreatic cancer cells and up-regulated by transforming growth factor (TGF)-ßbeta;1 in 2 of 8 of these cell lines. In conclusion, PMP22 expression is present in both CP and PDAC tissues. Its expression in PanIN lesions and some pancreatic cancer cells in vitro and in vivo suggests a role of PMP22 in the neoplastic transformation process from the normal pancreas to pre-malignant lesions to pancreatic cancer.

(J Histochem Cytochem 53:885–893, 2005)

PMP22 is a structural protein of Schwann cells, but it also influences cell proliferation. In the present study, quantitative RT-PCR (QRT-PCR) and immunohistochemistry were used to determine PMP22 mRNA levels and to localize PMP22 in the normal pancreas (n=20), chronic pancreatitis (CP) (n=22), pancreatic ductal adenocarcinoma (PDAC) (n=31), intraductal papillary mucinous neoplasms (IPMN) (n=9), mucinous cystic tumors (MCN) (n=4), and in a panel of PanIN lesions (n=29). PMP22 mRNA levels were significantly higher in CP (3-fold) and PDAC (2.5-fold), compared to normal pancreatic tissues. PMP22 expression was restricted to nerves in the normal pancreas, while in CP and PDAC PMP22 was also expressed in PanIN lesions and in a small percentage of pancreatic cancer cells. PMP22 was weak to absent in the tumor cells of IPMNs and MCNs. PMP22 mRNA was present at different levels in cultured pancreatic cancer cells and up-regulated by transforming growth factor (TGF)-ßbeta;1 in 2 of 8 of these cell lines. In conclusion, PMP22 expression is present in both CP and PDAC tissues. Its expression in PanIN lesions and some pancreatic cancer cells in vitro and in vivo suggests a role of PMP22 in the neoplastic transformation process from the normal pancreas to pre-malignant lesions to pancreatic cancer.

(J Histochem Cytochem 53:885–893, 2005)

Cellular prion protein (PrP^c) is a glycoprotein expressed at low to moderate levels within the nervous system. Recent studies suggest that PrP^c may possess neuroprotective functions and that its expression is upregulated in certain neurodegenerative disorders. We investigated whether PrP^c expression is altered in the frontal and occipital cortex in two well-characterized neurodegenerative disorders—Alzheimer's disease (AD) and diffuse Lewy body disease (DLBD)—compared with that in normal human brain using immunohistochemistry and computerized image analysis. The distribution of PrP^c was further tested for correlation with glial reactivity. We found that PrP^c was localized mainly in the gray matter (predominantly in neurons) and expressed at higher levels within the occipital cortex in the normal human brain. Image analysis revealed no significant variability in PrP^c expression between DLBD and control cases. However, blood vessels within the white matter of DLBD cases showed immunoreactivity to PrP^c. By contrast, this protein was differentially expressed in the frontal and occipital cortex of AD cases; it was markedly overexpressed in the former and significantly reduced in the latter. Epitope specificity of antibodies appeared important when detecting PrP^c. The distribution of PrP^c did not correlate with glial immunoreactivity. In conclusion, this study supports the proposal that regional changes in expression of PrP^c may occur in certain neurodegenerative disorders such as AD, but not in other disorders such as DLBD. (J Histochem Cytochem 53:929–940, 2005)

Cellular prion protein (PrP^c) is a glycoprotein expressed at low to moderate levels within the nervous system. Recent studies suggest that PrP^c may possess neuroprotective functions and that its expression is upregulated in certain neurodegenerative disorders. We investigated whether PrP^c expression is altered in the frontal and occipital cortex in two well-characterized neurodegenerative disorders—Alzheimer's disease (AD) and diffuse Lewy body disease (DLBD)—compared with that in normal human brain using immunohistochemistry and computerized image analysis. The distribution of PrP^c was further tested for correlation with glial reactivity. We found that PrP^c was localized mainly in the gray matter (predominantly in neurons) and expressed at higher levels within the occipital cortex in the normal human brain. Image analysis revealed no significant variability in PrP^c expression between DLBD and control cases. However, blood vessels within the white matter of DLBD cases showed immunoreactivity to PrP^c. By contrast, this protein was differentially expressed in the frontal and occipital cortex of AD cases; it was markedly overexpressed in the former and significantly reduced in the latter. Epitope specificity of antibodies appeared important when detecting PrP^c. The distribution of PrP^c did not correlate with glial immunoreactivity. In conclusion, this study supports the proposal that regional changes in expression of PrP^c may occur in certain neurodegenerative disorders such as AD, but not in other disorders such as DLBD. (J Histochem Cytochem 53:929–940, 2005)

Hypertension is a common complication in children with autosomal recessive polycystic kidney disease (ARPKD) who have survived the neonatal period. No information is available regarding the mechanism of hypertension in this condition. The renin-angiotensin system (RAS) is thought to play a role in hypertension associated with the more common autosomal dominant polycystic kidney disease (ADPKD). Occasional reports have documented increased activity of the intrarenal RAS in ADPKD, with ectopic renin expression within cysts and dilated tubules. Because of similarities between ARPKD and ADPKD, we hypothesized that increased intrarenal RAS activity might also be found in ARPKD. We performed immunohistochemical studies on kidney tissues from two infants with ARPKD and two control kidneys. The cystic dilated tubules showed staining with the peanut lectin arachis hypogaea, a marker of distal tubules and collecting ducts, but not with lotus tetragonolobus, a marker of proximal tubules. Strong renin staining was seen in many cysts and tubules of ARPKD kidneys, but only in the afferent arterioles of the normal control kidneys. Angiotensinogen staining was also observed in some cysts and in proximal tubules. Staining for angiotensin-converting enzyme, angiotensin II type 1 receptor, and angiotensin II peptide was present in many cystic dilated tubules. These immunohistochemical studies document for the first time ectopic expression of components of the RAS in cystic-dilated tubules of ARPKD and suggest that overactivity of RAS could result in increased intrarenal angiotensin II production, which may contribute to the development of hypertension in ARPKD. (J Histochem Cytochem 53:979–988, 2005)

Hypertension is a common complication in children with autosomal recessive polycystic kidney disease (ARPKD) who have survived the neonatal period. No information is available regarding the mechanism of hypertension in this condition. The renin-angiotensin system (RAS) is thought to play a role in hypertension associated with the more common autosomal dominant polycystic kidney disease (ADPKD). Occasional reports have documented increased activity of the intrarenal RAS in ADPKD, with ectopic renin expression within cysts and dilated tubules. Because of similarities between ARPKD and ADPKD, we hypothesized that increased intrarenal RAS activity might also be found in ARPKD. We performed immunohistochemical studies on kidney tissues from two infants with ARPKD and two control kidneys. The cystic dilated tubules showed staining with the peanut lectin arachis hypogaea, a marker of distal tubules and collecting ducts, but not with lotus tetragonolobus, a marker of proximal tubules. Strong renin staining was seen in many cysts and tubules of ARPKD kidneys, but only in the afferent arterioles of the normal control kidneys. Angiotensinogen staining was also observed in some cysts and in proximal tubules. Staining for angiotensin-converting enzyme, angiotensin II type 1 receptor, and angiotensin II peptide was present in many cystic dilated tubules. These immunohistochemical studies document for the first time ectopic expression of components of the RAS in cystic-dilated tubules of ARPKD and suggest that overactivity of RAS could result in increased intrarenal angiotensin II production, which may contribute to the development of hypertension in ARPKD. (J Histochem Cytochem 53:979–988, 2005)

Expression of CXCR4 is associated with axillary lymph node status in patients with early breast cancer.

Breast. 2005 Oct 17;

Authors: Su YC, Wu MT, Huang CJ, Hou MF, Yang SF, Chai CY

Recent studies have discovered that CXCR4 is associated with tumor metastasis. It is worth understanding the association between CXCR4 expression and axillary lymph node involvement in early breast cancer. Eighty-five patients with early breast cancers were divided into three groups based on their axillary lymph node status. CXCR4 expression was assessed by immunohistochemistry in all cases and its correlation with axillary lymph node involvement was evaluated. There was a significant difference in nuclear CXCR4 expression among these three groups and high nuclear expression of CXCR4 was associated with cases with no lymph node involvement. However, high cytoplasmic expression of CXCR4 was associated with patients who developed high-level axillary lymph node involvement. In conclusion, the different staining locations of CXCR4 have varying biological significance for the metastatic potential of axillary lymph nodes. In particular, it provided information that high cytoplasmic expression of CXCR4 was related to axillary internodal metastasis, and adjuvant radio-chemotherapy was suggested.

PMID: 16239110 [PubMed - as supplied by publisher]

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Expression of D2-40 in lymphatic endothelium of normal tissues and in vascular tumours.

Histopathology. 2005 Apr;46(4):396-402

Authors: Fukunaga M

AIMS: To evaluate the expression of D2-40 in normal lymphatic endothelium and vascular tumours or tumour-like lesions of the skin and soft tissue. D2-40 is a novel monoclonal antibody to a Mr 40 000 O-linked sialoglycoprotein that reacts with a fixation-resistant epitope in lymphatic endothelium. METHODS AND RESULTS: Formalin-fixed paraffin-embedded sections from 30 normal tissue samples, including skin, soft tissue, stomach, and colon, and 84 vascular tumours or vascular tumour-like lesions were immunostained with monoclonal antibodies to D2-40 and CD31. Normal lymphatic endothelial cells in all normal tissues expressed D2-40. Its positive staining delineated flattened channels or open spaces lined by a single layer of endothelial cells whose lumena were sometimes filled with lymphocytes. Ten of 10 cases of lymphangioma, nine of 10 Kaposi's sarcomas (KSs), one of five spindle cell haemangiomas, one of one reactive angioenodotheliomatosis, one of one vascular transformation of lymph node sinuses, three of three Dabska tumours, one of 10 epithelioid haemangioendotheliomas (HEs) and seven of 15 angiosarcomas were positive for D2-40. Positively staining angiosarcomas were characterized by epithelioid or papillary endothelial cells. Twenty-two non-spindle cell haemangiomas, one retiform HE and one Kaposiform HE, and five glomus tumours were negative for D2-40. In comparison, CD31 was expressed in five of 10 lymphangiomas, nine of 10 KSs, 27 of 27 haemangiomas, three of three Dabska tumours, 10 of 10 epithelioid HEs, 15 of 15 angiosarcomas and one of one each of retiform HE, Kaposiform HE, reactive angioendotheliomatosis, and vascular transformation of node sinuses. Five glomus tumours were negative for CD31. CONCLUSIONS: The monoclonal antibody D2-40 is a highly sensitive and specific marker of lymphatic endothelium in normal tissue and a subset of vascular lesions, including KS, Dabska tumour, and lymphangioma. The findings support the concept that these tumours show at least partial lymphatic endothelial differentiation. Subsets of angiosarcomas and HEs show both vascular and lymphatic endothelial differentiation. D2-40 can be used in a panel of markers to classify vascular tumours. There is no requirement for epitope retrieval. This novel monoclonal antibody also has the potential for increasing the accuracy of detection of lymphatic invasion in primary tumours and could be widely applied for this purpose in surgical pathology.

PMID: 15810951 [PubMed - indexed for MEDLINE]

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Expression of HBsAg and HBcAg in the ovaries and ova of patients with chronic hepatitis B.

World J Gastroenterol. 2005 Sep 28;11(36):5718-20

Authors: Ye F, Yue YF, Li SH, Chen TY, Zhang SL, Bai GQ, Liu M

AIM: To investigate the expression and distribution of HBV in the ovaries and ova. METHODS: The immunohistochemistry method was used to detect the HBsAg and HBcAg in the ovaries of patients with chronic hepatitis B. RESULTS: Expression of HBsAg in the ova, granular and interstitial cells of the ovaries was located in the cytomembrane and cytoplasm. Expression of HBcAg in the ova, granular, interstitial and endothelial cells of interstitial blood vessels of the ovaries was found in the cytomembrane, cytoplasm, and nuclei. CONCLUSION: HBV can infect the ova at different stages of development and replicate in it.

PMID: 16237773 [PubMed - in process]

The Nkx6 gene family of homeodomain transcription factors consists of three members. For two, Nkx6.1 and Nkx6.2, important developmental roles in the central nervous system and pancreas have been demonstrated. Here we introduce the third member of the Nkx6 gene family, Nkx6.3, and identify similar and distinct patterns of expression for all three Nkx6 genes in the hindbrain and gut of the developing mouse embryo.

(J Histochem Cytochem 53:787–790, 2005)

The Nkx6 gene family of homeodomain transcription factors consists of three members. For two, Nkx6.1 and Nkx6.2, important developmental roles in the central nervous system and pancreas have been demonstrated. Here we introduce the third member of the Nkx6 gene family, Nkx6.3, and identify similar and distinct patterns of expression for all three Nkx6 genes in the hindbrain and gut of the developing mouse embryo.

(J Histochem Cytochem 53:787–790, 2005)

The Nkx6 gene family of homeodomain transcription factors consists of three members. For two, Nkx6.1 and Nkx6.2, important developmental roles in the central nervous system and pancreas have been demonstrated. Here we introduce the third member of the Nkx6 gene family, Nkx6.3, and identify similar and distinct patterns of expression for all three Nkx6 genes in the hindbrain and gut of the developing mouse embryo.

(J Histochem Cytochem 53:787–790, 2005)

Cyclic nucleotide phosphodiesterase 11A (PDE11A) is the newest member in the PDE family. Although the tissue distribution of PDE11A mRNA has been shown, its protein expression pattern has not been well studied. The goal of this report is to investigate the distribution of PDE11A proteins in a wide range of normal and malignant human tissues. We utilized a polyclonal antibody that recognized all four PDE11A isoforms. Its specificity was demonstrated by Western blot analysis on a recombinant human PDE11A protein and native PDE11A proteins in various human tissues. Immunohistochemistry showed that PDE11A is widely expressed. Various degrees of immunoreactivity were observed in the epithelial cells, endothelial cells, and smooth muscle cells of all tissues examined. The highest expression was in the epithelial, endothelial, and smooth muscle cells of the prostate, Leydig, and spermatogenic cells of the testis, the tubule epithelial cells in the kidney, the epithelial and endothelial cells in the adrenal, the epithelial cells and macrophages in the colon, and the epidermis in the skin. Furthermore, PDE11A expression was also detected in several human carcinomas. Our results suggest that PDE11A might be involved in multiple physiological processes in various organs via its ability to modulate intracellular cAMP and cGMP levels. (J Histochem Cytochem 53:895–903, 2005)

Cyclic nucleotide phosphodiesterase 11A (PDE11A) is the newest member in the PDE family. Although the tissue distribution of PDE11A mRNA has been shown, its protein expression pattern has not been well studied. The goal of this report is to investigate the distribution of PDE11A proteins in a wide range of normal and malignant human tissues. We utilized a polyclonal antibody that recognized all four PDE11A isoforms. Its specificity was demonstrated by Western blot analysis on a recombinant human PDE11A protein and native PDE11A proteins in various human tissues. Immunohistochemistry showed that PDE11A is widely expressed. Various degrees of immunoreactivity were observed in the epithelial cells, endothelial cells, and smooth muscle cells of all tissues examined. The highest expression was in the epithelial, endothelial, and smooth muscle cells of the prostate, Leydig, and spermatogenic cells of the testis, the tubule epithelial cells in the kidney, the epithelial and endothelial cells in the adrenal, the epithelial cells and macrophages in the colon, and the epidermis in the skin. Furthermore, PDE11A expression was also detected in several human carcinomas. Our results suggest that PDE11A might be involved in multiple physiological processes in various organs via its ability to modulate intracellular cAMP and cGMP levels. (J Histochem Cytochem 53:895–903, 2005)

This study demonstrated determination of fetal gender from nucleated red blood cells (NRBCs) in maternal blood and attempted to apply prenatal diagnosis of hemophilia A using BclI DNA polymorphism. Venous blood was drawn from 20 pregnant women, and NRBCs were recovered by magnetic activated cell sorting and anti-GPA (glycophorin A) immunostaining. After microdissector isolation of the NRBCs, primer extension preamplification (PEP) and nested PCR of the amelogenin gene were performed to determine fetal gender. We also performed PEP and nested PCR of BclI polymorphism to verify the validity of prenatal diagnosis of hemophilia A. DNA amplification was achieved in 107 cells (51.9%) and fetal gender determined with 65.0% accuracy. Unfortunately, we could not verify the validity within the scope of this study. However, in a larger number of cases that are informative in BclI polymorphism, we will be able to identify patients affected by hemophilia A using fetal NRBCs in maternal blood. (J Histochem Cytochem 53:323–327, 2005)

The present study investigated potential age-related changes in human muscle spindles with respect to the intrafusal fiber-type content and myosin heavy chain (MyHC) composition in biceps brachii muscle. The total number of intrafusal fibers per spindle decreased significantly with aging, due to a significant reduction in the number of nuclear chain fibers. Nuclear chain fibers in old spindles were short and some showed novel expression of MyHC -cardiac. The expression of MyHC -cardiac in bag₁ and bag₂ fibers was greatly decreased in the A region. The expression of slow MyHC was increased in nuclear bag₁ fibers and that of fetal MyHC decreased in bag₂ fibers whereas the patterns of distribution of the remaining MyHC isoforms were generally not affected by aging. We conclude that aging appears to have an important impact on muscle spindle composition. These changes in muscle spindle phenotype may reflect an age-related deterioration in sensory and motor innervation and are likely to have an impact in motor control in the elderly. (J Histochem Cytochem 53:445–454, 2005)

The present study investigated potential age-related changes in human muscle spindles with respect to the intrafusal fiber-type content and myosin heavy chain (MyHC) composition in biceps brachii muscle. The total number of intrafusal fibers per spindle decreased significantly with aging, due to a significant reduction in the number of nuclear chain fibers. Nuclear chain fibers in old spindles were short and some showed novel expression of MyHC -cardiac. The expression of MyHC -cardiac in bag₁ and bag₂ fibers was greatly decreased in the A region. The expression of slow MyHC was increased in nuclear bag₁ fibers and that of fetal MyHC decreased in bag₂ fibers whereas the patterns of distribution of the remaining MyHC isoforms were generally not affected by aging. We conclude that aging appears to have an important impact on muscle spindle composition. These changes in muscle spindle phenotype may reflect an age-related deterioration in sensory and motor innervation and are likely to have an impact in motor control in the elderly. (J Histochem Cytochem 53:445–454, 2005)

The present study investigated potential age-related changes in human muscle spindles with respect to the intrafusal fiber-type content and myosin heavy chain (MyHC) composition in biceps brachii muscle. The total number of intrafusal fibers per spindle decreased significantly with aging, due to a significant reduction in the number of nuclear chain fibers. Nuclear chain fibers in old spindles were short and some showed novel expression of MyHC -cardiac. The expression of MyHC -cardiac in bag₁ and bag₂ fibers was greatly decreased in the A region. The expression of slow MyHC was increased in nuclear bag₁ fibers and that of fetal MyHC decreased in bag₂ fibers whereas the patterns of distribution of the remaining MyHC isoforms were generally not affected by aging. We conclude that aging appears to have an important impact on muscle spindle composition. These changes in muscle spindle phenotype may reflect an age-related deterioration in sensory and motor innervation and are likely to have an impact in motor control in the elderly. (J Histochem Cytochem 53:445–454, 2005)

First results of chronic wasting disease (CWD) surveillance in the south-eastern part of Belgium.

Vet Q. 2005 Sep;27(3):98-104

Authors: Roels S, Saegerman C, De Bosschere H, Berkvens D, Gregoire F, Hoyoux A, Mousset B, Desmecht D, Vanopdenbosch E, Linden A

Chronic wasting disease (CWD) has not been reported in Europe, whereas it is considered to be enzootic in free-ranging mule deer, Rocky mountain elk and white-tailed deer in the area of Colorado, Wyoming, and Nebraska, and new foci of CWD have been detected in other parts of the United States. However, no large-scale active epidemiosurveillance of European wild cervids has been installed in Europe. In accordance with the opinion of the European Scientific Steering Committee, a preliminary (active) surveillance scheme was installed, in order to improve the knowledge of the CWD status of the Belgian free-ranging cervids (roe deer and red deer). Spleen samples (n=866) of roe deer and red deer collected in the south-eastern part of Belgium, were examined for CWD using a enzyme-linked immunosorbent assay of Bio-Rad. Afterwards, the ELISA was systematically confirmed by immunohistochemistry using three antibodies, namely R524, 2G11 and 12F10. There were no indications on the occurrence of transmissible spongiform enncephalopathy (TSE) in any of the samples. A Bayesian framework was used for the estimation of the true prevalence of CWD in south-eastern part of Belgium that was estimated to have a median value of zero with a 95% percentile value of 0.00115.

PMID: 16238109 [PubMed - in process]

We present the first data on our comparative genomic hybridization (CGH)–based strategy for the analysis of ancient DNA (aDNA) samples extracted from fetuses preserved in the Meckel Anatomical Collection in Halle, Germany. The collection contains numerous differently fixed ancient samples of fetal malformations collected from the middle of the 18th to the early 19th century. The main objective of this study is to establish a "standard" aDNA extraction and amplification protocol as a prerequisite for successful CGH analyses to detect or exclude chromosomal imbalances possibly causative for the malformations described for the fetuses. (J Histochem Cytochem 53:381–384, 2005)

This study aimed to determine whether the number of nucleated red blood cells (NRBCs) in maternal circulation during the first trimester of pregnancy could identify pregnancies that will have an anomalous Doppler in the second trimester. A total of 85 blood samples were obtained at 11–14 weeks of gestation with mean uterine arterial perfusion index >1.6, as noted by Doppler ultrasonography. NRBCs were enriched by magnetic automated cell sorting using anti-CD71 and were stained with May/Grunwald/Giemsa. A total of 4.8 NRBCs (range 1–75) were identified in 68 cases. Follow-up scans at 22–24 weeks were available in 46 cases. In 39 women, blood flow in the uterine arteries normalized, whereas in seven, high resistance was noted. One woman in the high-resistance group developed preeclampsia (PET; four NRBCs) and another delivered an intrauterine growth restriction (IUGR) baby (75 NRBCs). The number of NRBCs in women whose Doppler indices later normalized and in those who continued to have increased impedance was similar. The study indicates that NRBC number in maternal circulation during the first trimester cannot be used to screen pregnancies at high risk for developing preeclampsia (PET)/IUGR. High-impedance blood flow in the uterine arteries in the first trimester may be due to an unfinished process of trophoblastic invasion, most likely to be completed successfully by 22–24 weeks. (J Histochem Cytochem 53:315–317, 2005)

An improvement in prenatal screening for chromosomal defects has been achieved by combining sonography and biochemical markers. Analyzing markers taken from maternal blood such as pregnancy-associated plasma protein A and free ßbeta;-human chorionic gonadotropin in combination with the ultrasound marker nuchal translucency provides detection rates of 90% for the most important chromosomal anomalies. In addition, nuchal translucency is a marker for severe heart defects. This report discusses the potential of new markers such as the nasal bone. (J Histochem Cytochem 53:281–283, 2005)

Current cytogenetic approaches in noninvasive prenatal diagnosis focus on fetal nucleated red blood cells in maternal blood. This practice may be too restrictive because a vast proportion of other fetal cells is ignored. Recent studies have indicated that fetal cells can be directly detected, without prior enrichment, in maternal blood samples by fluorescence in situ hybridization (FISH) analysis for chromosomes X and Y (XY-FISH). In our blinded analysis of 40 maternal blood samples, we therefore examined all fetal cells without any enrichment. Initial examinations using conventional XY-FISH indicated a low specificity of 69.4%, which could be improved to 89.5% by the use of two different Y-chromosome-specific probes (YY-FISH) with only a slight concomitant decrease in sensitivity (52.4% vs 42.9%). On average, 12–20 male fetal cells/ml of maternal blood were identified by XY- and YY-FISH, respectively. (J Histochem Cytochem 53:319–322, 2005)

We investigated the frequencies of abnormalities involving either chromosome 1, 16, 18, or 21 in failed-fertilized human oocytes. Although abnormalities involving chromosome 16 showed an age-dependent increase, results for the other chromosomes did not show statistically significant differences among the three age groups, <35 years, 35–39 years, and >39 years. The scoring of four chromosomes is likely to underestimate the true rate of aneuploid cells. Therefore, for a pilot study investigating a more-comprehensive analysis of oocytes and their corresponding first polar bodies, we developed a novel eight-probe chromosome enumeration scheme using fluorescence in situ hybridization and spectral imaging analysis. (J Histochem Cytochem 53:269–272, 2005)

Fluorescence in situ hybridization, comparative genomic hybridization, and other molecular biology techniques in the analysis of effusions.

Diagn Cytopathol. 2005 Oct 20;33(5):294-299

Authors: Reis-Filho JS, de Landér Schmitt FC

Over the last 20 yr, the introduction of immunocytochemistry as a diagnostic tool has dramatically revolutionized diagnostic pathology. With the introduction of molecular methods as part of the diagnostic armamentarium, the practicing pathologist is facing the new challenge of grasping novel concepts of the molecular cytogenetics era. Herein, we review the diagnostic contribution of ancillary molecular techniques, including fluorescent and chromogenic in situ hybridization, telomerase assays, loss of heterozygosity, comparative genomic hybridization (CGH), and microarray-based CGH, for the practicing cytopathologists and discuss how these techniques will help pathologists in decision-making. Diagn. Cytopathol. 2005;33:294-299. (c) 2005 Wiley-Liss, Inc.

PMID: 16240394 [PubMed - as supplied by publisher]

Fluorescent two-color whole mount in situ hybridization in Platynereis dumerilii (Polychaeta, Annelida), an emerging marine molecular model for evolution and development.

Biotechniques. 2005 Oct;39(4):460, 462, 464

Authors: Tessmar-Raible K, Steinmetz PR, Snyman H, Hassel M, Arendt D

PMID: 16235555 [PubMed - in process]

Formation of nonculturable Escherichia coli in drinking water.

J Appl Microbiol. 2005 Nov;99(5):1090-1098

Authors: Bjergbæk LA, Roslev P

Aims: To examine whether incubation of Escherichia coli in nondisinfected drinking water result in development of cells that are not detectable using standard procedures but maintain a potential for metabolic activity and cell division. Methods and Results: Survival and detectability of four different E. coli strains were studied using drinking water microcosms and samples from contaminated drinking water wells. Recovery of E. coli was compared using different cultivation-dependent methods, fluorescence in situ hybridization (FISH) using specific oligonucleotide probes, direct viable counts (DVC), and by enumeration of gfp-tagged E. coli (green fluorescent protein, GFP). Two levels of stress responses were observed after incubation of E. coli in nondisinfected drinking water: (i) the presence of cells that were not detected using standard cultivation methods but could be cultivated after gentle resuscitation on nonselective nutrient-rich media, and (ii) the presence of cells that responded to nutrient addition but could only be detected by cultivation-independent methods (DVC, FISH and GFP). Collectively, the experiments demonstrated that incubation for 20-60 days in nondisinfected drinking water resulted in detection of only 0.7-5% of the initial E. coli population using standard cultivation methods, whereas 1-20% could be resuscitated to a culturable state, and 17-49% could be clearly detected using cultivation-independent methods. Conclusions: Resuscitation of stressed E. coli on nonselective nutrient-rich media increased cell counts in drinking water using both traditional (CFU), and cultivation-independent methods (DVC, FISH and GFP). The cultivation-independent methods resulted in detection of 10-20 times more E. coli than the traditional methods. The results indicate that a subpopulation of substrate-responsive but apparent nonculturable E. coli may develop in drinking water during long-term starvation survival. Significance and Impact of the Study: The existence of substrate-responsive but nonculturable cells should be considered when evaluating the survival potential of E. coli in nondisinfected drinking water.

PMID: 16238739 [PubMed - as supplied by publisher]

Tartrate-resistant acid phosphatase (TRAP) is essential for elimination of Staphylococcus aureus, the main infectious agent responsible for osteomyelitis. This in vitro study investigated uptake and processing of fluorescence-labeled S. aureus by human osteoclasts and dendritic cells. The cells were stained for TRAP and the acidic compartment using a fluorescence-based protocol. In dendritic cells, TRAP and bacteria were colocalized. In osteoclasts, there was no colocalization of bacteria, TRAP, or the acidic compartment, indicating that there are three distinct vesicular compartments: the apical phago-lysosomal compartment, the basal secretory compartment, and the basolateral transcytotic compartment. Dissociation of the TRAP-containing transcytotic vesicles from the apical phago-lysosomal compartment may restrain osteoclasts from eliminating S. aureus.

(J Histochem Cytochem 53:665–670, 2005)

Tartrate-resistant acid phosphatase (TRAP) is essential for elimination of Staphylococcus aureus, the main infectious agent responsible for osteomyelitis. This in vitro study investigated uptake and processing of fluorescence-labeled S. aureus by human osteoclasts and dendritic cells. The cells were stained for TRAP and the acidic compartment using a fluorescence-based protocol. In dendritic cells, TRAP and bacteria were colocalized. In osteoclasts, there was no colocalization of bacteria, TRAP, or the acidic compartment, indicating that there are three distinct vesicular compartments: the apical phago-lysosomal compartment, the basal secretory compartment, and the basolateral transcytotic compartment. Dissociation of the TRAP-containing transcytotic vesicles from the apical phago-lysosomal compartment may restrain osteoclasts from eliminating S. aureus.

(J Histochem Cytochem 53:665–670, 2005)

Tartrate-resistant acid phosphatase (TRAP) is essential for elimination of Staphylococcus aureus, the main infectious agent responsible for osteomyelitis. This in vitro study investigated uptake and processing of fluorescence-labeled S. aureus by human osteoclasts and dendritic cells. The cells were stained for TRAP and the acidic compartment using a fluorescence-based protocol. In dendritic cells, TRAP and bacteria were colocalized. In osteoclasts, there was no colocalization of bacteria, TRAP, or the acidic compartment, indicating that there are three distinct vesicular compartments: the apical phago-lysosomal compartment, the basal secretory compartment, and the basolateral transcytotic compartment. Dissociation of the TRAP-containing transcytotic vesicles from the apical phago-lysosomal compartment may restrain osteoclasts from eliminating S. aureus.

(J Histochem Cytochem 53:665–670, 2005)

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Galanin-mediated anxiolytic effect in rat central amygdala is not a result of corelease from noradrenergic terminals.

Synapse. 2005 Oct 19;59(1):27-40

Authors: Barrera G, Hernandez A, Poulin JF, Laforest S, Drolet G, Morilak DA

Galanin is colocalized extensively with norepinephrine in brain. Although this suggests possible activity-dependent neurotransmitter interactions, the functional significance of such colocalization remains elusive. Previously, we showed that enhancing stress-activation of the noradrenergic system by yohimbine pretreatment released galanin in central amygdala, attenuating the anxiety-like behavioral response to stress on the elevated plus-maze. The present study was conducted to determine, in this context, whether galanin was indeed coreleased from noradrenergic terminals, or instead from another galanin afferent or local stress-responsive galanin neurons in the amygdala. In experiment 1, galanin-mediated anxiolytic effects on the plus-maze following yohimbine + stress were unaltered by lesioning the noradrenergic innervation of central amygdala. In experiment 2, combining immunohistochemistry and in situ hybridization, galanin neurons specifically activated by yohimbine + stress treatment were found only in the locus coeruleus and intraamygdalar bed nucleus of the stria terminalis, adjacent to central amygdala. In experiment 3, retrograde tracing combined with in situ hybridization revealed few if any galanin cells projecting to central amygdala in locus coeruleus or nucleus tractus solitarius, sources of noradrenergic innervation. Indeed, few retrogradely-labeled galanin neurons were observed anywhere in the brain, including a small number in the intraamygdalar bed nucleus. Together, these results suggest that stress following yohimbine may have induced galanin release from an afferent to central amygdala originating in the bed nucleus, or from local neurons in the intraamygdalar bed nucleus, but that anxiolytic effects exerted by galanin in this context of elevated noradrenergic activity were not the result of corelease from noradrenergic terminals innervating central amygdala. Synapse 59:27-40, 2006. (c) 2005 Wiley-Liss, Inc.

PMID: 16237681 [PubMed - as supplied by publisher]

Related Articles

Galanin-mediated anxiolytic effect in rat central amygdala is not a result of corelease from noradrenergic terminals.

Synapse. 2005 Oct 19;59(1):27-40

Authors: Barrera G, Hernandez A, Poulin JF, Laforest S, Drolet G, Morilak DA

Galanin is colocalized extensively with norepinephrine in brain. Although this suggests possible activity-dependent neurotransmitter interactions, the functional significance of such colocalization remains elusive. Previously, we showed that enhancing stress-activation of the noradrenergic system by yohimbine pretreatment released galanin in central amygdala, attenuating the anxiety-like behavioral response to stress on the elevated plus-maze. The present study was conducted to determine, in this context, whether galanin was indeed coreleased from noradrenergic terminals, or instead from another galanin afferent or local stress-responsive galanin neurons in the amygdala. In experiment 1, galanin-mediated anxiolytic effects on the plus-maze following yohimbine + stress were unaltered by lesioning the noradrenergic innervation of central amygdala. In experiment 2, combining immunohistochemistry and in situ hybridization, galanin neurons specifically activated by yohimbine + stress treatment were found only in the locus coeruleus and intraamygdalar bed nucleus of the stria terminalis, adjacent to central amygdala. In experiment 3, retrograde tracing combined with in situ hybridization revealed few if any galanin cells projecting to central amygdala in locus coeruleus or nucleus tractus solitarius, sources of noradrenergic innervation. Indeed, few retrogradely-labeled galanin neurons were observed anywhere in the brain, including a small number in the intraamygdalar bed nucleus. Together, these results suggest that stress following yohimbine may have induced galanin release from an afferent to central amygdala originating in the bed nucleus, or from local neurons in the intraamygdalar bed nucleus, but that anxiolytic effects exerted by galanin in this context of elevated noradrenergic activity were not the result of corelease from noradrenergic terminals innervating central amygdala. Synapse 59:27-40, 2006. (c) 2005 Wiley-Liss, Inc.

PMID: 16237681 [PubMed - as supplied by publisher]

Related Articles

Gene expression in human keloids is altered from dermal to chondrocytic and osteogenic lineage.

Genes Cells. 2005 Nov;10(11):1081-91

Authors: Naitoh M, Kubota H, Ikeda M, Tanaka T, Shirane H, Suzuki S, Nagata K

Keloids are a dermal fibrotic disease whose etiology remains totally unknown and for which there is no successful treatment. Here, we employed cDNA microarray analysis to examine gene expression in keloid lesions and control skin. We found that 32 genes among the 9000 tested were strongly up-regulated in keloid lesions, of which 21 were confirmed by Northern blotting. These included at least seven chondrocyte/osteoblast marker genes, and RT-PCR analysis revealed that transcription factors specific for these genes, SOX9 and CBFA1, were induced. Immunostaining and in situ hybridization further supported that these markers are expressed in keloid lesions. Intriguingly, scleraxis, a transcription factor known as a marker of tendons and ligaments, was also induced in keloid fibroblasts. We propose that reprogramming of gene expression or disordered differentiation from a dermal pattern to that of a chondrocytic/osteogenic lineage, probably closer to that of tendon/ligament lineage, may be involved in the etiology of keloids.

PMID: 16236136 [PubMed - in process]

Heterogeneous chloride homeostasis and GABAA responses in the median preoptic nucleus of the rat.

J Physiol. 2005 Oct 20;

Authors: Grob M, Mouginot D

The median preoptic nucleus (MnPO) is an integrative structure of the hypothalamus receiving periphery-derived information pertinent to hydromineral and cardiovascular homeostasis. In this context, excitability of MnPO neurons is controlled by fast GABAergic, glutamatergic and angiotensinergic projection from the subfornical organ (SFO). Taking advantage of a brain slice preparation preserving synaptic connection between the SFO and the MnPO and appropriate bicarbonate-free artificial CSF, we investigated the role of the neuron-specific K(+)/Cl(-) cotransporter, KCC2, in regulating efficacy of the GABAA receptor-mediated inhibitory response at the SFO-MnPO synapse. When somata of the MnPO neurons was loaded with 18 mM chloride, stimulation of the SFO evoked outward IPSCs in 81% of the MnPO neurons held at -60 mV. Accordingly, EIPSC was found 25 mV hyperpolarized from the theoretical value calculated from the Nernst equation, indicating that IPSC polarity and amplitude were driven by an active Cl(-) extrusion system in these neurons. EIPSC estimated with gramicidin-based perforated-patch recordings amounted -89.2 +/- 4.3 mV. Furosemide (100microM), a pharmacological compound known to block the activity of the neuron-specific K(+)/Cl(-) cotransporter, KCC2, reversed IPSC polarity and shifted EIPSC towards theoretical value. Presence of the KCC2 protein in the MnPO was further detected with immunohistochemistry, revealing a dense network of KCC2 positive intermingled fibers. In the presence of a GABAB receptor antagonist, high frequency stimulation (5Hz) of the SFO evoked a train of IPSCs or IPSPs, whose amplitude was maintained throughout the sustained stimulation. Contrastingly, similar 5Hz stimulation carried out in the presence of furosemide (50microM) evoked IPSCs/IPSPs, whose amplitude collapsed during the high-frequency stimulation. Similar reduction in inhibitory neurotransmission was also observed in MnPO neurons lacking the functional Cl(-) extrusion mechanism. We conclude that a majority of MnPO neurons were characterized by a functional Cl(-) transporter that insured efficient activity-dependent Cl(-) transport rate, allowing sustained synaptic inhibition of these neurons. Pharmacological and anatomical data strongly suggested the involvement of KCC2, as an essential postsynaptic determinant of the inhibitory neurotransmission afferent to the MnPO, a key-structure in the physiology of the hydromineral and cardiovascular homeostasis.

PMID: 16239278 [PubMed - as supplied by publisher]

Pompe disease is an autosomal recessive lysosomal storage disorder caused by a deficiency of the lysosomal enzyme acid -glucosidase, responsible for the degradation of lysosomal glycogen. Absent or low levels of the enzyme leads to lysosomal glycogen accumulation in cardiac and skeletal muscle cells, resulting in progressive muscle weakness and death from cardiac or respiratory failure. Recombinant enzyme replacement and gene therapy are now being investigated as treatment modalities for this disease. A knockout mouse model for Pompe disease, induced by the disruption of exon 6 within the acid -glucosidase gene, mimics the human disease and has been used to evaluate the efficacy of treatment modalities for clearing glycogen. However, for accurate histopathological assessment of glycogen clearance, maximal preservation of in situ lysosomal glycogen is essential. To improve retention of glycogen in Pompe tissues, several fixation and embedding regimens were evaluated. The best glycogen preservation was obtained when tissues fixed with 3% glutaraldehyde and postfixed with 1% osmium tetroxide were processed into epon–araldite. Preservation was confirmed by staining with the Periodic acid–Schiff's reaction and by electron microscopy. This methodology resulted in high-resolution light microscopy (HRLM) sections suitable for digital quantification of glycogen content in heart and skeletal muscle. Combining this method of tissue fixation with computer-assisted histomorphometry has provided us with what we believe is the most objective and reproducible means of evaluating histological glycogen load in Pompe disease.

(J Histochem Cytochem 53:63–73, 2005)

The power for appreciating complex cellular interactions during embryonic development using green fluorescent protein (GFP) as a visual histological marker has not been applied to adult tissues due to loss of GFP signal during paraffin embedding and a high autofluorescent background, particularly in section of bone and bone marrow. Here we demonstrate that the GFP signal is well preserved in frozen sections of adult decalcified bone. Using a tape-transfer system that preserves histological relationships, GFP expression can be related to standard histological stains used in bone biology research. The choice of a dual-filter cube and a strong GFP signal makes it possible to readily distinguish at least four different GFP colors that are distinctly different from the autofluorescent background. An additional advantage of the frozen sections is better preservation of immunological epitopes that allow colocalization of an immunostained section with an endogenous GFP and a strong lacZ signal emanating from a ßbeta;-gal marker gene. We present an approach for recording multiple images from the same histological section that allows colocalization of a GFP signal with subsequent stains and procedures that destroy GFP. Examples that illustrate the flexibility for dual imaging of various fluorescent signals are described in this study. The same imaging approach can serve as a vehicle for archiving, retrieving, and sharing histological images among research groups. (J Histochem Cytochem 53:593–602, 2005)

The power for appreciating complex cellular interactions during embryonic development using green fluorescent protein (GFP) as a visual histological marker has not been applied to adult tissues due to loss of GFP signal during paraffin embedding and a high autofluorescent background, particularly in section of bone and bone marrow. Here we demonstrate that the GFP signal is well preserved in frozen sections of adult decalcified bone. Using a tape-transfer system that preserves histological relationships, GFP expression can be related to standard histological stains used in bone biology research. The choice of a dual-filter cube and a strong GFP signal makes it possible to readily distinguish at least four different GFP colors that are distinctly different from the autofluorescent background. An additional advantage of the frozen sections is better preservation of immunological epitopes that allow colocalization of an immunostained section with an endogenous GFP and a strong lacZ signal emanating from a ßbeta;-gal marker gene. We present an approach for recording multiple images from the same histological section that allows colocalization of a GFP signal with subsequent stains and procedures that destroy GFP. Examples that illustrate the flexibility for dual imaging of various fluorescent signals are described in this study. The same imaging approach can serve as a vehicle for archiving, retrieving, and sharing histological images among research groups. (J Histochem Cytochem 53:593–602, 2005)

The power for appreciating complex cellular interactions during embryonic development using green fluorescent protein (GFP) as a visual histological marker has not been applied to adult tissues due to loss of GFP signal during paraffin embedding and a high autofluorescent background, particularly in section of bone and bone marrow. Here we demonstrate that the GFP signal is well preserved in frozen sections of adult decalcified bone. Using a tape-transfer system that preserves histological relationships, GFP expression can be related to standard histological stains used in bone biology research. The choice of a dual-filter cube and a strong GFP signal makes it possible to readily distinguish at least four different GFP colors that are distinctly different from the autofluorescent background. An additional advantage of the frozen sections is better preservation of immunological epitopes that allow colocalization of an immunostained section with an endogenous GFP and a strong lacZ signal emanating from a ßbeta;-gal marker gene. We present an approach for recording multiple images from the same histological section that allows colocalization of a GFP signal with subsequent stains and procedures that destroy GFP. Examples that illustrate the flexibility for dual imaging of various fluorescent signals are described in this study. The same imaging approach can serve as a vehicle for archiving, retrieving, and sharing histological images among research groups. (J Histochem Cytochem 53:593–602, 2005)

Proteomic analysis of clinical tissue specimens is a difficult undertaking. Described here is a multiplex study of protein expression levels in histological sections of human prostate that addresses many of the associated challenges. Whole-mount sections from 10 prostatectomy specimens were studied using 15 antibodies, immunohistochemical staining, digital imaging, and mathematical analysis of the data sets. The approach was successful in stratifying cell lineages present in the samples based on proteomic patterns, including differentiating normal epithelium from cancer. This strategy likely will be a useful method for extending the number of proteins that can be analyzed in clinical cancer specimens using currently available laboratory techniques.

(J Histochem Cytochem 53:177–185, 2005)

The epithelial barrier of the upper respiratory tract plays a crucial role in host defense. In this study, to elucidate whether there is antigen monitoring by dendritic cells (DCs) beyond the epithelial tight-junction barrier in allergic rhinitis, we investigated the expression and function of tight junctions and characterized DCs in the epithelium of nasal mucosa from patients with allergic rhinitis. In reverse transcription-polymerase chain reaction, mRNAs of tight-junction proteins occludin, JAM-1, ZO-1, and claudin-1, -4, -7, -8, -12, -13, and -14 were detected in the nasal mucosa. Occludin, JAM-1, and ZO-1 were colocalized in the uppermost layer in the pseudostratified epithelium of the nasal mucosa, whereas claudin-1, -4, and -7 were found throughout the epithelium. In freeze-fracture replicas of the nasal mucosa, continuous tight-junction strands formed well-developed networks. Epithelial barrier function measured by a dye tracer was well maintained in occludin-positive tight junctions in the epithelium of the nasal mucosa. HLA-DR- and CD11c-positive DCs expressed claudin-1 and penetrated beyond occludin in the epithelium of the nasal mucosa with, but not without, allergic rhinitis. These results indicate that DCs may easily access antigens beyond epithelial tight junctions in the human nasal mucosa of allergic rhinitis.

(J Histochem Cytochem 53:611–619, 2005)

The epithelial barrier of the upper respiratory tract plays a crucial role in host defense. In this study, to elucidate whether there is antigen monitoring by dendritic cells (DCs) beyond the epithelial tight-junction barrier in allergic rhinitis, we investigated the expression and function of tight junctions and characterized DCs in the epithelium of nasal mucosa from patients with allergic rhinitis. In reverse transcription-polymerase chain reaction, mRNAs of tight-junction proteins occludin, JAM-1, ZO-1, and claudin-1, -4, -7, -8, -12, -13, and -14 were detected in the nasal mucosa. Occludin, JAM-1, and ZO-1 were colocalized in the uppermost layer in the pseudostratified epithelium of the nasal mucosa, whereas claudin-1, -4, and -7 were found throughout the epithelium. In freeze-fracture replicas of the nasal mucosa, continuous tight-junction strands formed well-developed networks. Epithelial barrier function measured by a dye tracer was well maintained in occludin-positive tight junctions in the epithelium of the nasal mucosa. HLA-DR- and CD11c-positive DCs expressed claudin-1 and penetrated beyond occludin in the epithelium of the nasal mucosa with, but not without, allergic rhinitis. These results indicate that DCs may easily access antigens beyond epithelial tight junctions in the human nasal mucosa of allergic rhinitis.

(J Histochem Cytochem 53:611–619, 2005)

The epithelial barrier of the upper respiratory tract plays a crucial role in host defense. In this study, to elucidate whether there is antigen monitoring by dendritic cells (DCs) beyond the epithelial tight-junction barrier in allergic rhinitis, we investigated the expression and function of tight junctions and characterized DCs in the epithelium of nasal mucosa from patients with allergic rhinitis. In reverse transcription-polymerase chain reaction, mRNAs of tight-junction proteins occludin, JAM-1, ZO-1, and claudin-1, -4, -7, -8, -12, -13, and -14 were detected in the nasal mucosa. Occludin, JAM-1, and ZO-1 were colocalized in the uppermost layer in the pseudostratified epithelium of the nasal mucosa, whereas claudin-1, -4, and -7 were found throughout the epithelium. In freeze-fracture replicas of the nasal mucosa, continuous tight-junction strands formed well-developed networks. Epithelial barrier function measured by a dye tracer was well maintained in occludin-positive tight junctions in the epithelium of the nasal mucosa. HLA-DR- and CD11c-positive DCs expressed claudin-1 and penetrated beyond occludin in the epithelium of the nasal mucosa with, but not without, allergic rhinitis. These results indicate that DCs may easily access antigens beyond epithelial tight junctions in the human nasal mucosa of allergic rhinitis.

(J Histochem Cytochem 53:611–619, 2005)

Related Articles

Identification of a new neuropeptide precursor reveals a novel source of extrinsic modulation in the feeding system of Aplysia.

J Neurosci. 2005 Oct 19;25(42):9637-48

Authors: Proekt A, Vilim FS, Alexeeva V, Brezina V, Friedman A, Jing J, Li L, Zhurov Y, Sweedler JV, Weiss KR

The Aplysia feeding system is advantageous for investigating the role of neuropeptides in behavioral plasticity. One family of Aplysia neuropeptides is the myomodulins (MMs), originally purified from one of the feeding muscles, the accessory radula closer (ARC). However, two MMs, MMc and MMe, are not encoded on the only known MM gene. Here, we identify MM gene 2 (MMG2), which encodes MMc and MMe and four new neuropeptides. We use matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to verify that these novel MMG2-derived peptides (MMG2-DPs), as well as MMc and MMe, are synthesized from the precursor. Using antibodies against the MMG2-DPs, we demonstrate that neuronal processes that stain for MMG2-DPs are found in the buccal ganglion, which contains the feeding network, and in the buccal musculature including the ARC muscle. Surprisingly, however, no immunostaining is observed in buccal neurons including the ARC motoneurons. In situ hybridization reveals only few MMG2-expressing neurons that are mostly located in the pedal ganglion. Using immunohistochemical and electrophysiological techniques, we demonstrate that some of these pedal neurons project to the buccal ganglion and are the likely source of the MMG2-DP innervation of the feeding network and musculature. We show that the MMG2-DPs are bioactive both centrally and peripherally: they bias egestive feeding programs toward ingestive ones, and they modulate ARC muscle contractions. The multiple actions of the MMG2-DPs suggest that these peptides play a broad role in behavioral plasticity and that the pedal-buccal projection neurons that express them are a novel source of extrinsic modulation of the feeding system of Aplysia.

PMID: 16237168 [PubMed - in process]

Adrenomedullin (AM) is a potent vasodilator peptide present in the lung of mammals where it is expressed mainly in the columnar epithelium and alveolar macrophages. AM increases the secretion of phosphatidylcholine by type II pneumocytes, which suggests a role as an autocrine modulator of surfactant secretion. In this study we show the expression of an AM-like protein in the lung of the pigeon, Columba livia. Using an antibody against its human ortholog, AM-like immunoreactivity was found to be associated with membranous structures of the multivesicular bodies of type II pneumocytes. We also studied the differential expression of AM-like peptide in the lung of pigeons exposed to polluted city air vs cleaner countryside conditions and found that AM-like expression was higher in city animals. Similar results were obtained in an experimental study in which pigeons were exposed to increasing concentrations of a single pollutant, ozone. Taken together, our findings support the implication of AM in the response of type II pneumocytes to air pollutants.

(J Histochem Cytochem 53:773–780, 2005)

Adrenomedullin (AM) is a potent vasodilator peptide present in the lung of mammals where it is expressed mainly in the columnar epithelium and alveolar macrophages. AM increases the secretion of phosphatidylcholine by type II pneumocytes, which suggests a role as an autocrine modulator of surfactant secretion. In this study we show the expression of an AM-like protein in the lung of the pigeon, Columba livia. Using an antibody against its human ortholog, AM-like immunoreactivity was found to be associated with membranous structures of the multivesicular bodies of type II pneumocytes. We also studied the differential expression of AM-like peptide in the lung of pigeons exposed to polluted city air vs cleaner countryside conditions and found that AM-like expression was higher in city animals. Similar results were obtained in an experimental study in which pigeons were exposed to increasing concentrations of a single pollutant, ozone. Taken together, our findings support the implication of AM in the response of type II pneumocytes to air pollutants.

(J Histochem Cytochem 53:773–780, 2005)

Adrenomedullin (AM) is a potent vasodilator peptide present in the lung of mammals where it is expressed mainly in the columnar epithelium and alveolar macrophages. AM increases the secretion of phosphatidylcholine by type II pneumocytes, which suggests a role as an autocrine modulator of surfactant secretion. In this study we show the expression of an AM-like protein in the lung of the pigeon, Columba livia. Using an antibody against its human ortholog, AM-like immunoreactivity was found to be associated with membranous structures of the multivesicular bodies of type II pneumocytes. We also studied the differential expression of AM-like peptide in the lung of pigeons exposed to polluted city air vs cleaner countryside conditions and found that AM-like expression was higher in city animals. Similar results were obtained in an experimental study in which pigeons were exposed to increasing concentrations of a single pollutant, ozone. Taken together, our findings support the implication of AM in the response of type II pneumocytes to air pollutants.

(J Histochem Cytochem 53:773–780, 2005)

Identification of Vascular Progenitor Cells in Pulmonary Arteries of Patients with COPD.

Am J Respir Cell Mol Biol. 2005 Oct 20;

Authors: Peinado VI, Ramirez J, Roca J, Rodriguez-Roisin R, Barbera JA

Progenitor cells of bone marrow origin migrate to injured vessels, where they may contribute to endothelial maintenance and vessel remodeling, through vascular endothelial growth factor (VEGF) related signals. To what extent progenitor cells may play a role in vascular changes occurring in patients with chronic obstructive pulmonary disease (COPD) remains unsettled. The study was addressed to identify vascular progenitor cells in pulmonary arteries of patients with COPD and to investigate whether the presence of these cells could be related to changes in endothelial function or the expression of VEGF. Pulmonary arteries of 9 patients with COPD and 6 controls were studied. Scanning electron microscopy demonstrated areas of denuded endothelium in arteries of COPD patients. Vascular progenitor cells were identified by immunohistochemistry and immunogold using antibodies against AC133, CD34 and CD45. AC133(+) cells were localized in the endothelial surface, close to denuded areas. The number of AC133(+) and CD45(+) cells in pulmonary arteries was greater in COPD patients than in controls. The number of AC133(+) cells correlated with the response of pulmonary artery rings to hypoxic stimulus. AC133(+) and CD45(+) cells were also identified in the intimal layer. The wall thickness correlated with the number of progenitor cells in the intima, as well as with VEGF and VEGF receptor-2 mRNA expression. We conclude that COPD patients show an increased number of bone marrow-derived progenitor cells in pulmonary arteries. These cells seem to contribute to ongoing endothelial repair, but they might also be involved in the pathogenesis of pulmonary vascular remodeling.

PMID: 16239642 [PubMed - as supplied by publisher]

We recently found that four peptide hormones synthesized by the same gene completely inhibit the growth of human pancreatic adenocarcinomas in athymic mice. The present immunocytochemical investigation was designed to determine where in the adenocarcinomas these peptide hormones localize. Atrial natriuretic peptide, vessel dilator, long-acting natriuretic peptide, and kaliuretic peptide localized to the cytoplasm and nucleus of the human pancreatic adenocarcinomas, which is consistent with their ability to decrease DNA synthesis in the nucleus of this cancer. In this first investigation of where these peptide hormones with anticancer effects localize in any cancer, these peptide hormones also localized to the endothelium of capillaries and fibroblasts within these cancers. This is the first demonstration of growth-inhibiting peptide hormones localizing to the nucleus, where they inhibit DNA synthesis and may interact with growth-promoting hormones that localize there as the etiology of their ability to inhibit the growth of adenocarcinomas both in vitro and in vivo. (J Histochem Cytochem 53:989–995, 2005)

We recently found that four peptide hormones synthesized by the same gene completely inhibit the growth of human pancreatic adenocarcinomas in athymic mice. The present immunocytochemical investigation was designed to determine where in the adenocarcinomas these peptide hormones localize. Atrial natriuretic peptide, vessel dilator, long-acting natriuretic peptide, and kaliuretic peptide localized to the cytoplasm and nucleus of the human pancreatic adenocarcinomas, which is consistent with their ability to decrease DNA synthesis in the nucleus of this cancer. In this first investigation of where these peptide hormones with anticancer effects localize in any cancer, these peptide hormones also localized to the endothelium of capillaries and fibroblasts within these cancers. This is the first demonstration of growth-inhibiting peptide hormones localizing to the nucleus, where they inhibit DNA synthesis and may interact with growth-promoting hormones that localize there as the etiology of their ability to inhibit the growth of adenocarcinomas both in vitro and in vivo. (J Histochem Cytochem 53:989–995, 2005)

An immunocytochemistry (ICC) for the anticancer antibiotic daunomycin (DM) was developed using a combination of anti-DM serum produced against N-(gamma-maleimidobutyryloxy)succinimide (GMBS)-conjugated DM, and DM-uptake human melanoma BD cells. The antiserum was demonstrated to be specific for DM and the structurally related analogs adriamicin and epirubicin by an ICC model system of the enzyme immunoassay (EIA) using glutaraldehyde (GA)-conjugated DM as a solid phase antigen. No cross-reaction occurred with any of the other antibiotics tested such as bleomycin, pepleomycin, and mitomycin C. Successful DM ICC required a series of processes prior to the immunocytochemical reaction: the cells were first fixed with GA, then reduced with NaBH₄, treated with hydrochloric acid, and finally digested with protease. The cell specimens were then subjected to immunoreaction with anti-DM serum followed by peroxidase-labeled goat anti-rabbit IgG/Fab', and in both immune reagents the detergent Triton X-100 was contained as well. The present ICC covering all these processes successfully stained for DM in the nucleus and in the perinuclear Golgi region of the cytoplasm of the BD cells, consistent with the results obtained by the DM autofluorescence method. This ICC was found to be three times as sensitive as the cytofluorometric method and applicable to the paraffin sections of the liver of rats 24 hr after an IV injection of DM. The principle used in the present study for developing DM ICC might be applied to other drugs containing the primary amino group(s) in the molecule. Thus, these ICCs for drugs are direct, precise and easy new methods that should have potential for pharmacology and toxicology studies of drugs, revealing the localization of a drug in cells and tissues. (J Histochem Cytochem 53:467–474, 2005)

An immunocytochemistry (ICC) for the anticancer antibiotic daunomycin (DM) was developed using a combination of anti-DM serum produced against N-(gamma-maleimidobutyryloxy)succinimide (GMBS)-conjugated DM, and DM-uptake human melanoma BD cells. The antiserum was demonstrated to be specific for DM and the structurally related analogs adriamicin and epirubicin by an ICC model system of the enzyme immunoassay (EIA) using glutaraldehyde (GA)-conjugated DM as a solid phase antigen. No cross-reaction occurred with any of the other antibiotics tested such as bleomycin, pepleomycin, and mitomycin C. Successful DM ICC required a series of processes prior to the immunocytochemical reaction: the cells were first fixed with GA, then reduced with NaBH₄, treated with hydrochloric acid, and finally digested with protease. The cell specimens were then subjected to immunoreaction with anti-DM serum followed by peroxidase-labeled goat anti-rabbit IgG/Fab', and in both immune reagents the detergent Triton X-100 was contained as well. The present ICC covering all these processes successfully stained for DM in the nucleus and in the perinuclear Golgi region of the cytoplasm of the BD cells, consistent with the results obtained by the DM autofluorescence method. This ICC was found to be three times as sensitive as the cytofluorometric method and applicable to the paraffin sections of the liver of rats 24 hr after an IV injection of DM. The principle used in the present study for developing DM ICC might be applied to other drugs containing the primary amino group(s) in the molecule. Thus, these ICCs for drugs are direct, precise and easy new methods that should have potential for pharmacology and toxicology studies of drugs, revealing the localization of a drug in cells and tissues. (J Histochem Cytochem 53:467–474, 2005)

An immunocytochemistry (ICC) for the anticancer antibiotic daunomycin (DM) was developed using a combination of anti-DM serum produced against N-(gamma-maleimidobutyryloxy)succinimide (GMBS)-conjugated DM, and DM-uptake human melanoma BD cells. The antiserum was demonstrated to be specific for DM and the structurally related analogs adriamicin and epirubicin by an ICC model system of the enzyme immunoassay (EIA) using glutaraldehyde (GA)-conjugated DM as a solid phase antigen. No cross-reaction occurred with any of the other antibiotics tested such as bleomycin, pepleomycin, and mitomycin C. Successful DM ICC required a series of processes prior to the immunocytochemical reaction: the cells were first fixed with GA, then reduced with NaBH₄, treated with hydrochloric acid, and finally digested with protease. The cell specimens were then subjected to immunoreaction with anti-DM serum followed by peroxidase-labeled goat anti-rabbit IgG/Fab', and in both immune reagents the detergent Triton X-100 was contained as well. The present ICC covering all these processes successfully stained for DM in the nucleus and in the perinuclear Golgi region of the cytoplasm of the BD cells, consistent with the results obtained by the DM autofluorescence method. This ICC was found to be three times as sensitive as the cytofluorometric method and applicable to the paraffin sections of the liver of rats 24 hr after an IV injection of DM. The principle used in the present study for developing DM ICC might be applied to other drugs containing the primary amino group(s) in the molecule. Thus, these ICCs for drugs are direct, precise and easy new methods that should have potential for pharmacology and toxicology studies of drugs, revealing the localization of a drug in cells and tissues. (J Histochem Cytochem 53:467–474, 2005)

Galectins are an evolutionarily conserved family of 15 different lectins found in various combinations in virtually every type of animal cell. One of the primary galectins expressed in intestinal epithelium is galectin-4, a tandem-repeat galectin with two carbohydrate-recognition domains in a single polypeptide chain. In the current study, we produced an anti-galectin-4 monoclonal antibody (MAb) for determining the distribution of galectin-4 in porcine small intestine to enhance our understanding of where galectin-4 performs its functions in the small intestine. In immunohistochemistry studies, this MAb detected galectin-4 primarily in the cytoplasm of absorptive epithelial cells lining intestinal villi. Mature epithelial cells at the villous tips stained the most intensely with this MAb, with progressively less intense staining observed along the sides of villi and into the crypts. In addition to its cytoplasmic localization, galectin-4 was also associated with nuclei in villous tip cells, indicating that some galectin-4 may migrate to the nucleus during terminal maturation of these cells. In intestinal crypts, a specific subset of cells, which may be enteroendocrine cells, expressed galectin-4 at a relatively high level. Galectin-4 distribution patterns were similar in all three regions (duodenum, jejunum, and ileum) of porcine small intestine. (J Histochem Cytochem 53:197–205, 2005)

The expression of the ßbeta;4 integrin subunit protein in pancreatic cancer was investigated using routine immunohistochemical methods on paraffin-embedded archival material. Forty-eight cases of pancreatic ductal adenocarcinoma were immunostained with a monoclonal antibody to the ßbeta;4 integrin subunit, and the extent of staining was compared with that seen in non-cancerous pancreatic tissues, including 15 separate cases of chronic pancreatitis and 6 sections from normal pancreas. We found that the ßbeta;4 integrin subunit protein was overexpressed in the majority of pancreatic carcinoma cases tested, whereas chronic pancreatitis and normal pancreas did not display substantial levels of expression. (J Histochem Cytochem 53:799–801, 2005)

The expression of the ßbeta;4 integrin subunit protein in pancreatic cancer was investigated using routine immunohistochemical methods on paraffin-embedded archival material. Forty-eight cases of pancreatic ductal adenocarcinoma were immunostained with a monoclonal antibody to the ßbeta;4 integrin subunit, and the extent of staining was compared with that seen in non-cancerous pancreatic tissues, including 15 separate cases of chronic pancreatitis and 6 sections from normal pancreas. We found that the ßbeta;4 integrin subunit protein was overexpressed in the majority of pancreatic carcinoma cases tested, whereas chronic pancreatitis and normal pancreas did not display substantial levels of expression. (J Histochem Cytochem 53:799–801, 2005)

The expression of the ßbeta;4 integrin subunit protein in pancreatic cancer was investigated using routine immunohistochemical methods on paraffin-embedded archival material. Forty-eight cases of pancreatic ductal adenocarcinoma were immunostained with a monoclonal antibody to the ßbeta;4 integrin subunit, and the extent of staining was compared with that seen in non-cancerous pancreatic tissues, including 15 separate cases of chronic pancreatitis and 6 sections from normal pancreas. We found that the ßbeta;4 integrin subunit protein was overexpressed in the majority of pancreatic carcinoma cases tested, whereas chronic pancreatitis and normal pancreas did not display substantial levels of expression. (J Histochem Cytochem 53:799–801, 2005)

Cytochrome c release from the intermembrane space of mitochondria is one of the triggers of apoptosis. There is no histochemical method available to demonstrate cytochrome c in cryostat sections, possibly because small cytosolic proteins diffuse readily into aqueous fixation media. This report shows that it is possible to demonstrate cytochrome c release in cardiomyocytes in failing myocardium using vapor fixation of cryostat sections and immunohistochemistry. The method is calibrated using sections from gelatin blocks containing known concentrations of cytochrome c. The method is applied to the hypertrophied right ventricular wall of rats in which pulmonary hypertension was induced by monocrotaline. Cytochrome c release is found in a fraction of the cardiomyocytes, leading to a mosaic-staining pattern. Cytochrome c release was found in myocytes over the full range of cross-sectional area (from 1 to 3.9 times control) in the hypertrophied myocardium. Cytosolic cytochrome c concentrations up to 0.4–0.5 mM occur frequently.

(J Histochem Cytochem 53:803–807, 2005)

Cytochrome c release from the intermembrane space of mitochondria is one of the triggers of apoptosis. There is no histochemical method available to demonstrate cytochrome c in cryostat sections, possibly because small cytosolic proteins diffuse readily into aqueous fixation media. This report shows that it is possible to demonstrate cytochrome c release in cardiomyocytes in failing myocardium using vapor fixation of cryostat sections and immunohistochemistry. The method is calibrated using sections from gelatin blocks containing known concentrations of cytochrome c. The method is applied to the hypertrophied right ventricular wall of rats in which pulmonary hypertension was induced by monocrotaline. Cytochrome c release is found in a fraction of the cardiomyocytes, leading to a mosaic-staining pattern. Cytochrome c release was found in myocytes over the full range of cross-sectional area (from 1 to 3.9 times control) in the hypertrophied myocardium. Cytosolic cytochrome c concentrations up to 0.4–0.5 mM occur frequently.

(J Histochem Cytochem 53:803–807, 2005)

Sphingosine kinase 1 (SK1) is a key enzyme critical to the sphingolipid metabolic pathway responsible for catalyzing the formation of the bioactive lipid sphingosine-1-phosphate. SK1-mediated production of sphingosine-1-phosphate has been shown to stimulate such biological processes as cell growth, differentiation, migration, angiogenesis, and inhibition of apoptosis. In this study, cell type–specific immunolocalization of SK1 was examined in the bronchus/terminal bronchiole of the lung. Strong immunopositive staining was evident at the apical surface of pseudostratified epithelial cells of the bronchus and underlying smooth muscle cells, submucosal serous glands, immature chondrocytes, type II alveolar cells, foamy macrophages, endothelial cells of blood vessels, and neural bundles. Immunohistochemical screening for SK1 expression was performed in 25 samples of normal/tumor patient matched non–small-cell lung cancer tissue and found that 25 of 25 tumor samples (carcinoid [5 samples], squamous [10 samples], and adenocarcinoma tumors [10 samples]), exhibited overwhelmingly positive immunostaining for SK1 as compared with patient-matched normal tissue. In addition, an approximately 2-fold elevation of SK1 mRNA expression was observed in lung cancer tissue versus normal tissue, as well as in several other solid tumors. Taken together, these findings define the localization of SK1 in lung and provide clues as to how SK1 may play a role in normal lung physiology and the pathophysiology of lung cancer. (J Histochem Cytochem 53:1159–1166, 2005)

Inactivation of the cell cycle inhibitor gene p16MTS1 seems to be involved in human papillomavirus (HPV)-related carcinogenesis because E6 and E7 oncoproteins may impair p16^INK4a and, indirectly, bcl-2 functions. In this study, we analyzed the role of immunohistochemical expression of p16^INK4a and bcl-2 in HPV-infected cervical biopsies as prognostic markers of the progression of squamous intraepithelial lesion (SIL). Sixty-five cervical biopsies were stratified into two subgroups according to the second biopsy: 27 of them maintained a low-grade (LG)-SIL diagnosis, and 38 progressed from LG-SIL to high-grade (HG)-SIL. p16^INK4a and bcl-2 quantitative expression levels were measured by the immunoperoxidase method. PCR-DNA techniques were used to detect and type HPV. The Wilcoxon and Fisher exact tests were employed for the statistical analysis. In the group with an LG-SIL diagnosis at the second biopsy, no significant associations were found between p16^INK4a and bcl-2 expression and presence of HPV16/18. In the group that progressed to HG-SIL, a significant association was observed between p16^INK4a overexpression and HPV16/18 presence (p=0.021), but none with bcl-2 levels. It is concluded that immunohistochemical bcl-2 expression may not be useful for predicting the progression of HPV-related SIL. In contrast, p16^INK4a overexpression seemed to be associated with HPV 16 and 18, suggesting that it may be a good marker for predicting SIL progression.

(J Histochem Cytochem 53:509–516, 2005)

Inactivation of the cell cycle inhibitor gene p16MTS1 seems to be involved in human papillomavirus (HPV)-related carcinogenesis because E6 and E7 oncoproteins may impair p16^INK4a and, indirectly, bcl-2 functions. In this study, we analyzed the role of immunohistochemical expression of p16^INK4a and bcl-2 in HPV-infected cervical biopsies as prognostic markers of the progression of squamous intraepithelial lesion (SIL). Sixty-five cervical biopsies were stratified into two subgroups according to the second biopsy: 27 of them maintained a low-grade (LG)-SIL diagnosis, and 38 progressed from LG-SIL to high-grade (HG)-SIL. p16^INK4a and bcl-2 quantitative expression levels were measured by the immunoperoxidase method. PCR-DNA techniques were used to detect and type HPV. The Wilcoxon and Fisher exact tests were employed for the statistical analysis. In the group with an LG-SIL diagnosis at the second biopsy, no significant associations were found between p16^INK4a and bcl-2 expression and presence of HPV16/18. In the group that progressed to HG-SIL, a significant association was observed between p16^INK4a overexpression and HPV16/18 presence (p=0.021), but none with bcl-2 levels. It is concluded that immunohistochemical bcl-2 expression may not be useful for predicting the progression of HPV-related SIL. In contrast, p16^INK4a overexpression seemed to be associated with HPV 16 and 18, suggesting that it may be a good marker for predicting SIL progression.

(J Histochem Cytochem 53:509–516, 2005)

Inactivation of the cell cycle inhibitor gene p16MTS1 seems to be involved in human papillomavirus (HPV)-related carcinogenesis because E6 and E7 oncoproteins may impair p16^INK4a and, indirectly, bcl-2 functions. In this study, we analyzed the role of immunohistochemical expression of p16^INK4a and bcl-2 in HPV-infected cervical biopsies as prognostic markers of the progression of squamous intraepithelial lesion (SIL). Sixty-five cervical biopsies were stratified into two subgroups according to the second biopsy: 27 of them maintained a low-grade (LG)-SIL diagnosis, and 38 progressed from LG-SIL to high-grade (HG)-SIL. p16^INK4a and bcl-2 quantitative expression levels were measured by the immunoperoxidase method. PCR-DNA techniques were used to detect and type HPV. The Wilcoxon and Fisher exact tests were employed for the statistical analysis. In the group with an LG-SIL diagnosis at the second biopsy, no significant associations were found between p16^INK4a and bcl-2 expression and presence of HPV16/18. In the group that progressed to HG-SIL, a significant association was observed between p16^INK4a overexpression and HPV16/18 presence (p=0.021), but none with bcl-2 levels. It is concluded that immunohistochemical bcl-2 expression may not be useful for predicting the progression of HPV-related SIL. In contrast, p16^INK4a overexpression seemed to be associated with HPV 16 and 18, suggesting that it may be a good marker for predicting SIL progression.

(J Histochem Cytochem 53:509–516, 2005)

Immunohistochemical localization of calcitonin gene-related peptide and substance P in the rat knee cartilage at birth.

Physiol Res. 2005;55(5):549-56

Authors: Oliva F, Tarantino U, Maffulli N

Substance P (SP) and calcitonin gene-related peptide (CGRP) have been found in the perichondrium and within the cartilage canals. It is still unknown whether they exert a direct effect on chondrocytes during joint development. We processed 28 knees of newborn Wistar rats in 7 different fashions to perform histology and immunohistochemistry studies. Positive immunoreactivity against CGRP and SP was found in the inner aspect of the perichondrium in a close contact with chondrocytes. The presence of CGRP and SP indicates the presence of nerves fibers, and precedes the development of cartilage canals. Nerve fibers may play a role in the development of synovial joints before and during the presence of cartilage canals. The presence of CGRP and SP in the cartilage at birth may be involved in the early postnatal maturation of synovial joints. It remains to be determined whether autonomic innervation is later involved in age-related degenerative joint disease.

PMID: 16238469 [PubMed - in process]

Monoamine oxidase (MAO) is regarded as a mitochondrial enzyme. This enzyme localizes on the outer membrane of mitochondria. There are two kinds of MAO isozymes, MAO type A (MAOA) and type B (MAOB). Previous studies have shown that MAOB activity is found in the pancreatic islets. This activity in the islets is increased by the fasting-induced decrease of plasma glucose level. Islet B cells contain monoamines in their secretory granules. These monoamines inhibit the secretion of insulin from the B cells. MAOB is active in degrading monoamines. Therefore, MAOB may influence the insulin-secretory process by regulating the stores of monoamines in the B cells. However, it has not been determined whether MAOB is localized on B cells or other cell types of the islets. In the present study, we used both double-labeling immunofluorescence histochemical and electron microscopic immunohistochemical methods to examine the subcellular localization of MAOB in rat pancreatic islets. MAOB was found in the mitochondrial outer membranes of glucagon-secreting cells (A cells), insulin-secreting cells (B cells), and some pancreatic polypeptide (PP)-secreting cells (PP cells), but no MAOB was found in somatostatin-secreting cells (D cells), nor in certain other PP cells. There were two kinds of mitochondria in pancreatic islet B cells: one contains MAOB on their outer membranes, but a substantial proportion of them lack this enzyme. Our findings indicate that pancreatic islet B cells contain MAOB on their mitochondrial outer membranes, and this enzyme may be involved in the regulation of monoamine levels and insulin secretion in the B cells.

(J Histochem Cytochem 53:1149–1158, 2005)

The MDR1 gene product, P-glycoprotein (P-gp), was shown to confer multidrug resistance to cancer cells, but its overexpression is also suggested to be involved in pharmacoresistance of epilepsy by acting as an energy-dependent drug-efflux pump in the blood–brain barrier (BBB). In normal brain tissue, P-gp is almost exclusively expressed by capillary endothelial cells (EC) of the BBB, whereas little or no expression is detected in other cell types. Increased P-gp expression was observed after seizures, but localization of this increase, i.e., within brain capillary EC or within parenchymal or perivascular astrocytes, which contribute to the BBB function, is controversial. To test whether these antithetic data arise from unusual properties of the antigen itself, we compared different immunohistochemical techniques and monoclonal or polyclonal antibodies to P-gp in normal rat brain and rat brain after kainate-induced seizures. Using acetone-fixed cryostat sections of snap-frozen tissue, strong P-gp labeling was detected in EC and, after seizures, in hippocampal neurons, but not in astrocytes. In contrast, EC and neuronal P-gp immunolabeling were not seen in paraformaldehyde-fixed sections, whereas both perivascular and parenchymal astrocytes exhibited strong P-gp labeling after seizures. The lack of P-gp labeling in EC by paraformaldehyde fixation, was reversed by treatment of the sections with acetate/ethanol. These experiments demonstrate that various fixation conditions have a striking effect on the immunohistochemical localization of P-gp in rat brain and detection of its increased expression by seizures. When data obtained from different immunohistochemical techniques are taken together, seizures seem to induce overexpression of P-gp in four different cell types, i.e., EC, perivascular astrocytes, parenchymal astrocytes, and neurons. (J Histochem Cytochem 53:517–531, 2005)

The MDR1 gene product, P-glycoprotein (P-gp), was shown to confer multidrug resistance to cancer cells, but its overexpression is also suggested to be involved in pharmacoresistance of epilepsy by acting as an energy-dependent drug-efflux pump in the blood–brain barrier (BBB). In normal brain tissue, P-gp is almost exclusively expressed by capillary endothelial cells (EC) of the BBB, whereas little or no expression is detected in other cell types. Increased P-gp expression was observed after seizures, but localization of this increase, i.e., within brain capillary EC or within parenchymal or perivascular astrocytes, which contribute to the BBB function, is controversial. To test whether these antithetic data arise from unusual properties of the antigen itself, we compared different immunohistochemical techniques and monoclonal or polyclonal antibodies to P-gp in normal rat brain and rat brain after kainate-induced seizures. Using acetone-fixed cryostat sections of snap-frozen tissue, strong P-gp labeling was detected in EC and, after seizures, in hippocampal neurons, but not in astrocytes. In contrast, EC and neuronal P-gp immunolabeling were not seen in paraformaldehyde-fixed sections, whereas both perivascular and parenchymal astrocytes exhibited strong P-gp labeling after seizures. The lack of P-gp labeling in EC by paraformaldehyde fixation, was reversed by treatment of the sections with acetate/ethanol. These experiments demonstrate that various fixation conditions have a striking effect on the immunohistochemical localization of P-gp in rat brain and detection of its increased expression by seizures. When data obtained from different immunohistochemical techniques are taken together, seizures seem to induce overexpression of P-gp in four different cell types, i.e., EC, perivascular astrocytes, parenchymal astrocytes, and neurons. (J Histochem Cytochem 53:517–531, 2005)

The MDR1 gene product, P-glycoprotein (P-gp), was shown to confer multidrug resistance to cancer cells, but its overexpression is also suggested to be involved in pharmacoresistance of epilepsy by acting as an energy-dependent drug-efflux pump in the blood–brain barrier (BBB). In normal brain tissue, P-gp is almost exclusively expressed by capillary endothelial cells (EC) of the BBB, whereas little or no expression is detected in other cell types. Increased P-gp expression was observed after seizures, but localization of this increase, i.e., within brain capillary EC or within parenchymal or perivascular astrocytes, which contribute to the BBB function, is controversial. To test whether these antithetic data arise from unusual properties of the antigen itself, we compared different immunohistochemical techniques and monoclonal or polyclonal antibodies to P-gp in normal rat brain and rat brain after kainate-induced seizures. Using acetone-fixed cryostat sections of snap-frozen tissue, strong P-gp labeling was detected in EC and, after seizures, in hippocampal neurons, but not in astrocytes. In contrast, EC and neuronal P-gp immunolabeling were not seen in paraformaldehyde-fixed sections, whereas both perivascular and parenchymal astrocytes exhibited strong P-gp labeling after seizures. The lack of P-gp labeling in EC by paraformaldehyde fixation, was reversed by treatment of the sections with acetate/ethanol. These experiments demonstrate that various fixation conditions have a striking effect on the immunohistochemical localization of P-gp in rat brain and detection of its increased expression by seizures. When data obtained from different immunohistochemical techniques are taken together, seizures seem to induce overexpression of P-gp in four different cell types, i.e., EC, perivascular astrocytes, parenchymal astrocytes, and neurons. (J Histochem Cytochem 53:517–531, 2005)

We aimed to examine the distribution of SEPT4, SEPT5, and SEPT8 in the human eye. For each septin, five to six normal human eyes were examined by immunohistochemical staining of paraffin sections using polyclonal antibodies against SEPT4, SEPT5, and SEPT8 and an avidin biotin complex immunodetection system. SEPT4 immunoreactivity (IR) was detected primarily in the epithelium of cornea, lens, and nonpigmented ciliary epithelium; in the endothelium of cornea and vessels of iris and retina; and in the retinal nerve fiber layer, the outer plexiform layer, the outer segments of the photoreceptor cells, the inner limiting membrane of the optic nerve head, and optic nerve axons. SEPT5-IR was present in corneal endothelial cells, iris tissue, nonpigmented ciliary epithelium, and epithelial cells of the lens. SEPT8-IR almost paralleled that of SEPT4, except for a lower SEPT8-IR of the outer photoreceptor segments and a positive staining of the meningothelial cell nests in the subarachnoidal space of the bulbar segment of the orbital optic nerve. In conclusion, SEPT4, SEPT5, and SEPT8 are expressed in various ocular tissues, each revealing a distinct expression pattern. Both physiological and potential pathophysiological role of septins in the human eye deserve further investigation. (J Histochem Cytochem 53:1139–1147, 2005)

A new method is described for performing fluorescence in situ hybridization (FISH). FISH signals are enhanced by microwave pulses applied during the DNA-DNA hybridization process. It is the first description of FISH with a single/low-copy probe done more efficiently by application of microwave; the latter leads to quick results or enhancement of weak signals. Microwave FISH has been compared systematically with normal FISH, and we could demonstrate the efficiency of microwave irradiation especially in the first 100 min of hybridization. (J Histochem Cytochem 53:1301–1303, 2005)

A new method is described for performing fluorescence in situ hybridization (FISH). FISH signals are enhanced by microwave pulses applied during the DNA-DNA hybridization process. It is the first description of FISH with a single/low-copy probe done more efficiently by application of microwave; the latter leads to quick results or enhancement of weak signals. Microwave FISH has been compared systematically with normal FISH, and we could demonstrate the efficiency of microwave irradiation especially in the first 100 min of hybridization. (J Histochem Cytochem 53:1301–1303, 2005)

Interaction between spontaneous and neurally-mediated regulation of smooth muscle tone in the rabbit corpus cavernosum.

J Physiol. 2005 Oct 20;

Authors: Hashitani H, Yanai Y, Shirasawa N, Soji T, Tomita A, Kohri K, Suzuki H

Interaction between spontaneous and neurally-mediated regulation of tone in the corpus cavernosum smooth muscle (CCSM) of the rabbit was investigated. Changes in isometric muscle tension, intracellular concentration of Ca(2+) ([Ca(2+)]i) and membrane potential were recorded. CCSM developed spontaneous contractions, transient increases in [Ca(2+)]i (Ca transients) and depolarizations. This spontaneous activity was abolished by blocking L-type Ca channels (nicardipine 1 microM), sarcoplasmic reticulum Ca pump activity (cyclopiazonic acid 10 microM), Ca-activated Cl channels (niflumic acid 10 microM) or cyclooxygenase-2 (COX-2; NS-398 1 microM). Transmural nerve stimulation initiated either alpha-adrenergic contractions or nitrergic relaxations of CCSM depending on the level of muscle tone. NS-398 suppressed nerve-evoked contractions by about 70 % but caused only a 40 % reduction in the corresponding Ca transient. Blocking nitric oxide synthase with N(omega)-nitro-L-arginine (LNA 100 microM) reinforced nerve-evoked Ca transients by about 150 %, whilst increasing the corresponding Ca transients by only 20 %. In CCSM preparations which had been pre-contracted with either noradrenaline (0.3 microM) or prostaglandin F2alpha (0.1 microM), nerve stimulation inhibited about 70% of the contraction and caused only a 20 % decrease in [Ca(2+)]i. Fluorescent immunohistochemistry with COX-2 antibodies and the reverse transcriptase (RT)-polymerase chain reaction (PCR) method showed that the enzyme and its mRNA were highly expressed in the CCSM. These results suggest that spontaneously produced prostaglandins (PGs) not only contribute to the generation of spontaneous contractions but also facilitate nerve-evoked contractions. Conversely, spontaneously released nitric oxide (NO) suppresses excitation. Thus, interaction between spontaneous and neurally-mediated regulation of CCSM tone may be fundamental to maintaining the muscle contractility. In addition, both PGs and NO appear to alter CCSM tone with only small changes in [Ca(2+)]i.

PMID: 16239265 [PubMed - as supplied by publisher]

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Intercellular junctions in Ewing sarcoma/primitive neuroectodermal tumor: additional evidence of epithelial differentiation.

Mod Pathol. 2005 Nov;18(11):1403-10

Authors: Schuetz AN, Rubin BP, Goldblum JR, Shehata B, Weiss SW, Liu W, Wick MR, Folpe AL

Ewing sarcoma/primitive neuroectodermal tumor (ES/PNET) has recently been shown to frequently express cytokeratins, suggesting partial epithelial differentiation. Older ultrastructural studies have documented primitive cell-cell junctions in ES/PNET, reportedly resembling poorly formed desmosomes. Recently, paraffin-reactive antibodies have become available to proteins found in a variety of intercellular junctions indicative of epithelial differentiation, including tight junctions, desmosomes and adherens junctions. We examined intercellular junction protein expression in a large number of genetically confirmed ES/PNET. Formalin-fixed, paraffin-embedded sections from 23 primary and seven recurrent or metastatic cases of genetically confirmed ES/PNET were immunostained for claudin-1 and occludin (tight junction structural proteins), zonula occludens-1 (ZO-1, tight junction linker protein), desmoglein 1/2 (desmosomal adherens protein), desmoplakin (desmosomal structural protein) and E-cadherin (epithelial adherens junction protein), using steam heat-induced epitope retrieval and the Dako Envision system. Cases with >5% positive cells were scored as 'positive'. Normal colonic epithelium and skin served as external positive controls. Claudin-1 was expressed by 19 of 30 specimens (63%), ZO-1 was expressed by 15 of 29 specimens (51%), and occludin was expressed by three of 28 specimens (11%). In 28 specimens all three tight junction markers were evaluable. In all, 15 samples (54%) expressed only one tight junction marker, and 10 samples (36%) expressed two tight junction markers. No case expressed all three tight junction markers. Desmoglein was expressed in one of 30 (3%) samples. Desmoplakin was expressed in two of 28 (7%) samples. E-cadherin was negative in all cases. Our data suggest that many of the previously described cell-cell junctions in ES/PNET are poorly formed tight junctions, given the high frequency of claudin-1 and ZO-1 expression. This may underestimate the true frequency of tight junction protein expression in ES/PNET, as there are at least 20 different claudins and other ZO proteins. These tight junctions are almost certainly abnormal, given the absence of occludin expression in most cases. Desmosomal and adherens junction protein expression was rare to absent. Our findings provide additional evidence that ES/PNET frequently show partial epithelial differentiation.Modern Pathology (2005) 18, 1403-1410. doi:10.1038/modpathol.3800435; published online 6 May 2005.

PMID: 15920547 [PubMed - in process]

Coexpression of desmosomal proteins and vimentin has been reported in a specific mesenchymal phenotype. This study investigated the expression of vimentin-binding desmosomal proteins in human dental pulp fibroblasts (DPF) and odontoblasts. The dental pulp has no cells expressing desmocollin (DSC) 1-3, desmoglein (DSG) 1-3, junction plakoglobin (JUP), or desmoplakin (DPK) 1 and 2 except for odontoblasts expressing DPK. A confocal image by laser-scanning microscopy demonstrated the diffuse distribution of DPK in the cytoplasm throughout the odontoblast processes. In culture, the mRNA expression of JUP and DPK1, but not DSC1-3 and DSG1-3, was detected in all DPF clones tested and also in odontoblast-like cells (OB) expressing osteocalcin and dentin sialophosphoprotein mRNAs established in the differentiation medium. The DPF having the potential to differentiate into OB expressed vimentin, but not DPK before culturing in the differentiation medium, whereas OB expressed vimentin-binding DPK1. These results suggest that DPF usually expresses DPK1 mRNA, and that the DPK1 production and the bonding of vimentin to DPK1 occur in DPF with the differentiation into odontoblasts.

(J Histochem Cytochem 53:1099–1108, 2005)

Recently, laser capture microdissection (LCM) has become a powerful tool for investigating the genome and protein contents of cells populating normal and pathologically altered tissues. The present work reports a technique for the preparation of tissue specimens for further LCM combined with electron microscopy. In this work, atherosclerotic plaque cells containing oxidized low-density lipoproteins (LDL) were microdissected from human carotid arteries and the intracellular distribution of oxidized LDL was examined, providing novel information about the association of microvesicles containing oxidized LDL with "lipid droplets" in macrophage foam cells.

(J Histochem Cytochem 53:793–797, 2005)

Recently, laser capture microdissection (LCM) has become a powerful tool for investigating the genome and protein contents of cells populating normal and pathologically altered tissues. The present work reports a technique for the preparation of tissue specimens for further LCM combined with electron microscopy. In this work, atherosclerotic plaque cells containing oxidized low-density lipoproteins (LDL) were microdissected from human carotid arteries and the intracellular distribution of oxidized LDL was examined, providing novel information about the association of microvesicles containing oxidized LDL with "lipid droplets" in macrophage foam cells.

(J Histochem Cytochem 53:793–797, 2005)

Recently, laser capture microdissection (LCM) has become a powerful tool for investigating the genome and protein contents of cells populating normal and pathologically altered tissues. The present work reports a technique for the preparation of tissue specimens for further LCM combined with electron microscopy. In this work, atherosclerotic plaque cells containing oxidized low-density lipoproteins (LDL) were microdissected from human carotid arteries and the intracellular distribution of oxidized LDL was examined, providing novel information about the association of microvesicles containing oxidized LDL with "lipid droplets" in macrophage foam cells.

(J Histochem Cytochem 53:793–797, 2005)

The tyramide signal amplification (TSA) technique has been shown to detect scarce tissue antigens in light and electron microscopy. In this study we applied the TSA technique at the electron microscopic level to pre-embedding immunocytochemistry. This protocol was compared to the non-amplified protocol. With the TSA protocol, the labeling of GM130, a cis-Golgi matrix protein, was tested in a cell line and found to be highly sensitive and more enhanced than that with the simple protocol. Moreover, the gold particles were well localized to the cis-side of the Golgi apparatus in both the TSA and the simple protocol. (J Histochem Cytochem 53:249–252, 2005)

Bromodeoxyuridine (BrdU) immunohistochemistry is the method of choice for labeling newly generated cells in the brain. Most BrdU studies utilize paraformaldehyde-fixed brain tissue because of its compatibility with both BrdU and other immunohistochemical methods. However, stronger fixation is required for electron microscopic studies, and unfixed tissue is needed for biochemical and molecular studies. Because there are no systematic studies comparing the effects of different fixatives on BrdU immunohistochemistry in brain tissue, we compared BrdU immunohistochemical methods in brain tissue fixed with 4% paraformaldehyde, a mixed glutaraldehyde–paraformaldehyde fixative for electron microscopy, and unfixed tissue from brains perfused only with buffer and flash frozen. After optimizing immunostaining protocols, qualitative assessments of light microscopic diaminobenzidine labeling and of double-label immunofluorescence with confocal microscopy demonstrated excellent BrdU labeling in each of the three groups. Quantitative stereological assessment of the number of BrdU-labeled cells in rat dentate gyrus showed no significant difference in the number of labeled cells detected with each perfusion protocol. Additionally, we developed a protocol to visualize BrdU-labeled cells in the electron microscope with adequate preservation of fine structure in both rat and monkey brain.

(J Histochem Cytochem 53:821–832, 2005)

Bromodeoxyuridine (BrdU) immunohistochemistry is the method of choice for labeling newly generated cells in the brain. Most BrdU studies utilize paraformaldehyde-fixed brain tissue because of its compatibility with both BrdU and other immunohistochemical methods. However, stronger fixation is required for electron microscopic studies, and unfixed tissue is needed for biochemical and molecular studies. Because there are no systematic studies comparing the effects of different fixatives on BrdU immunohistochemistry in brain tissue, we compared BrdU immunohistochemical methods in brain tissue fixed with 4% paraformaldehyde, a mixed glutaraldehyde–paraformaldehyde fixative for electron microscopy, and unfixed tissue from brains perfused only with buffer and flash frozen. After optimizing immunostaining protocols, qualitative assessments of light microscopic diaminobenzidine labeling and of double-label immunofluorescence with confocal microscopy demonstrated excellent BrdU labeling in each of the three groups. Quantitative stereological assessment of the number of BrdU-labeled cells in rat dentate gyrus showed no significant difference in the number of labeled cells detected with each perfusion protocol. Additionally, we developed a protocol to visualize BrdU-labeled cells in the electron microscope with adequate preservation of fine structure in both rat and monkey brain.

(J Histochem Cytochem 53:821–832, 2005)

Light microscopic demonstration of the microlumen of ependymoma: a study of the usefulness of antigen retrieval for epithelial membrane antigen (EMA) immunostaining.

Brain Tumor Pathol. 2004;21(1):17-21

Authors: Kawano N, Yasui Y, Utsuki S, Oka H, Fujii K, Yamashina S

To determine the origin of dotlike epithelial membrane antigen (EMA) immunoreactivity of ependymoma, which is consistent with the eosinophilic globular body in hematoxylin and eosin (H&E) stain, an immunoelectron microscopic study was undertaken. The usefulness of antigen retrieval pretreatment in detecting the dotlike EMA immunoreactivity in ependymomas was also studied. The materials were 29 ependymomas, 7 autopsy brains as a normal control, and 50 brain tumors of various types. The study confirmed that most of the brown dots in EMA immunostain in ependymoma represented microlumina of tumor cells. In ependymomas, plain EMA immunostaining showed dotlike positivity in only six cases (21%), and antigen retrieval pretreatment increased the number of positives up to 26 cases (90%). Antigen retrieved CD99 detected 23 positive cases (80%) in ependymomas. On the basis of the results, although some false positive findings were raised by antigen retrieval pretreatment, the authors positively recommend adoption of the technique, especially when ependymoma remains as one of the differential diagnoses of the tumor.

PMID: 15696964 [PubMed - indexed for MEDLINE]

CD44 is a multifunctional adhesion molecule that binds to hyaluronan (HA), type I collagen, and fibronectin. We investigated localization of CD44 and HA in mandibular condylar cartilage compared with the growth plate and the articular cartilage, to clarify the characteristics of chondrocytes. We also performed Western blotting using a lysate of mandibular condyle. In mandibular condyle, CD44-positive cells were seen in the surface region of the fibrous cell layer and in the proliferative cell layer. Western blotting revealed that the molecular weight of CD44 in condyle was 78 to 86 kD. Intense reactivity for HA was detected on the surface of the condyle and the lacunae of the hypertrophic cell layer. Moderate labeling was seen in cartilage matrix of the proliferative and maturative layer. Weak labeling was also seen in the fibrous cell layer. In growth plate and articular cartilage, HA was detected in all cell layers. However, chondrocytes of these cartilages did not exhibit reactivity for CD44. These results suggest that chondrocytes in the mandibular condylar cartilage differ in expression of CD44 from those in tibial growth plate and articular cartilage. Cell-matrix interaction between CD44 and HA may play an important role in the proliferation of chondrocytes in the mandibular condyle.

(J Histochem Cytochem 53:113–120, 2005)

CYP2S1 is a recently discovered dioxin-inducible member of the cytochrome P450 superfamily. It has been shown to be involved in the metabolism of some aromatic hydrocarbons as well as retinoic acid, suggesting a role in biotransformation of both exogenous and endogenous compounds. In this study, we used mRNA in situ hybridization and immunohistochemistry to investigate the cellular localization of CYP2S1 in various human tissues using tissue microarrays. High expression levels were observed mainly in epithelial cell types, especially in the epithelia frequently exposed to xenobiotics. In the respiratory tract, the expression was strong in nasal cavity, bronchi, and bronchioli, whereas it was low in the alveolar lining cells. Similarly, CYP2S1 was highly expressed in the epithelial cells throughout the gastrointestinal tract. Strong epithelial expression was also observed in uterine cervix, urinary bladder, and skin. In many exocrine glands (e.g., adrenal gland and pancreas), secretory epithelial cells showed moderate to strong expression levels. In the liver, the expression was low. CYP2S1 was highly expressed in epithelial cells that are major targets for carcinogen exposure and common progenitor cells to tumor development. Indeed, we found strong CYP2S1 expression in many tumors of epithelial origin.

(J Histochem Cytochem 53:549–556, 2005)

CYP2S1 is a recently discovered dioxin-inducible member of the cytochrome P450 superfamily. It has been shown to be involved in the metabolism of some aromatic hydrocarbons as well as retinoic acid, suggesting a role in biotransformation of both exogenous and endogenous compounds. In this study, we used mRNA in situ hybridization and immunohistochemistry to investigate the cellular localization of CYP2S1 in various human tissues using tissue microarrays. High expression levels were observed mainly in epithelial cell types, especially in the epithelia frequently exposed to xenobiotics. In the respiratory tract, the expression was strong in nasal cavity, bronchi, and bronchioli, whereas it was low in the alveolar lining cells. Similarly, CYP2S1 was highly expressed in the epithelial cells throughout the gastrointestinal tract. Strong epithelial expression was also observed in uterine cervix, urinary bladder, and skin. In many exocrine glands (e.g., adrenal gland and pancreas), secretory epithelial cells showed moderate to strong expression levels. In the liver, the expression was low. CYP2S1 was highly expressed in epithelial cells that are major targets for carcinogen exposure and common progenitor cells to tumor development. Indeed, we found strong CYP2S1 expression in many tumors of epithelial origin.

(J Histochem Cytochem 53:549–556, 2005)

CYP2S1 is a recently discovered dioxin-inducible member of the cytochrome P450 superfamily. It has been shown to be involved in the metabolism of some aromatic hydrocarbons as well as retinoic acid, suggesting a role in biotransformation of both exogenous and endogenous compounds. In this study, we used mRNA in situ hybridization and immunohistochemistry to investigate the cellular localization of CYP2S1 in various human tissues using tissue microarrays. High expression levels were observed mainly in epithelial cell types, especially in the epithelia frequently exposed to xenobiotics. In the respiratory tract, the expression was strong in nasal cavity, bronchi, and bronchioli, whereas it was low in the alveolar lining cells. Similarly, CYP2S1 was highly expressed in the epithelial cells throughout the gastrointestinal tract. Strong epithelial expression was also observed in uterine cervix, urinary bladder, and skin. In many exocrine glands (e.g., adrenal gland and pancreas), secretory epithelial cells showed moderate to strong expression levels. In the liver, the expression was low. CYP2S1 was highly expressed in epithelial cells that are major targets for carcinogen exposure and common progenitor cells to tumor development. Indeed, we found strong CYP2S1 expression in many tumors of epithelial origin.

(J Histochem Cytochem 53:549–556, 2005)

The distribution of P2X and P2Y receptor subtypes in upper lumbosacral cat dorsal root ganglia (DRG) has been investigated using immunohistochemistry. Intensity of immunoreactivity for six P2X receptors (P2X₅ receptors were immuno-negative) and the three P2Y receptors examined in cat DRG was in the order of P2Y₂ = P2Y₄>P2X₃>P2X₂ = P2X₇>P2X₆>P2X₁ = P2X₄>P2Y₁. P2X₃, P2Y₂, and P2Y₄ receptor polyclonal antibodies stained 33.8%, 35.3%, and 47.6% of DRG neurons, respectively. Most P2Y₂, P2X₁, P2X₃, P2X₄, and P2X₆ receptor staining was detected in small- and medium-diameter neurons. However, P2Y₄, P2X₂, and P2X₇ staining was present in large- and small-diameter neurons. Double-labeling immunohistochemistry showed that 90.8%, 32.1%, and 2.4% of P2X₃ receptor-positive neurons coexpressed IB₄, CGRP, and NF200, respectively; whereas 67.4%, 41.3%, and 39.1% of P2Y₄ receptor-positive neurons coexpressed IB₄, CGRP, and NF200, respectively. A total of 18.8%, 16.6%, and 63.5% of P2Y₂ receptor-positive neurons also stained for IB₄, CGRP, and NF200, respectively. Only 30% of DRG neurons in cat were P2X₃-immunoreactive compared with 90% in rat and in mouse. A further difference was the low expression of P2Y₁ receptors in cat DRG neurons compared with more than 80% of the neurons in rat. Many small-diameter neurons were NF200-positive in cat, again differing from rat and mouse. (J Histochem Cytochem 53:1273–1282, 2005)

The distribution of P2X and P2Y receptor subtypes in upper lumbosacral cat dorsal root ganglia (DRG) has been investigated using immunohistochemistry. Intensity of immunoreactivity for six P2X receptors (P2X₅ receptors were immuno-negative) and the three P2Y receptors examined in cat DRG was in the order of P2Y₂ = P2Y₄>P2X₃>P2X₂ = P2X₇>P2X₆>P2X₁ = P2X₄>P2Y₁. P2X₃, P2Y₂, and P2Y₄ receptor polyclonal antibodies stained 33.8%, 35.3%, and 47.6% of DRG neurons, respectively. Most P2Y₂, P2X₁, P2X₃, P2X₄, and P2X₆ receptor staining was detected in small- and medium-diameter neurons. However, P2Y₄, P2X₂, and P2X₇ staining was present in large- and small-diameter neurons. Double-labeling immunohistochemistry showed that 90.8%, 32.1%, and 2.4% of P2X₃ receptor-positive neurons coexpressed IB₄, CGRP, and NF200, respectively; whereas 67.4%, 41.3%, and 39.1% of P2Y₄ receptor-positive neurons coexpressed IB₄, CGRP, and NF200, respectively. A total of 18.8%, 16.6%, and 63.5% of P2Y₂ receptor-positive neurons also stained for IB₄, CGRP, and NF200, respectively. Only 30% of DRG neurons in cat were P2X₃-immunoreactive compared with 90% in rat and in mouse. A further difference was the low expression of P2Y₁ receptors in cat DRG neurons compared with more than 80% of the neurons in rat. Many small-diameter neurons were NF200-positive in cat, again differing from rat and mouse. (J Histochem Cytochem 53:1273–1282, 2005)

Sphingosine kinase (SPHK) catalyzes sphingosine phosphorylation to form a bioactive lipid mediator, sphingosine-1-phosphate (S1P). In the current study, we report the presence of SPHK-1 in mouse spermatozoa. SPHK-1 was localized to the acrosomes of spermatozoa, and its expression was proven by RT-PCR and Western blot analysis. SPHK activity of mouse spermatozoa was 18.1 pmol/min/mg protein. Furthermore, we identified the presence of the S1P receptors S1P₁, S1P₂, S1P₃, and S1P₅, in mouse spermatozoa by RT-PCR. These results suggest that S1P produced by SPHK-1 would play a role in the acrosomal reaction through S1P receptors. (J Histochem Cytochem 53:243–247, 2005)

The enzyme type 8 17ßbeta;-hydroxysteroid dehydrogenase (17ßbeta;-HSD) selectively catalyzes the conversion of estradiol (E2) to estrone (E1). To obtain detailed information on the sites of action of type 8 17ßbeta;-HSD, we have studied the cellular localization of type 8 17ßbeta;-HSD mRNA in mouse tissues using in situ hybridization. In the ovary, hybridization signal was detected in granulosa cells of growing follicles and luteal cells. In the uterus, type 8 17ßbeta;-HSD mRNA was found in the epithelial (luminal and glandular) and stromal cells. In the female mammary gland, the enzyme mRNA was seen in ductal epithelial cells and stromal cells. In the testis, hybridization signal was observed in the seminiferous tubule. In the prostate, type 8 17ßbeta;-HSD was detected in the epithelial cells of the acini and stromal cells. In the clitoral and preputial glands, labeling was detected in the epithelial cells of acini and small ducts. The three lobes of the pituitary gland were labeled. In the adrenal gland, hybridization signal was observed in the three zones of the cortex, the medulla being unlabeled. In the kidney, the enzyme mRNA was found to be expressed in the epithelial cells of proximal convoluted tubules. In the liver, all the hepatocytes exhibited a positive signal. In the lung, type 8 17ßbeta;-HSD mRNA was detected in bronchial epithelial cells and walls of pulmonary arteries. The present data suggest that type 8 17ßbeta;-HSD can exert its action to downregulate E2 levels in a large variety of tissues. (J Histochem Cytochem 53:1257–1271, 2005)

The enzyme type 8 17ßbeta;-hydroxysteroid dehydrogenase (17ßbeta;-HSD) selectively catalyzes the conversion of estradiol (E2) to estrone (E1). To obtain detailed information on the sites of action of type 8 17ßbeta;-HSD, we have studied the cellular localization of type 8 17ßbeta;-HSD mRNA in mouse tissues using in situ hybridization. In the ovary, hybridization signal was detected in granulosa cells of growing follicles and luteal cells. In the uterus, type 8 17ßbeta;-HSD mRNA was found in the epithelial (luminal and glandular) and stromal cells. In the female mammary gland, the enzyme mRNA was seen in ductal epithelial cells and stromal cells. In the testis, hybridization signal was observed in the seminiferous tubule. In the prostate, type 8 17ßbeta;-HSD was detected in the epithelial cells of the acini and stromal cells. In the clitoral and preputial glands, labeling was detected in the epithelial cells of acini and small ducts. The three lobes of the pituitary gland were labeled. In the adrenal gland, hybridization signal was observed in the three zones of the cortex, the medulla being unlabeled. In the kidney, the enzyme mRNA was found to be expressed in the epithelial cells of proximal convoluted tubules. In the liver, all the hepatocytes exhibited a positive signal. In the lung, type 8 17ßbeta;-HSD mRNA was detected in bronchial epithelial cells and walls of pulmonary arteries. The present data suggest that type 8 17ßbeta;-HSD can exert its action to downregulate E2 levels in a large variety of tissues. (J Histochem Cytochem 53:1257–1271, 2005)

Two distinct chitinases have been identified in mammals: a phagocyte-specific enzyme named chitotriosidase and an acidic mammalian chitinase (AMCase) expressed in the lungs and gastrointestinal tract. Increased expression of both chitinases has been observed in different pathological conditions: chitotriosidase in lysosomal lipid storage disorders like Gaucher disease and AMCase in asthmatic lung disease. Recently, it was reported that AMCase activity is involved in the pathogenesis of asthma in an induced mouse model. Inhibition of chitinase activity was found to alleviate the inflammation-driven pathology. We studied the tissue-specific expression of both chitinases in mice and compared it to the situation in man. In both species AMCase is expressed in alveolar macrophages and in the gastrointestinal tract. In mice, chitotriosidase is expressed only in the gastrointestinal tract, the tongue, fore-stomach, and Paneth cells in the small intestine, whereas in man the enzyme is expressed exclusively by professional phagocytes. This species difference seems to be mediated by distinct promoter usage. In conclusion, the pattern of expression of chitinases in the lung differs between mouse and man. The implications for the development of anti-asthma drugs with chitinases as targets are discussed.

(J Histochem Cytochem 53:1283–1292, 2005)

Two distinct chitinases have been identified in mammals: a phagocyte-specific enzyme named chitotriosidase and an acidic mammalian chitinase (AMCase) expressed in the lungs and gastrointestinal tract. Increased expression of both chitinases has been observed in different pathological conditions: chitotriosidase in lysosomal lipid storage disorders like Gaucher disease and AMCase in asthmatic lung disease. Recently, it was reported that AMCase activity is involved in the pathogenesis of asthma in an induced mouse model. Inhibition of chitinase activity was found to alleviate the inflammation-driven pathology. We studied the tissue-specific expression of both chitinases in mice and compared it to the situation in man. In both species AMCase is expressed in alveolar macrophages and in the gastrointestinal tract. In mice, chitotriosidase is expressed only in the gastrointestinal tract, the tongue, fore-stomach, and Paneth cells in the small intestine, whereas in man the enzyme is expressed exclusively by professional phagocytes. This species difference seems to be mediated by distinct promoter usage. In conclusion, the pattern of expression of chitinases in the lung differs between mouse and man. The implications for the development of anti-asthma drugs with chitinases as targets are discussed.

(J Histochem Cytochem 53:1283–1292, 2005)

In this study, we examined the mechanism of heat-induced antigen retrieval using analytical procedures involving SDS-PAGE, Western blotting, and immunohistochemistry. Five proteins were treated with 4% formaldehyde in the presence or absence of 25 mM CaCl₂, then heated under various conditions after removal of formaldehyde and analyzed on SDS-PAGE. Formaldehyde produced inter- and intramolecular cross-links in the proteins. Heating at high temperatures cleaved these cross-links at all pH ranges examined (pH 3.0, 6.0, 7.5, 9.0) and produced almost the same electrophoregrams as the native proteins. Proteins treated with formaldehyde containing CaCl₂ showed similar electrophoretic patterns, observed without heating or after heating at pH 6.0 and pH 9.0 in the presence or absence of 10 mM EDTA. Western blot analyses demonstrated that the soluble forms of ßbeta;-actin (monomer and oligomers) and fibronectin were present in extracts from deparaffinized mouse uterine sections autoclaved for 15 min but not in extracts from unheated specimens. Nine of ten antigens, independent of their isoelectric points, exhibited much stronger immunoreaction in the sections heated at pH 9.0 than in those heated at pH 6.0. The second heating at pH 6.0 significantly decreased the immunostaining of the antigens that had been boiled at pH 9.0, but the immunostaining was recovered after a third heating at pH 9.0. These results suggest that the main mechanism of heat-induced antigen retrieval is disruption of the cross-links and that pH is an essential factor for a proper refolding of epitopes. (J Histochem Cytochem 53:13–21, 2005)

Related Articles

Mechanisms of heat-induced antigen retrieval: analyses in vitro employing SDS-PAGE and immunohistochemistry.

J Histochem Cytochem. 2005 Jan;53(1):13-21

Authors: Yamashita S, Okada Y

In this study, we examined the mechanism of heat-induced antigen retrieval using analytical procedures involving SDS-PAGE, Western blotting, and immunohistochemistry. Five proteins were treated with 4% formaldehyde in the presence or absence of 25 mM CaCl2, then heated under various conditions after removal of formaldehyde and analyzed on SDS-PAGE. Formaldehyde produced inter- and intramolecular cross-links in the proteins. Heating at high temperatures cleaved these cross-links at all pH ranges examined (pH 3.0, 6.0, 7.5, 9.0) and produced almost the same electrophoregrams as the native proteins. Proteins treated with formaldehyde containing CaCl2 showed similar electrophoretic patterns, observed without heating or after heating at pH 6.0 and pH 9.0 in the presence or absence of 10 mM EDTA. Western blot analyses demonstrated that the soluble forms of beta-actin (monomer and oligomers) and fibronectin were present in extracts from deparaffinized mouse uterine sections autoclaved for 15 min but not in extracts from unheated specimens. Nine of ten antigens, independent of their isoelectric points, exhibited much stronger immunoreaction in the sections heated at pH 9.0 than in those heated at pH 6.0. The second heating at pH 6.0 significantly decreased the immunostaining of the antigens that had been boiled at pH 9.0, but the immunostaining was recovered after a third heating at pH 9.0. These results suggest that the main mechanism of heat-induced antigen retrieval is disruption of the cross-links and that pH is an essential factor for a proper refolding of epitopes.

PMID: 15637334 [PubMed - indexed for MEDLINE]

The multicolor-banding (mcb) technique is a fluorescence in situ hybridization (FISH)-banding approach, which is based on region-specific microdissection libraries producing changing fluorescence intensity ratios along the chromosomes. The latter are used to assign different pseudocolors to specific chromosomal regions. Here we present the first three available mcb-probe sets for the Mus musculus chromosomes 3, 6, and 18. In the present work, the creation of the microdissection libraries was done for the first time on mouse/human somatic cell hybrids. During creation of the mcb-probes, the latter enabled an unambiguous identification of the, otherwise in GTG-banding, hardly distinguishable murine chromosomes. (J Histochem Cytochem 53:791–792, 2005)

The multicolor-banding (mcb) technique is a fluorescence in situ hybridization (FISH)-banding approach, which is based on region-specific microdissection libraries producing changing fluorescence intensity ratios along the chromosomes. The latter are used to assign different pseudocolors to specific chromosomal regions. Here we present the first three available mcb-probe sets for the Mus musculus chromosomes 3, 6, and 18. In the present work, the creation of the microdissection libraries was done for the first time on mouse/human somatic cell hybrids. During creation of the mcb-probes, the latter enabled an unambiguous identification of the, otherwise in GTG-banding, hardly distinguishable murine chromosomes. (J Histochem Cytochem 53:791–792, 2005)

The multicolor-banding (mcb) technique is a fluorescence in situ hybridization (FISH)-banding approach, which is based on region-specific microdissection libraries producing changing fluorescence intensity ratios along the chromosomes. The latter are used to assign different pseudocolors to specific chromosomal regions. Here we present the first three available mcb-probe sets for the Mus musculus chromosomes 3, 6, and 18. In the present work, the creation of the microdissection libraries was done for the first time on mouse/human somatic cell hybrids. During creation of the mcb-probes, the latter enabled an unambiguous identification of the, otherwise in GTG-banding, hardly distinguishable murine chromosomes. (J Histochem Cytochem 53:791–792, 2005)

Microdissected rat proximal straight tubules (PST) and inner medullary collecting ducts (IMCD) highly produce urea from l-arginine, supporting the expression of the mitochondrial arginase II. However, IMCD contain a very low density of mitochondria compared with PST. Recently, arginase II has been localized by immunohistochemistry in rat PST but not IMCD. This study was designed to verify whether rat IMCD express arginase II and to identify its subcellular localization. We developed an antibody raised against arginase II that allowed the detection of a band of 38 kDa corresponding to arginase II on immunoblots. In male and female rat kidneys, Western blot analyses revealed that arginase II was highly expressed in the inner medulla (IM), the outer stripe of the outer medulla (osOM), and the deep cortex. Immunocytochemistry demonstrated that arginase II was homogeneously expressed in IMCD. Proteins of the cytosolic and mitochondrial fractions extracted from osOM and IM and analyzed by Western blot showed that 86% of arginase II was associated with mitochondria. The molecular weight of arginase II was similar in the cytosolic and mitochondrial fractions. Immunoelectron microscopy confirmed the presence of arginase II in the mitochondria of IMCD. In conclusion, arginase II is expressed in mitochondria of male and female rat IMCD. (J Histochem Cytochem 53:533–541, 2005)

Microdissected rat proximal straight tubules (PST) and inner medullary collecting ducts (IMCD) highly produce urea from l-arginine, supporting the expression of the mitochondrial arginase II. However, IMCD contain a very low density of mitochondria compared with PST. Recently, arginase II has been localized by immunohistochemistry in rat PST but not IMCD. This study was designed to verify whether rat IMCD express arginase II and to identify its subcellular localization. We developed an antibody raised against arginase II that allowed the detection of a band of 38 kDa corresponding to arginase II on immunoblots. In male and female rat kidneys, Western blot analyses revealed that arginase II was highly expressed in the inner medulla (IM), the outer stripe of the outer medulla (osOM), and the deep cortex. Immunocytochemistry demonstrated that arginase II was homogeneously expressed in IMCD. Proteins of the cytosolic and mitochondrial fractions extracted from osOM and IM and analyzed by Western blot showed that 86% of arginase II was associated with mitochondria. The molecular weight of arginase II was similar in the cytosolic and mitochondrial fractions. Immunoelectron microscopy confirmed the presence of arginase II in the mitochondria of IMCD. In conclusion, arginase II is expressed in mitochondria of male and female rat IMCD. (J Histochem Cytochem 53:533–541, 2005)

Microdissected rat proximal straight tubules (PST) and inner medullary collecting ducts (IMCD) highly produce urea from l-arginine, supporting the expression of the mitochondrial arginase II. However, IMCD contain a very low density of mitochondria compared with PST. Recently, arginase II has been localized by immunohistochemistry in rat PST but not IMCD. This study was designed to verify whether rat IMCD express arginase II and to identify its subcellular localization. We developed an antibody raised against arginase II that allowed the detection of a band of 38 kDa corresponding to arginase II on immunoblots. In male and female rat kidneys, Western blot analyses revealed that arginase II was highly expressed in the inner medulla (IM), the outer stripe of the outer medulla (osOM), and the deep cortex. Immunocytochemistry demonstrated that arginase II was homogeneously expressed in IMCD. Proteins of the cytosolic and mitochondrial fractions extracted from osOM and IM and analyzed by Western blot showed that 86% of arginase II was associated with mitochondria. The molecular weight of arginase II was similar in the cytosolic and mitochondrial fractions. Immunoelectron microscopy confirmed the presence of arginase II in the mitochondria of IMCD. In conclusion, arginase II is expressed in mitochondria of male and female rat IMCD. (J Histochem Cytochem 53:533–541, 2005)

We have proposed to divide intestinal metaplasia (IM) into two categories, i.e., a mixed gastric and intestinal (GI) type, and a solely intestinal (I) type, based on the residual gastric phenotype cells. The GI-mixed-type IM can be identified by the presence of both cells with either gastric or intestinal phenotypes in a single gland. This study is conducted to elucidate whether cells in the GI-mixed-type IM glands can simultaneously present both gastric and intestinal phenotypes. MUC5AC, MUC2, CD10 and villin expressions were investigated in 20 samples from five gastric cancer cases, directly using either AlexaFluor 488- or 568-labeled specific monoclonal antibodies and observed by fluorescent microscopy and confocal laser-scanning microscopy. GI-mixed IM glands comprise a population expressing MUC5AC and MUC2, MUC5AC and villin, and MUC5AC and CD10. MUC2 and villin expressions were reciprocally increased with decreasing MUC5AC expression, while CD10 expression was limited to cells with only a residual MUC5AC expression or no expression. These results suggest that a heterogeneous cell population with both gastric and intestinal phenotypes would develop into a single intestinal phenotype, as reflected in the progression of intestinal metaplasia from GI-mixed-type- to I-type IM-type glands.

(J Histochem Cytochem 53:75–85, 2005)

Array CGH (comparative genomic hybridization) enables the identification of chromosomal copy number changes. The availability of clone sets covering the human genome opens the possibility for the widespread use of array CGH for both research and diagnostic purposes. In this manuscript we report on the parameters that were critical for successful implementation of the technology, assess quality criteria, and discuss the potential benefits and pitfalls of the technology for improved pre- and postnatal constitutional genetic diagnosis. We propose to name the genome-wide array CGH "molecular karyotyping," in analogy with conventional karyotyping that uses staining methods to visualize chromosomes. (J Histochem Cytochem 53:413–422, 2005)

Two rat monoclonal antibodies have been generated to plant cell wall (1->4)-ßbeta;-D-xylans using a penta-1,4-xylanoside–containing neoglycoprotein as an immunogen. The monoclonal antibodies, designated LM10 and LM11, have different specificities to xylans in relation to the substitution of the xylan backbone as indicated by immunodot assays and competitive-inhibition ELISAs. LM10 is specific to unsubstituted or low-substituted xylans, whereas LM11 binds to wheat arabinoxylan in addition to unsubstituted xylans. Immunocytochemical analyses indicated the presence of both epitopes in secondary cell walls of xylem but differences in occurrence in other cell types.

(J Histochem Cytochem 53:543–546, 2005)

Two rat monoclonal antibodies have been generated to plant cell wall (1->4)-ßbeta;-D-xylans using a penta-1,4-xylanoside–containing neoglycoprotein as an immunogen. The monoclonal antibodies, designated LM10 and LM11, have different specificities to xylans in relation to the substitution of the xylan backbone as indicated by immunodot assays and competitive-inhibition ELISAs. LM10 is specific to unsubstituted or low-substituted xylans, whereas LM11 binds to wheat arabinoxylan in addition to unsubstituted xylans. Immunocytochemical analyses indicated the presence of both epitopes in secondary cell walls of xylem but differences in occurrence in other cell types.

(J Histochem Cytochem 53:543–546, 2005)

Two rat monoclonal antibodies have been generated to plant cell wall (1->4)-ßbeta;-D-xylans using a penta-1,4-xylanoside–containing neoglycoprotein as an immunogen. The monoclonal antibodies, designated LM10 and LM11, have different specificities to xylans in relation to the substitution of the xylan backbone as indicated by immunodot assays and competitive-inhibition ELISAs. LM10 is specific to unsubstituted or low-substituted xylans, whereas LM11 binds to wheat arabinoxylan in addition to unsubstituted xylans. Immunocytochemical analyses indicated the presence of both epitopes in secondary cell walls of xylem but differences in occurrence in other cell types.

(J Histochem Cytochem 53:543–546, 2005)

Related Articles

Morphologic and immunophenotypic diversity in Ewing family tumors: a study of 66 genetically confirmed cases.

Am J Surg Pathol. 2005 Aug;29(8):1025-33

Authors: Folpe AL, Goldblum JR, Rubin BP, Shehata BM, Liu W, Dei Tos AP, Weiss SW

More than 85% of Ewing sarcoma/primitive neuroectodermal tumor (ES/PNET), or "Ewing family of tumors" (EFTs), have the translocation, t (11;22) (q24;q12), with others having variant translocations. Identification of these by cytogenetic and/or molecular genetic techniques is specific for EFT and is increasingly recognized as the "gold standard" for diagnosis. However, these techniques are not universally available. We therefore studied a large group of genetically confirmed EFTs to more completely understand the morphologic and immunophenotypic spectrum of this rare sarcoma. Sixty-six cytogenetically, FISH or RT-PCR proven-EFTs were retrieved. In 56 cases, immunohistochemistry (IHC) was performed for pan-cytokeratins (PanCK), high molecular weight cytokeratins (HMWCK), desmin (DES), CD99, CD117, and FLI1 protein using heat-induced epitope retrieval and the Dako Envision system. The cases arose chiefly in children and young adults (median 18 years; range, 3-65 years) of both sexes (male, 32; female, 31; unknown, 3) in a variety of bone (N = 39) and soft tissue (N = 27) sites. Histologically, 46 cases (73%) showed only typical features of ES, 9 cases (16%) showed features of PNET, 3 cases (5%) showed "adamantinoma-like" features, 3 cases (5%) corresponded to "atypical Ewing sarcoma," 3 cases (5%) showed principally intersecting fascicles of spindled cells, and 2 cases had abundant hyalinized matrix. IHC results were as follows: PanCK (18 of 56, 32%), HMWCK (3 of 55, 5%), DES (1 of 56, 2%), CD99 (52 of 52, 100%), CD117 (13 of 54, 24%), and FLI1 (44 of 47, 94%). HMWCK was expressed only in "adamantinoma-like" EFTs, none of which expressed DES. In conclusion, most, but not all, EFTs can be accurately diagnosed using time-honored morphologic criteria and ancillary immunohistochemistry. However, genetic confirmation remains essential for the diagnosis of unusual morphologic variants of EFT, including "adamantinoma-like," spindled, sclerosing, and clear cell/anaplastic variants. Therefore, to exclude or confirm the diagnosis of Ewing's sarcoma in round cell sarcomas having a variety of patterns but not specifically conforming to a tumor of known lineage (eg, rhabdomyosarcoma), cytogenetics, and/or molecular analysis is required.

PMID: 16006796 [PubMed - indexed for MEDLINE]

Mosquitoes mount strong humoral and cellular immune responses against foreign organisms. Two components of the mosquito immune response that have received much attention are the phenoloxidase cascade that leads to melanization and antimicrobial peptides. The purpose of the current study was to use immunocytochemistry and transmission electron microscopy to identify the location of the melanization rate-limiting enzyme phenoloxidase and the antimicrobial peptide defensin in innate immune reactions against Escherichia coli and Micrococcus luteus by the mosquito Aedes aegypti. Our results show that both phenoloxidase and defensin are present at the sites of melanin biosynthesis in immune reactions against bacteria. Furthermore, both proteins are often present inside the same melanotic capsules. When hemocytes were analyzed, phenoloxidase was present in the cytosol of oenocytoids, but no significant amounts of defensin were detected inside any hemocytes. In summary, these data show that phenoloxidase and defensin colocalize in melanization reactions against bacteria and argue for further studies into the potential role of defensin in phenoloxidase-based melanization innate immune responses in mosquitoes.

(J Histochem Cytochem 53:689–698, 2005)

Mosquitoes mount strong humoral and cellular immune responses against foreign organisms. Two components of the mosquito immune response that have received much attention are the phenoloxidase cascade that leads to melanization and antimicrobial peptides. The purpose of the current study was to use immunocytochemistry and transmission electron microscopy to identify the location of the melanization rate-limiting enzyme phenoloxidase and the antimicrobial peptide defensin in innate immune reactions against Escherichia coli and Micrococcus luteus by the mosquito Aedes aegypti. Our results show that both phenoloxidase and defensin are present at the sites of melanin biosynthesis in immune reactions against bacteria. Furthermore, both proteins are often present inside the same melanotic capsules. When hemocytes were analyzed, phenoloxidase was present in the cytosol of oenocytoids, but no significant amounts of defensin were detected inside any hemocytes. In summary, these data show that phenoloxidase and defensin colocalize in melanization reactions against bacteria and argue for further studies into the potential role of defensin in phenoloxidase-based melanization innate immune responses in mosquitoes.

(J Histochem Cytochem 53:689–698, 2005)

Mosquitoes mount strong humoral and cellular immune responses against foreign organisms. Two components of the mosquito immune response that have received much attention are the phenoloxidase cascade that leads to melanization and antimicrobial peptides. The purpose of the current study was to use immunocytochemistry and transmission electron microscopy to identify the location of the melanization rate-limiting enzyme phenoloxidase and the antimicrobial peptide defensin in innate immune reactions against Escherichia coli and Micrococcus luteus by the mosquito Aedes aegypti. Our results show that both phenoloxidase and defensin are present at the sites of melanin biosynthesis in immune reactions against bacteria. Furthermore, both proteins are often present inside the same melanotic capsules. When hemocytes were analyzed, phenoloxidase was present in the cytosol of oenocytoids, but no significant amounts of defensin were detected inside any hemocytes. In summary, these data show that phenoloxidase and defensin colocalize in melanization reactions against bacteria and argue for further studies into the potential role of defensin in phenoloxidase-based melanization innate immune responses in mosquitoes.

(J Histochem Cytochem 53:689–698, 2005)

Live cell imaging methods were used to characterize goblet cells expressing a MUC5AC domain fused to enhanced green fluorescent protein that labels the granule lumen. Golgi complex and endosome/lysosome elements largely resided in the periphery of the granular mass. On the contrary, a tubular meshwork of endoplasmic reticulum (ER) was in close contact with the mucin granules. This meshwork could be identified in fixed native human bronchial goblet cells labeled with an anti-calreticulin antibody. The potential biological significance of this ER network for mucin secretion is discussed.

(J Histochem Cytochem 53:1305–1309, 2005)

Live cell imaging methods were used to characterize goblet cells expressing a MUC5AC domain fused to enhanced green fluorescent protein that labels the granule lumen. Golgi complex and endosome/lysosome elements largely resided in the periphery of the granular mass. On the contrary, a tubular meshwork of endoplasmic reticulum (ER) was in close contact with the mucin granules. This meshwork could be identified in fixed native human bronchial goblet cells labeled with an anti-calreticulin antibody. The potential biological significance of this ER network for mucin secretion is discussed.

(J Histochem Cytochem 53:1305–1309, 2005)

Dermal elastic fibers are believed to have a primary role in providing elastic stretch and recoil to the skin. Here we compare the structural arrangement of dermal elastic fibers of chick skin and different animal species. Most elastic fibers in chick skin are derived from cells that line the feather follicle and/or smooth muscle that connects the pterial and apterial muscle bundles to feather follicles. Elastic fibers in the dermis of animals with single, primary hair follicles are derived from cells lining the hair follicle or from the ends of the pili muscle, which anchors the muscle to the matrix or to the hair follicle. Each follicle is interconnected with elastic fibers. Follicles of animals with primary and secondary (wool) hair follicles are also interconnected by elastic fibers, yet only the elastic fibers derived from the primary follicle are connected to each primary follicle. Only the primary hair follicles are connected to the pili muscle. Human skin, but not the skin of other primates, is significantly different from other animals with respect to elastic fiber organization and probably cell of origin. The data suggest that the primary role for elastic fibers in animals, with the possible exception of humans, is movement and/or placement of feathers or hair. (J Histochem Cytochem 53:431–443, 2005)

Dermal elastic fibers are believed to have a primary role in providing elastic stretch and recoil to the skin. Here we compare the structural arrangement of dermal elastic fibers of chick skin and different animal species. Most elastic fibers in chick skin are derived from cells that line the feather follicle and/or smooth muscle that connects the pterial and apterial muscle bundles to feather follicles. Elastic fibers in the dermis of animals with single, primary hair follicles are derived from cells lining the hair follicle or from the ends of the pili muscle, which anchors the muscle to the matrix or to the hair follicle. Each follicle is interconnected with elastic fibers. Follicles of animals with primary and secondary (wool) hair follicles are also interconnected by elastic fibers, yet only the elastic fibers derived from the primary follicle are connected to each primary follicle. Only the primary hair follicles are connected to the pili muscle. Human skin, but not the skin of other primates, is significantly different from other animals with respect to elastic fiber organization and probably cell of origin. The data suggest that the primary role for elastic fibers in animals, with the possible exception of humans, is movement and/or placement of feathers or hair. (J Histochem Cytochem 53:431–443, 2005)

Dermal elastic fibers are believed to have a primary role in providing elastic stretch and recoil to the skin. Here we compare the structural arrangement of dermal elastic fibers of chick skin and different animal species. Most elastic fibers in chick skin are derived from cells that line the feather follicle and/or smooth muscle that connects the pterial and apterial muscle bundles to feather follicles. Elastic fibers in the dermis of animals with single, primary hair follicles are derived from cells lining the hair follicle or from the ends of the pili muscle, which anchors the muscle to the matrix or to the hair follicle. Each follicle is interconnected with elastic fibers. Follicles of animals with primary and secondary (wool) hair follicles are also interconnected by elastic fibers, yet only the elastic fibers derived from the primary follicle are connected to each primary follicle. Only the primary hair follicles are connected to the pili muscle. Human skin, but not the skin of other primates, is significantly different from other animals with respect to elastic fiber organization and probably cell of origin. The data suggest that the primary role for elastic fibers in animals, with the possible exception of humans, is movement and/or placement of feathers or hair. (J Histochem Cytochem 53:431–443, 2005)

The endothelin/endothelin–receptor system is a key player in the regulation of vascular tone in mammals. We raised and characterized an antiserum against rat ET_B receptor and investigated the distribution of ET_B receptors in different vascular beds during postnatal development (day 0 through day 28) and in the adult rat. We report the tissue-specific and age-dependent presence of vasoconstrictor ET_B receptors. At the time of birth, vascular smooth muscle cells from all tissues examined did not exhibit ET_B receptor immunoreactivity. The occurrence of ET_B receptor immunoreactivity in the postnatal development was time dependent and started in small coronary and meningeal arteries at day 5, followed by small mesenteric arteries as well as brachial artery and vein at day 14. At day 21, ET_B receptors were present in the media of muscular segments of pulmonary artery, large coronary arteries, and intracerebral arterioles. At day 28, ET_B receptor immunoreactivity was evident in interlobular renal arteries, vas afferens, and efferens. Large renal arteries, mesenteric artery, and elastic segments of pulmonary arteries, as well as coronary and mesenteric veins, did not exhibit ET_B receptor immunoreactivity. These data demonstrate the age-dependent and tissue-specific presence of ET_B receptors, mainly on arterial smooth muscle cells in the vascular system of the rat. (J Histochem Cytochem 53:187–196, 2005)

Muscle side population (SP) cells have demonstrated hematopoietic and myogenic activities in vivo upon intravenous (IV) injection into lethally irradiated mdx mice. In contrast, muscle main population (MP) cells were unable to rescue the bone marrow of lethally irradiated mice and, consequently, their in vivo myogenic potential could not be assessed using this method. In the current study, muscle SP or MP cells derived from syngeneic wild-type male mice were delivered to sub-lethally irradiated mdx female mice by single or serial IV injections. Recipient mice were euthanized 12 weeks after transplantation at which time the quadriceps and diaphragm muscles were analyzed for the presence of donor-derived cells. Mice injected with 10⁴ muscle SP cells or with 10⁶ MP cells appeared to have similar numbers of dystrophin-positive myofibers containing fused donor nuclei. Analysis of the remaining tissue via real-time quantitative PCR indicated that mice injected with muscle SP cells had a higher percentage of donor-derived Y-DNA in the quadriceps than mice injected with MP cells, suggesting that muscle SP cells may be enriched for progenitors able to engraft dystrophic skeletal muscles from the circulation. Although the overall engraftment did not reach therapeutically significant levels, these results indicate that further optimization of cell delivery techniques may lead to improved efficacy of cell-mediated therapy using muscle SP cells. (J Histochem Cytochem 53:861–873, 2005)

Muscle side population (SP) cells have demonstrated hematopoietic and myogenic activities in vivo upon intravenous (IV) injection into lethally irradiated mdx mice. In contrast, muscle main population (MP) cells were unable to rescue the bone marrow of lethally irradiated mice and, consequently, their in vivo myogenic potential could not be assessed using this method. In the current study, muscle SP or MP cells derived from syngeneic wild-type male mice were delivered to sub-lethally irradiated mdx female mice by single or serial IV injections. Recipient mice were euthanized 12 weeks after transplantation at which time the quadriceps and diaphragm muscles were analyzed for the presence of donor-derived cells. Mice injected with 10⁴ muscle SP cells or with 10⁶ MP cells appeared to have similar numbers of dystrophin-positive myofibers containing fused donor nuclei. Analysis of the remaining tissue via real-time quantitative PCR indicated that mice injected with muscle SP cells had a higher percentage of donor-derived Y-DNA in the quadriceps than mice injected with MP cells, suggesting that muscle SP cells may be enriched for progenitors able to engraft dystrophic skeletal muscles from the circulation. Although the overall engraftment did not reach therapeutically significant levels, these results indicate that further optimization of cell delivery techniques may lead to improved efficacy of cell-mediated therapy using muscle SP cells. (J Histochem Cytochem 53:861–873, 2005)

Previous studies have indicated that ErbB receptors for neuregulins play an important role in cardiac development and muscle spindle formation during embryogenesis; however, little is known about their functions in adulthood. Recent reports indicate that breast cancer therapy with humanized monoclonal ErbB2 antibody induces cardiomyopathy, suggesting that ErbB2 serves as a crucial signaling receptor, even in the adult heart. Here, we examine ErbB2 expression and localization in both cardiac and skeletal muscle of adult mice via immunoblot and immunohistochemistry. ErbB2 was detected as a band ~185 kD molecular mass in each cardiac and skeletal muscle extraction. Confocal images of double labeling showed that ErbB2 was colocalized with caveolin-3 in cardiac muscle and with dihydropyridine receptor in skeletal muscle, suggesting that ErbB2 was localized at the T-tubule. In addition, immunoelectron micrographs clearly demonstrated that ErbB2 was located at the T-tubule in both types of muscle. Taken together, the results of the present study suggest that neuregulin-ErbB2 signaling plays a role in the physiological function of cardiac and skeletal muscle, even in adulthood.

(J Histochem Cytochem 53:87–91, 2005)

Related Articles

Neuroanatomy of sea spiders implies an appendicular origin of the protocerebral segment.

Nature. 2005 Oct 20;437(7062):1144-8

Authors: Maxmen A, Browne WE, Martindale MQ, Giribet G

Independent specialization of arthropod body segments has led to more than a century of debate on the homology of morphologically diverse segments, each defined by a lateral appendage and a ganglion of the central nervous system. The plesiomorphic composition of the arthropod head remains enigmatic because variation in segments and corresponding appendages is extreme. Within extant arthropod classes (Chelicerata, Myriapoda, Crustacea and Hexapoda--including the insects), correspondences between the appendage-bearing second (deutocerebral) and third (tritocerebral) cephalic neuromeres have been recently resolved on the basis of immunohistochemistry and Hox gene expression patterns. However, no appendage targets the first ganglion, the protocerebrum, and the corresponding segmental identity of this anterior region remains unclear. Reconstructions of stem-group arthropods indicate that the anteriormost region originally might have borne an ocular apparatus and a frontal appendage innervated by the protocerebrum. However, no study of the central nervous system in extant arthropods has been able to corroborate this idea directly, although recent analyses of cephalic gene expression patterns in insects suggest a segmental status for the protocerebral region. Here we investigate the developmental neuroanatomy of a putative basal arthropod, the pycnogonid sea spider, with immunohistochemical techniques. We show that the first pair of appendages, the chelifores, are innervated at an anterior position on the protocerebrum. This is the first true appendage shown to be innervated by the protocerebrum, and thus pycnogonid chelifores are not positionally homologous to appendages of extant arthropods but might, in fact, be homologous to the 'great appendages' of certain Cambrian stem-group arthropods.

PMID: 16237442 [PubMed - in process]

Laser microdissection has opened a window to new technologies. The scientific fields of genomics, transcriptomics, and proteomics need pure samples for rendering reliable results. Homogeneous sample preparation is a prerequisite for modern molecular analyses, both qualitative and quantitative. Laser microdissection and pressure catapulting (LMPC) is a tool for isolating specific cells from complex tissues in a non-contact and contamination-free manner. Because LMPC technology is an optimal method for obtaining fast and reliable access to single cells, the possibility of automatic isolation of single fetal cells has the promise of being a big step forward in developing protocols for non-invasive prenatal diagnosis. (J Histochem Cytochem 53:409–412, 2005)

An important aspect of the image analysis of immunocytochemical preparations is the evaluation of colocalization of different molecules. The aim of the present study is to introduce image analysis methods to identify double-labeled locations exhibiting the highest association of two fluorophores and to characterize their pattern of distribution. These methods will be applied to the analysis of the cotrafficking of adenosine A_2A and dopamine D₂ receptors belonging to the G protein–coupled receptor family and visualized by means of fluorescence immunocytochemistry in Chinese hamster ovary cells after agonist treatment. The present procedures for colocalization have the great advantage that they are, to a large extent, insensitive to the need for a balanced staining with the two fluorophores. Thus, these procedures involve image processing, visualization, and analysis of colocalized events, using a covariance method and a multiply method and the evaluation of the identified colocalization patterns. Moreover, the covariance method offers the possibility of detecting and quantitatively characterizing anticorrelated patterns of intensities, whereas the immediate detection of colocalized clusters with a high concentration of labeling is a possibility offered by the multiply method. The present methods offer a new and sensitive approach to detecting and quantitatively characterizing strongly associated fluorescence events, such as those generated by receptor–receptor interaction, and their distribution patterns in dual-color confocal laser microscopy.

(J Histochem Cytochem 53:941–953, 2005)

An important aspect of the image analysis of immunocytochemical preparations is the evaluation of colocalization of different molecules. The aim of the present study is to introduce image analysis methods to identify double-labeled locations exhibiting the highest association of two fluorophores and to characterize their pattern of distribution. These methods will be applied to the analysis of the cotrafficking of adenosine A_2A and dopamine D₂ receptors belonging to the G protein–coupled receptor family and visualized by means of fluorescence immunocytochemistry in Chinese hamster ovary cells after agonist treatment. The present procedures for colocalization have the great advantage that they are, to a large extent, insensitive to the need for a balanced staining with the two fluorophores. Thus, these procedures involve image processing, visualization, and analysis of colocalized events, using a covariance method and a multiply method and the evaluation of the identified colocalization patterns. Moreover, the covariance method offers the possibility of detecting and quantitatively characterizing anticorrelated patterns of intensities, whereas the immediate detection of colocalized clusters with a high concentration of labeling is a possibility offered by the multiply method. The present methods offer a new and sensitive approach to detecting and quantitatively characterizing strongly associated fluorescence events, such as those generated by receptor–receptor interaction, and their distribution patterns in dual-color confocal laser microscopy.

(J Histochem Cytochem 53:941–953, 2005)

We assessed the feasibility of fetal RHD and RHCE genotyping by analysis of DNA extracted from plasma samples of RhD-negative pregnant women using real-time PCR and primers and probes targeted toward RHD and RHCE genes. We analyzed 45 pregnant women in the 11th to 40th weeks of pregnancy and correlated the results with serological analysis of cord blood after delivery. Non-invasive prenatal fetal RHD exon 7, RHD exon 10, RHCE exon 2 (C allele), and RHCE exon 5 (E allele) genotyping analysis of maternal plasma samples was correctly performed in 45 out of 45 RhD-negative pregnant women delivering 24 RhD-, 17 RhC-, and 7 RhE-positive newborns. Detection of fetal RHD and the C and E alleles of RHCE gene from maternal plasma is highly accurate and enables implementation into clinical routine. We recommend performing fetal RHD and RHCE genotyping together with fetal sex determination in alloimmunized D-negative pregnancies at risk of hemolytic disease of the newborn. In case of D-negative fetus, amplification of another paternally inherited allele (SRY and/or RhC and/or RhE positivity) proves the presence of fetal DNA in maternal circulation. (J Histochem Cytochem 53:301–305, 2005)