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Used Immunohistochemistry (used + immunohistochemistry)
Selected AbstractsNMDA receptor subunits GluR,1, GluR,3 and GluR,1 are enriched at the mossy fibre,granule cell synapse in the adult mouse cerebellumEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 11 2001Kazuyuki Yamada Abstract Cerebellar N -methyl- d -aspartate (NMDA) receptors are concentrated in the granular layer and are involved in motor coordination and the induction of long-term potentiation at mossy fibre,granule cell synapses. In the present study, we used immunohistochemistry to examine the distribution of NMDA receptor subunits in the adult mouse cerebellum. We found that appropriate pepsin pretreatment of sections greatly enhanced the sensitivity and specificity of immunohistochemical detection. As a result, intense immunolabelling for GluR,1 (NR2A), GluR,3 (NR2C), and GluR,1 (NR1) all appeared in synaptic glomeruli of the granular layer. Double immunofluorescence showed that these subunits were colocalized in individual synaptic glomeruli. Within the glomerulus, NMDA receptor subunits were located between centrally-located huge mossy fibre terminals and peripherally-located tiny Golgi axon terminals. By immunoelectron microscopy, all three subunits were detected at the postsynaptic junction in granule cell dendrites, forming synapses with mossy fibre terminals. Consistent with the known functional localization, GluR,1, GluR,3, and GluR,1 are, thus, anatomically concentrated at the mossy fibre,granule cell synapse. By contrast, immunohistochemical signals were very low in Purkinje cell somata and dendrites in the molecular layer. The lack of GluR,1 immunolabelling in Purkinje cells was unexpected because the cells express GluR,1 mRNA at high levels and high levels of GluR,1 protein in the molecular layer were revealed by immunoblot. As Purkinje cells are exceptionally lacking GluR, expression, the discrepant result may provide in vivo evidence suggesting the importance of accompanying GluR, subunits in synaptic localization of GluR,1. [source] Deletions removing the last exon of TACSTD1 constitute a distinct class of mutations predisposing to Lynch syndrome,HUMAN MUTATION, Issue 2 2009Marietta E. Kovacs Abstract Several different genetic alterations in the etiology of Lynch syndrome (hereditary nonpolyposis colorectal cancer [HNPCC]) are known, mostly point mutations and genomic rearrangements in 1 of at least 3 mismatch-repair (MMR) genes. However, no susceptibility factor has yet been identified in a significant part (30,50%) of clinicopathologically well-defined HNPCC families, suggesting the presence of other predisposing mechanisms. In a set of probands from 27 Lynch syndrome families who lacked evidence of a germline mutation in either the MSH2 or MLH1 gene, we performed genomic deletion screening with the use of multiplex ligation-dependent probe amplification (MLPA) and sequencing. We used immunohistochemistry (IHC) and microsatellite instability (MSI) analyses on samples of the probands of all families. Comparative analysis of mRNA transcripts was performed on blood leukocyte,derived samples from mutation carriers and noncarrier controls. We report that large germline deletions encompassing the last exons of the TACSTD1 gene, upstream of MSH2, cosegregate with the HNPCC phenotype in 19% (5/27) of families tested. The tumors of the carriers show high-level MSI and MSH2 protein loss. We show that these deletions, by removing the transcriptional termination sequences of the upstream gene, give rise to multiple TACSTD1/MSH2 fusion transcripts. Our results provide evidence that deletions removing the last exon of TACSTD1 constitute a distinct class of mutations predisposing to Lynch syndrome. Thus, analysis of the 3, region of the TACSTD1 gene should be included in the routine mutation screening protocols for HNPCC. Hum Mutat 30, 197,203, 2009. © 2009 Wiley-Liss, Inc. [source] Observations of serotonin and FMRFamide-like immunoreactivity in palp sensory structures and the anterior nervous system of spionid polychaetesJOURNAL OF MORPHOLOGY, Issue 5 2008David L. Forest Abstract Evidence suggests that ciliated sensory structures on the feeding palps of spionid polychaetes may function as chemoreceptors to modulate deposit-feeding activity. To investigate the probable sensory nature of these ciliated cells, we used immunohistochemistry, epi-fluorescence, and confocal laser scanning microscopy to label and image sensory cells, nerves, and their organization relative to the anterior central nervous system in several spionid polychaete species. Antibodies directed against acetylated ,tubulin were used to label the nervous system and detail the innervation of palp sensory cells in all species. In addition, the distribution of serotonin (5-HT) and FMRFamide-like immunoreactivity was compared in the spionid polychaetes Dipolydora quadrilobata and Pygospio elegans. The distribution of serotonin immunoreactivity was also examined in the palps of Polydora cornuta and Streblospio benedicti. Serotonin immunoreactivity was concentrated in cells underlying the food groove of the palps, in the palp nerves, and in the cerebral ganglion. FMRFamide-like immunoreactivity was associated with the cerebral ganglia, nuchal organs and palp nerves, and also with the perikarya of ciliated sensory cells on the palps. J. Morphol., 2008. © 2007 Wiley-Liss, Inc. [source] Nociceptin/Orphanin FQ Peptide in Hypothalamic Neurones Associated with the Control of Feeding BehaviourJOURNAL OF NEUROENDOCRINOLOGY, Issue 2 2010N. Maolood Nociceptin/orphanin FQ (N/OFQ), an endogenous peptide agonist of the opioid N/OFQ receptor, has been implicated in the regulation of energy balance. In the present study, we have used immunohistochemistry to investigate the cellular localisation and colocalisation of N/OFQ-immunoreactive cell bodies in hypothalamic regions containing neurones producing orexigenic or anorexigenic transmitters. In colchicine-treated rats, N/OFQ immunoreactivity was demonstrated in many cell bodies of the arcuate nucleus (Arc), paraventricular nucleus (PVN) and lateral hypothalamic area (LHA). Double-labelling revealed that N/OFQ was present in some neurones located in the ventrolateral part of the Arc producing pro-opiomelanocortin, as shown by the presence of the anorexigenic peptides ,-melanocyte-stimulating hormone (,-MSH) and cocaine- and amphetamine-regulated transcript and, occasionally, in single neurones of the ventrolateral Arc producing orexigenic agouti-related peptide, but not neuropeptide Y. N/OFQ immunoreactivity was also demonstrated in a few tyrosine hydroxylase- or dynorphin (DYN)-containing neurones in the dorsomedial part of the Arc. In the parvocellular PVN, N/OFQ was demonstrated in some thyrotrophin-releasing hormone- or DYN-, but not corticotrophin-releasing hormone-containing neurones. Most N/OFQ-immunoreactive neurones in the LHA contained orexin- and DYN, but not melanin-concentrating hormone. The results obtained, demonstrating the presence of N/OFQ in some ,-MSH- and in many orexin-containing neurones, suggest a functional relationship between these neuropeptides and N/OFQ in the control of feeding behaviour and body weight. [source] Interactive roles of fibroblast growth factor 2 and neurotrophin 3 in the sequence of migration, process outgrowth, and axonal differentiation of mouse cochlear ganglion cellsJOURNAL OF NEUROSCIENCE RESEARCH, Issue 11 2008Waheeda A. Hossain Abstract A growth factor may have different actions depending on developmental stage. We investigated this phenomenon in the interactions of fibroblast growth factor 2 (FGF2) and neurotrophins on cochlear ganglion (CG) development. The portions of the otocyst fated to form the CG and cochlear epithelium were cocultured at embryonic day 11 (E11). Cultures were divided into groups fed with defined medium, with or without FGF2 and neurotrophin supplements, alone or in combination, for 7 days. We measured the number of migrating neuroblasts and distances migrated, neurite outgrowth, and axonlike processes. We used immunohistochemistry to locate neurotrophin 3 (NT3) and its high-affinity receptor (TrkC) in the auditory system, along with FGF2 and its R1 receptor, at comparable developmental stages in vitro and in situ from E11 until birth (P1) in the precursors of hair cells, support cells, and CG cells. Potential sites for interaction were localized to the nucleus, perikaryal cytoplasm, and cell surfaces, including processes and growth cones. Time-lapse imaging and quantitative measures support the hypothesis that FGF2 alone or combined with neurotrophins promotes migration and neurite outgrowth. Synergism or antagonism between NT3 and other factors suggest interactions at the receptor level. Formation of axons, endings, and synaptic vesicle protein 2 were increased by interactions of NT3 and FGF2. Similar experiments with a mutant overexpressor for FGF2 suggest that endogenous FGF2 supports migration and neurite outgrowth of CG neuroblasts as well as proliferation, leading to accelerated development. The findings suggest interactive and sequential roles for FGF2 and NT3. © 2008 Wiley-Liss, Inc. [source] CC Chemokine ligand 17 in periodontal diseases: expression in diseased tissues and production by human gingival fibroblastsJOURNAL OF PERIODONTAL RESEARCH, Issue 4 2008Y. Hosokawa Background and Objective:, It has been reported that T helper 2 (Th2) cells are related to exacerbation of periodontal disease. However, it is uncertain how the migration of Th2 cells is controlled. In this study, we examined the expression of CC chemokine ligand 17 (CCL17), which is a Th2 chemokine, in periodontal tissues. Moreover, we investigated the effects of cytokines and toll-like receptor (TLR) ligands on the production of CCL17 by human gingival fibroblasts (HGFs). Material and Methods:, We used immunohistochemistry and reverse transcriptase-polymerase chain reaction (RT-PCR) to detect CCL17 in periodontal tissues. HGFs were exposed to cytokines and TLR ligands. Expression of CCL17 was examined by RT-PCR and enzyme-linked immunosorbent assay. We used signal transduction inhibitors in some experiments. Results:, Both CCL17 and its receptor, CC chemokine receptor 4 (CCR4), were expressed in diseased periodontal tissues. A combination of tumour necrosis factor , (TNF-,) and interleukin (IL)-4/IL-13 increased CCL17 expression. Moreover, treatment of HGFs with a low dose of interferon-, (IFN-,) in combination with TNF-, and IL-4 or IL-13 had synergistic effects on the production of CCL17, whereas a high dose of IFN-, inhibited CCL17 production. Furthermore, Escherichia coli (E. coli) lipopolysaccharide (TLR4 ligand) and Pam3CSK4 (TLR2 ligand) inhibited CCL17 production by TNF-, + IL-4-stimulated HGFs, while CpG DNA (TLR9 ligand) enhanced TNF-, + IL-4 induced-CCL17 production by HGFs. Furthermore, a c-Jun NH2 terminal kinase (JNK) inhibitor, a phosphatidylinositol-3-kinase (PI3K) inhibitor and a nuclear factor ,B (NF-,B) inhibitor inhibited CCL17 production by HGFs. Conclusion:, These results suggest that the CCL17 produced by HGFs may be involved in the migration of Th2 cells into inflamed tissues, and provide evidence that CCL17 production is controlled by cytokines and TLR ligands in periodontal disease. [source] Localization of Sepiapterin Reductase in Pigment Cells of Oryzias latipesPIGMENT CELL & MELANOMA RESEARCH, Issue 5 2003Sumiko Negishi Body colors of poikilothermal vertebrates are derived from three distinct types of pigment cells, melanophores, erythro/xanthophores and irido/leucophores. It is well known that melanin in melanophores is synthesized by tyrosinase within a specific organelle termed the melanosome. Although sepiapterin reductase (SPR) is an important enzyme involved in metabolizing biopterin and sepiapterin (a conspicuous pteridine as a coloring pigment in xanthophores) the distribution of SPR has not been shown in pigment cells. An antibody raised in rabbits against rat SPR was used to demonstrate the presence of SPR in pigment cells of Oryzias latipes. This study, which used immunohistochemistry with fluorescence or peroxidase/diaminobenzidine as markers, revealed that SPR could be detected readily in xanthophores, but only faintly in melanophores. These results suggest that sepiapterin is metabolized within xanthophores. Moreover, these experiments show that a protein sharing immunological cross-reactivity with rat SPR is located in teleost O. latipes xanthophores, which is significant considering the relationship of pteridine metabolism between poikilothermal vertebrates and mammals. Further progress in investigations of the roles of pteridines in vertebrates will be promoted by using these fish which can be bred in mass rather easily in the laboratory. [source] Immunohistochemical localization of CNTFR, in adult mouse retina and optic nerve following intraorbital nerve crush: Evidence for the axonal loss of a trophic factor receptor after injuryTHE JOURNAL OF COMPARATIVE NEUROLOGY, Issue 2 2007Jill A. Miotke Abstract Ciliary neurotrophic factor (CNTF) is important for the survival and outgrowth of retinal ganglion cells (RGCs) in vitro. However, in vivo adult RGCs fail to regenerate and subsequently die following axotomy, even though there are high levels of CNTF in the optic nerve. To address this discrepancy, we used immunohistochemistry to analyze the expression of CNTF receptor , (CNTFR,) in mouse retina and optic nerve following intraorbital nerve crush. In normal mice, RGC perikarya and axons were intensely labeled for CNTFR,. At 24 hours after crush, the immunoreactivity normally seen on axons in the nerve was lost near the lesion. This loss radiated from the crush site with time. At 2 days postlesion, labeled axons were not detected in the proximal nerve, and at 2 weeks were barely detectable in the retina. In the distal nerve, loss of axonal staining progressed to the optic chiasm by 7 days and remained undetectable at 2 weeks. Interfascicular glia in the normal optic nerve were faintly labeled, but by 24 hours after crush they became intensely labeled near the lesion. Double labeling showed these to be both astrocytes and oligodendrocytes. At 7 days postlesion, darkly labeled glia were seen throughout the optic nerve, but at 14 days labeling returned to normal. It is suggested that the loss of CNTFR, from axons renders RGCs unresponsive to CNTF, thereby contributing to regenerative failure and death, while its appearance on glia may promote glial scarring. J. Comp. Neurol. 500:384,400, 2007. © 2006 Wiley-Liss, Inc. [source] Role of canine basal cells in postnatal prostatic development, induction of hyperplasia, and sex hormone-stimulated growth; and the ductal origin of carcinoma,THE PROSTATE, Issue 3 2001Irwin Leav Abstract Background The canine prostate has often been proposed as a model for abnormal growth of the human gland. Hyperplasia of the prostate is common in aging men and has been estimated to be present in 100% of old intact dogs. While prostatic carcinoma is common in older men, it appears to be rare in dogs and unlike the disease in humans, it occurs with relatively high frequency in castrated animals. Since basal cells are thought to be key participants in normal and abnormal growth of the human gland, we used immunohistochemistry to investigate the role that they may play in canine prostatic development, the evolution of hyperplasia and carcinoma, and the effects of sex hormones on these cells. Methods Prostate specimens were obtained at autopsy from seven sexually immature dogs, autopsy and biopsy samples from 14 sexually mature intact animals, from four castrates, and from19 dogs with prostatic carcinoma. In addition, we also studied the prostates from two intact dogs treated with 5,-dihydrotestosterone (DHT) for 6 months and two castrated dogs that were subsequently treated with 5,-androstane-3, diol and estradiol-17,, as well as specimens from two sexually ablated animals given DHT for 2 weeks. All specimens were immunostained for high molecular weight cytokeratin (HMC), pancytokeratin, androgen receptor (AR), and the proliferative marker KI-67. Results We find that basal cells are the major proliferative cell type in the neonatal and adult canine prostate and that the expression of HMC staining, which defines these cells, may be regulated by androgens. In the adult gland, ductal basal cells formed a contiguous layer, whereas those lining acini were discontinuous. Populations of both basal cell types were variably AR positive, but while HMC immunostaining was abolished in acinar cells following long-term castration, staining remained in ductal cell counterparts. Paralleling the histological development of hyperplasia, the acinar basal cell population increased with age and were the major cell type that expressed KI -67. In contrast, ductal basal cell populations did not expand in the prostates of older dogs and were seldom positively stained for KI -67. The numbers of HMC and KI-67-stained acinar basal cells were dramatically increased in the prostates of intact dogs treated with DHT when compared with glands of untreated controls. This was not the case with ductal basal cells. Androgens given alone or together with estrogen to castrated dogs induced widespread HMC and KI -67 immunostaining in both populations of basal cells. In addition, our results indicate that the majority of canine prostatic carcinomas likely arise exclusively from ductal epithelium. Only one of the 19 cases of carcinoma contained cells that expressed AR, which suggests that androgens may not be required for the initiation or progression of these cancers. Conclusions Our findings indicate that two biologically distinct populations of basal cells may exist in the canine prostate. In this regard, the age-related expansion of proliferating acinar basal cell populations, probably mediated by sex steroids, is a key factor in the pathogenesis of canine prostatic hyperplasia. Additionally, we find that prostatic carcinoma in the dog likely arises from ductal cells. Taken together, these findings may indicate that canine acinar basal cells and ductal epithelium have separate susceptibilities to factors that promote hyperplastic or neoplastic development. Prostate 48:210,224, 2001. © 2001 Wiley-Liss, Inc. [source] BS12 PREDICTIVE MARKERS FOR BREAST CANCER NEOADJUVANT CHEMOTHERAPYANZ JOURNAL OF SURGERY, Issue 2007S. Syed Purpose Although neoadjuvant chemotherapy (NACT) is routinely used in the management of breast cancer, there is no definitive way of predicting which patients are more likely to respond to a particular therapy. The aim of this study was to identify markers that can be used to predict tumor response to chemotherapy in breast cancer. Methodology We used immunohistochemistry to evaluate blood microvessel density (MVD) (CD31), tumor cell proliferation (Ki-67), anti-apoptotic marker (Bcl-2), ER and PR expression, and HER-2/neu expression in core biopsy samples (taken before chemotherapy) from patients with locally advanced breast cancer (n = 20), receiving neo-adjuvant chemotherapy {anthracycline-based regimen (FEC100) (n = 10) vs single agent taxane regimen (docetaxel) (n = 10), and correlated these factors with tumor response (as assessed clinically and by tumor imaging) after 4 cycles of treatment. Results Tumors expressing low levels of Bcl-2 showed significantly greater reduction in size to both taxane (P < 0.05) and anthracycline-based (P < 0.01) regimens, compared to tumors expressing high levels of Bcl-2. Further, HER-2/neu positive tumors showed significantly greater reduction in size to taxane regimen (P < 0.05), while estrogen receptor (ER) negative tumors showed a trend of greater reduction in size to anthracycline-based regimen (P = 0.06). Conclusions Bcl-2 and HER-2/neu expression may be useful markers to predict response to neoadjuvant chemotherapy in breast cancer. While subject numbers are still too low to draw firm conclusions, the current data indicates that HER-2/neu may specifically predict a positive tumor response to taxane regimen, and high Bcl-2 is a marker of chemoresistance. [source] The local immune response in ulcerative lesions of Buruli diseaseCLINICAL & EXPERIMENTAL IMMUNOLOGY, Issue 3 2006A. E. Kiszewski Summary Buruli disease (BU) is a progressive necrotic and ulcerative disease of the skin and subcutaneous tissue caused by Mycobacterium ulcerans. BU is considered the third most common mycobacterial disease after tuberculosis and leprosy. Three clinical stages of the cutaneous lesions have been described in BU: pre-ulcerative, ulcerative and healed lesions. In this study we used immunohistochemistry and automated morphometry to determine the percentage of macrophages and of CD4/CD8 lymphocytes and their expression of interferon (IFN)-,, interleukin (IL)-10, tumour necrosis factor (TNF)-, and transforming growth factor (TGF)-,. Expression of these cytokines was correlated with the inflammatory response evaluated by histopathology. All the studied BU ulcerative cases showed extensive necrosis and chronic inflammation. The most important feature was the presence or absence of granulomas co-existing with a mixed pro-inflammatory/anti-inflammatory cytokine balance. When granulomas were present significantly higher expression of IFN-, was seen, whereas in ulcerative lesions without granulomas there was increased expression of IL-10 and significantly higher bacillary counts. These features correlated with the chronicity of the lesions; longer-lasting lesions showed granulomas. Thus, granulomas were absent from relatively early ulcerative lesions, which contained more bacilli and little IFN-,, suggesting that at this stage of the disease strong suppression of the protective cellular immune response facilitates proliferation of bacilli. [source] | |||||||||||||