丁型病毒性肝炎的原位分子杂交及免疫组化研究

利用原位分子杂交和免疫组化方法对１４２例乙型肝炎活检肝组织进行丁型肝炎病毒ＲＮＡ及其抗原的定位研究。２８／１４２例丁型肝炎病毒标记阳性。其中慢性重症型６例；慢性活动性１７例；慢性持续性５例。慢性活动性乙肝重叠丁型肝炎病毒感染组发生早期肝硬变的比例明显高于无重叠感染组（Ｐ＜０．０５）。２８例丁型肝炎病毒感染肝组织１９例ＨＢｃＡｇ阳性，并以核浆型为主，提示活动性ＨＢＶ复制与ＨＤＶ感染的正相关性，两者相加作用导致肝损害加重并加速发展为肝纤维化。ＨＤＶＲＮＡ在肝细胞内大量蓄积，ＨＤＡｇ在碎屑状坏死边缘肝细胞或气球样变肝细胞内呈浆膜型分布，提示ＨＤＶ直接细胞毒在丁型肝炎发病学中的作用。     

Expression of interleukin-6 and monocyte chemoattractant protein-1 by peritoneal sub-mesothelial cells during abdominal operations

Cytokines and chemokines including interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1) are secreted in response to major abdominal operations. The aim of this study was to identify the peritoneal cells that produce IL-6 and MCP-1. Samples of peritoneal tissue were taken from patients at the beginning and end of major abdominal operations. The samples were incubated in culture medium on microtitre plates for 5 h. The concentrations of IL-6 and MCP-1 were measured in culture supernatants by enzyme-linked immunosorbent assay (ELISA). In paraffin sections, cells that expressed IL-6 or MCP-1 were identified by combined in situ hybridization and immunohistochemistry. Antibodies against CD68, CD34, actin, and calretinin were included in these experiments. The median production of IL-6 increased significantly from 6256 pg/ml at the start of the operation to 20,000 pg/ml at the end. Production of MCP-1 rose from 7700 pg/ml to 11,820 pg/ml. IL-6 mRNA was mainly confined to endothelial cells. MCP-1 was expressed by a broader range of cells, consisting of actin-positive smooth muscle cells and endothelial cells, fibroblast-like cells, as well as occasional macrophages and mesothelial cells. Peritoneal endothelial cells contribute to the transient increase in concentrations of IL-6 in the circulation after surgical trauma. Recruitment of monocytes to the site of the trauma seems to be mainly effected by actin-positive smooth muscle cells and endothelial cells.     

In situ localization of type III and type IV collagen-expressing cells in human diabetic nephropathy

Nodular intercapillary glomerulosclerosis is the most typical lesion of diabetic nephropathy (DN) and is characterized by increased extracellular matrix (ECM) and amorphous masses of mesangial matrix. The local exaggeration of these deposits results in the formation in the typical diabetic nodule. To clarify the composition of the ECM of sclerotic lesions in DN, we investigated the distribution of type III and type IV collagens and their mRNAs by immunohistochemistry and in situ hybridization, respectively. In normal renal tissues, there was no intraglomerular immunostaining for type III collagen, while strongly positive staining was found in the extraglomerular interstitum. Positive immunostaining for type IV collagen was also present in the mesangium, glomerular basement membrane (GBM), Bowman's capsule, and the vascular pole of the normal glomerulus. In DN, the nodular lesions were negative for type III collagen and strongly positive for type IV collagen. On the other hand, in the late stage of global sclerosis, both type III and type IV collagens were diffusely present in the sclerotic matrix. To determine the origins of these type III and type IV collagens in the sclerotic matrix, in situ hybridization was performed, utilizing thymine-thymine (T-T) dimerized synthetic oligonucleotides complementary to either proα(III) chain or pro α1 (IV) chain mRNAs as probes. The signals were detected by enzyme immunohistochemistry using an anti-T-T antibody. Intraglomerular cells (glomerular epithelial and mesangil cells) containing type III collagen mRNA were found in DN with sclerotic lesions, but not in normal glomeruli. At this stage of sclerosis, intraglomerular cells (mainly glomerular epithelial cells and infrequently mesangial cells) were positive for type IV collagen mRNA, but there were few positive cells in globally sclerotic glomeruli. This study provides evidence that both type III and type IV collagens are synthesized by intraglomerular cells during sclerosis and become significant constituents of the sclerotic matrix in DN.     

生物素标记探针原位杂交法检测细胞中c-myc基因的表达

本文采用生物素标记的c-mye基因作探针，通过细胞原位分子杂交技术，对人舌癌、膀胱癌和正常细胞中c-myc基因的转录量进行了定性和半定量分析。结果表明与正常细胞相比，舌癌和膀胱癌细胞中c-myc基因异常高度表达。     

Detection of enterovirus in the thyroid tissue of patients with graves' disease

The etiology and pathogenesis of Graves' disease (GD) are still unknown, although it is thought that both genetic and environmental factors are important. Some indirect evidence implies that a viral infection may be a possible etiologic factor in autoimmunity. The main objective of this study was to examine direct evidence of the presence of enteroviruses (EVs) in the thyroid tissue of patients with GD. Thyroid tissue from 22 patients with newly diagnosed GD was obtained by core needle biopsy, while tissue from 24 patients with chronic GD and 24 control subjects without any autoimmune thyroid diseases was collected during neck surgery. Formalin‐fixed, paraffin‐embedded thyroid tissue samples were examined for the presence of enterovirus capsid protein using immunohistochemistry and for enterovirus RNA using in situ hybridization. Enterovirus capsid protein was detected in 17 (37%) patients and in 4 (17%) control subjects (P = 0.103). Enterovirus RNA was identified in thyroid tissue from nine (20%) patients, but in none of the control subjects (P = 0.016). Eight (90%) of the nine virus RNA positive patients were also positive for enterovirus protein. This is the first study to analyze thyroid tissue for EVs, including patients with untreated, newly diagnosed GD. The results suggest that EVs are more frequently present in thyroid tissue of patients than controls. Further studies are indicated to explore this association to find out if a low‐grade chronic enteroviral infection might be involved in the pathogenesis of GD and if this could offer new therapeutic and preventive opportunities. J. Med. Virol. 85:512–518, 2013. © 2012 Wiley Periodicals, Inc.     

Detection of human herpes virus 6 in AIDS-associated retinitis by means of in situ hybridization,polymerase chain reaction and immunohistochemistry

The ubiquitous nature of HHV-6 and its genomic relationship with cytomegalovirus led us to evaluate an etiological link between HHV-6 and AIDS-associated retinitis in a prospective study. HHV-6 infection was studied in patients with AIDS-associated retinitis and in two control populations. Eye pairs were obtained at necropsy from nine patients with AIDS-associated retinitis, four human immunodeficiency virus (HIV)-seropositive patients with normal fundus examination and three HIV-seronegative patients. HHV-6 infection was detected by polymerase chain reaction (PCR), in situ hybridization and immunohistochemistry. Human cytomegalovirus (CMV) and HIV-1 infections were detected in parallel by the same methods. HHV-6 infection was detected in three cases of AIDS-associated retinitis. In two of these patients, HHV-6 infection was detected both by immunohistochemistry and PCR while in the third case it was detected by in situ hybridization and PCR. In the three patients, fundus examination showed bilateral retinitis in two of them and unilateral retinitis in one of them. HHV-6 infection was not detected in the retina of the two control groups. CMV was also detected in the three cases positive for HHV-6 by all three methods. HIV DNA was detected by PCR in two of three cases and was confirmed in one of these cases by in situ hybridization. These results confirm that HHV-6 infects the retina but suggests that HHV-6 does not have an exclusive causative role in AIDS-associated retinitis, since CMV coinfection of the retina was detected in all three of the patients positive for HHV-6. © 1996 Wiley-Liss, Inc.     

Detection of human papillomavirus deoxyribonucleic acid by filter in situ hybridization during pregnancy

Samples taken from 101 healthy pregnant women (49 over and 52 under the 20-week gestational period) and 108 healthy nonpregnant women were tested for human papillomavirus (HPV) types. Using 6, 11, 16, and 18 HPV DNA probes, 3-5 x 10(5) exfoliated cells scraped from the cervix were tested by filter in situ hybridization (FISH). Thirty-five of the pregnant women (34.6%) had evidence of the presence of HPV DNA: with 11.8% (12/101) HPV 6; 7.9% (8/101) HPV 11; 8.9% (9/101) HPV 16; and 5.9% (6/101) HPV 18 positivity. HPV DNA was detected in 20.4% (22/108) of the non-pregnant women. Compared with the healthy, nonpregnant group, the higher level of asymptomatic cervical HPV infection was mainly due to the accumulation of HPV 16 and 18 nucleic acids during the gestational period: with detection of HPV 16 in 8/49 cases (16.3%) and of HPV 18 DNA sequences in 4/49 (7.6%) cases. Screening 6-8 weeks after delivery indicated a decline of HPV positivity. Of the 4/12 HPV type 16 positive mothers, only one retained the presence of HPV 16 DNA, whereas neither of the 2/12 type 18 positive women reacted after birth with the type 18 radioactive probe.     

Detection and localization of human papillomavirus DNA in human genital condylomas by in situ hybridization with biotinylated probes

We have examined the distribution of human papillomavirus (HPV) DNA in paraffin sections of humans warts by in situ hybridization with biotin-labeled DNA probes. Recombinant plasmid DNAs (HPV-1, -6, -11, -16) were labeled by nick translation with biotinylated deoxyuridine triphosphate. Paraffin sections were hybridized with the probes for 18 h in stringent or non-stringent conditions, and DNA-DNA hybrids were detected by immunocytochemistry. Paraffin sections of warts were also examined for the presence of HPV capsid antigen with the avidin-biotin peroxidase complex method for immunocytochemistry. HPV DNA was detected and localized in paraffin sections from a plantar wart, a laryngeal papilloma, and seven anogenital condylomas. The specific HPV type present in each lesion was determined by hybridization under stringent conditions with the homologous DNA probe. The papillomas were found to contain many more cells with replicating virus DNA, as demonstrated by in situ hybridization, than was apparent from the number of cells containing detectable virus antigen. In situ hybridization with biotin-labeled probes is an effective technique for the identification of HPV infection in routinely collected and processed tissue specimens.     

Detection of human immunodeficiency virus type 1 RNA by in situ hybridization in oral mucosa epithelial cells from anti-HIV-1 positive patients

Several in vitro studies have shown that HIV-1 can infect CD4 negative epithelial cells of different origin including normal human oral keratinocytes, but whether this infection of mucosal epithelial cells occurs in vivo is still unclear. In this report, the presence and cell types infected by HIV-1 in paraffin embedded oral mucosa biopsies from 17 anti-HIV-1 positive patients have been examined by in situ hybridization and immunohistochemistry. As controls, oral mucosa biopsies from eight patients without HIV-1 infection markers were also analyzed. The results showed that 8 out of the 17 anti-HIV-1 positive patients had HIV-1 RNA detectable in plasma. Positive hybridization signals were observed in the mucosa biopsies from 14 of the 17 anti-HIV-1 patients (82.3%). The mean percentage of cells showing HIV-1 RNA was 2.64% +/- 1.77% (range: 1% to 5.5%). No differences in the mean percentage of HIV-1 infected cells were found between patients with and without HIV-1 RNA in plasma (3.01% +/- 1.57% vs. 3.4% +/- 1.27% respectively), or between untreated patients and patients under antiretroviral therapy (2.83% +/- 1.63% vs. 3.42% +/- 1.29% respectively). Immunohistochemical detection of S-100 antigen, cytokeratin and CD4 showed that hybridization signals appeared in cytokeratin positive cells and CD4 positive cells but not in S-100 positive cells. In conclusion, this study has demonstrated that HIV-1 infects and replicates in oral mucosa epithelial cells in vivo and that these cells could represent a reservoir of the virus that may escape to the currently used antiretroviral therapy.     

Regulation and production of IL-8 by human proximal tubular epithelial cells in vitro

A number of inflammatory kidney diseases are associated with interstitial nephritis and influx of leucocytes in the renal interstitium. Potentially the influx of neutrophils in the interstitium may be induced by the chemotactic cytokine IL-8. In the present study we have analysed the production of IL-8 by cultured human proximal tubular epithelial cells (PTEC) in response to a number of proinflammatory cytokines. Primary cell lines of proximal tubular epithelium obtained from ten different kidneys, and cultured under serum-free conditions, were found to produce IL-8 to different degrees from not detectable levels up to 10·8±1·5 ng IL-8 per 1×10⁵ cells in 72 h. Gel filtration chromatography of PTEC supernatant indicated that the size of IL-8 of PTEC is 15·1 and 8·1 kD, and is chemotactically active for polymorphonuclear neutrophils (PMN). Addition of 0·5 ng/ml rIL-1α or 1000 U/ml recombinant tumour necrosis factor-alpha (TNF-α) to the culture media of PTEC induced an up-regulation of IL-8 production up to 6·3-fold and 3·0-fold, respectively. The up-regulation by IL-1α and TNF-α was dose- and time-dependent. In contrast, 500 U/ml recombinant interferon-gamma (rIFN-γ) down-regulated the production of IL-8 3·4-fold. Northern blot analysis showed that IL-1α and TNF-α increased the expression of IL-8 mRNA, whereas IFN-γ reduced IL-8 mRNA expression. Taken together, these experiments indicate that human PTEC are a potential source of IL-8 in the kidney, and that IL-8 produced in the proximal tubule can be induced by various proinflammatory cytokines.     

Site of production of IGF1 in the normal and stimulated mouse thyroid

Insulin-like growth factor 1 (IGF1) has emerged as an essential factor in the follicular cell growth response in vitro to TSH, although its source within the thyroid in vivo is not clear. We have studied the localisation of IGF1 mRNA by in situ hybridization using digoxigenin labelled oligoprobes in tissue sections of mouse thyroid. Our results show that in the thyroid IGF1 mRNA is predominantly present in follicular cells and C cells rather than the stroma. Follicular cell levels are higher during postnatal thyroid growth and during the growth response to goitrogen administration, but decrease in the mature animal. This decrease in production is limited to the follicular cells, as IGF1 mRNA is still easily demonstrable in C cells and in the parathyroid. Immunocytochemistry for IGF1 peptide shows a weak and variable follicular cell content in both juvenile and mature mice, but a more uniform distribution during growth in response to a goitrogenic stimulus. These studies show that the follicular cells are the main source of IGF1 in the thyroid, and suggest that the role of IGF1 in follicular cell growth is as an autocrine factor.     

Detection and typing of human papillomaviruses by in situ hybridization with biotinylated oligonucleotide mixtures

The value of biotinylated Oligonucleotide probes for screening and typing by in situ hybridization of the most frequent genital human papillomavirus infections (HPVs 6,11,16,18, 31, and 33) was assessed. Optimal hybridization conditions were defined on a panel of paraffin-embedded tissue sections previously characterized with HPV full genome probes. Mixtures of oligonucleotides rather than single oligonucleotides were used to improve sensitivity and specificity. All HPV-positive specimens were detected by the screening mixture with a sensitivity and specificity similar to that of full genome probes. Typing mixtures were highly specific for each HPV type. This study confirms the potential of Oligonucleotide probes for detecting and typing HPV infections. © 1995 Wiley-Liss, Inc.     

In situ hybridization analysis of isodicentric X-chromosomes with short arm fusion

We present here an alternative approach to the study of mosaic cell lines containing dicentric chromosomes. The approach is based on chromosome-specific non-radioactive in situ hybridization with centromere (alpha satellite DNA) probes. The hybridization analysis may be used as an alternative to the C-band analysis, while at the same time to some extent replacing the Q-band analysis as well. The advantage of using in situ hybridization is mainly that it allows the very fast screening of a large number of metaphases. We illustrate this new application of the technique by using it for the analysis of two cases of isodicentric X-chromosomes. The approach is expected to be generally applicable, so that it may be applied to the scoring of other types of chromosomal mosaicism as well.     

应用荧光原位杂交技术检测肺癌患者染色体数目的改变

目的应用荧光原位杂交(FISH)技术研究肺癌患者染色体数目改变及其意义。方法用人的全套染色体特异探针与肺癌患者中期染色体进行杂交检测。结果肺癌患者细胞为异倍体,染色体数目以亚二倍体居多,常见2、5、7、8、10、11、14和17号染色体增多及1、4号染色体的丢失。结论 FISH技术可检测肺癌患者染色体数目的改变,肺癌遗传学变异主要是2、5、7、8、10、11、14、17号染色体的增加,并对肺癌的发生、发展和预后有一定的提示作用。     

Interleukin-6 production by thyroid epithelial cells. Enhancement by interleukin-1.

Interleukin-1 is a potent inhibitor of thyroglobulin and cAMP production in human thyroid cells and the inhibitory effect is enhanced by tumor necrosis factor-alpha and interferon-gamma. In the present study secondary cultures of human thyroid cells produced interleukin-6 and the production was significantly increased after exposure of the cells to recombinant interleukin-1 alpha and -1 beta. This increase was dose-dependent and concomitant of the IL-1 induced decrease in cAMP and thyroglobulin production. Both tumor necrosis factor-alpha and -beta also augmented interleukin-6 production, but less potently than interleukin-1. Interferon-gamma did not affect the production of interleukin-6. The rat thyroid cell line FRTL-5 produced interleukin-6 spontaneously, and the production was enhanced after addition of recombinant interleukin-1 beta. A pathogenetic role of interleukin-6 in autoimmune thyroid disease is suggested.     

An immunohistochemical and in situ hybridization study of c-myc and c-erbB-2 expression in primary human breast carcinomas

In previous studies of the expression and organization of proto-oncogenes in human breast a significant correlation has been found between amplification of c-myc and c-erbB-2 genes in carcinomas and poor short-term prognosis. Gene expression was estimated by analysis of total RNA from tissues, and similarly assessment of gene organization relied upon extraction of DNA from tissues. The present study has compared the expression of c-myc and c-erbB-2 mRNA as determined by in situ hybridization, and c-myc and c-erbB-2 protein expression detected by immunohistochemistry in a group of carcinomas for which there was knowledge of genomic organization and/or expression. Formalin-fixed, paraffin-embedded tissues of 38 carcinomas were assessed for the presence of c-myc protein, and 13 of these were examined for c-myc mRNA by in situ hybridization. Similarly processed tissue from 14 tumours was tested for c-erbB-2 protein using the antiserum 21N and ten of these carcinomas studied for c-erbB-2 mRNA localization. There was a good correlation between gene amplification, the presence of c-erbB-2 protein and mRNA: both the latter were detected in six of the seven carcinomas with an amplification but in none without. For some carcinomas there was a good correlation between c-myc protein and mRNA levels. Three carcinomas with gene amplification had a lower percentage of cells with detectable protein than showed hybridization for mRNA. Other carcinomas had a lower level of mRNA expression than protein. Neither approach could predict which carcinomas had amplification of the c-myc gene.(ABSTRACT TRUNCATED AT 250 WORDS)     

Detection of numerical alterations of chromosome 1 in cytopathological specimens of breast tumors by chromogen in situ hybridization

To investigate the effectiveness of chromogen in situ hybridization (CISH) in the diagnosis of breast tumors, numerical alterations of chromosome 1 were examined by CISH and fluorescence in situ hybridization (FISH) methods, and the presence of der(16)t(1;16) was also examined by FISH in imprinted cytology specimens from resected tissues of 14 carcinomas and five non-malignant lesions. The modal signal counts of chromosome 1 were compared between the specimens processed by CISH and FISH for each case. Aneusomies of the long arm of chromosome 1 were detected in 10 (71%) carcinomas as the major clones by both methods. In addition, one atypical papilloma demonstrated tetrasomy of 1q12 as a major clone by CISH, but such a clone was at first overlooked by FISH. Four other benign lesions showed disomic 1q12 signals as a major clone by both CISH and FISH. As additional information from FISH, eight cancers showed structural or numerical alterations of chromosome 16, and four showed der(16)t(1;16). In total, 10 carcinomas showed chromosome 16 alterations, and all of these overlapped with the carcinomas with 1q12 aneusomies. The CISH method provided almost the same results as the FISH method, and both methods were considered applicable in supportive diagnosis of cytological specimens of breast tumors. In addition, the CISH method was superior in the detection of numerical alterations in carcinoma cells by referring to the morphology of cells and in the detection of significant clones which might be missed under dark-field microscopy.     

Detection of the Epstein-Barr virus in primary gastric lymphoma by in situ hybridization

The Epstein-Barr virus (EBV) has been shown to be associated with numerous human malignancies including Burktt's lymphoma and nasopharyngeal lymphoepithelioma. In addition, some typical gastric adenocarcinomas were also recently reported to demonstrate EBV relevance. The present study was designed to detect EBV in primary gastric lymphoma, using the in situ hybridization (ISH) method, in which oligo-nucleotide probes for the EBERl RNA and the EBV DNA W region have been used. Of the 49 cases of primary gastric lymphoma studied, which all showed B cell immunopheno-type, EBER1 sequences could only be found in four cases, including two low-grade cases and two high-grade cases of histological subtypes while the number of positive cells was less than 50% of the tumor cells. In one case of low-grade mucosa associated lymphoid tissue (MALT) lymphoma, the EBER1 -positive neoplastic cells were found in the regional lymph node, but the primary site of the stomach showed no positive signals. The EBV presence was further confirmed by the EBV DNA ISH. Using the ISH method, rare or occasional positive lymphoid cells (probably non-tumorous bystander cells) could be detected in 10 other cases including all histological subtypes. The present study shows that only a small proportion of primary gastric lymphoma is associated with EBV, and such positive cases could be found in both high- and low-grade histological subtypes. It is also suggested that the EBV presence in the neoplastic cells of some cases of primary gastric lymphoma is most likely a secondary phenomenon.     

Detection and localization of an extra HLA locus in a karyotypically normal male by chromosomal in situ hybridization

The codominant expression of three HLA haplotypes was found in a healthy 21-year-old Black male, whose prometaphase karyotype was normal by light microscopy. He was the sibling of an antenatally diagnosed female fetus with a partial duplication of 6p. The duplication arose from a complex presumably balanced maternal chromosome rearrangement: 46,XX,dir ins(14;6)(14pter----14p11::6p22----6p21.1::14 p11----14qter; 6pter----6p22::6p21.1----6qter). Chromosomal in situ hybridization using a tritium-labeled genomic clone corresponding to a class I HLA gene revealed two sites of hybridization: at 6p21.3, the band to which this probe has been assigned in normal individuals (Morton et al. 1984a) and a second site at 6p11. We postulate that a recombinational event during meiotic pairing in the mother led to the reintroduction into the normal chromosome 6 homolog of a small segment of the original insertion in chromosome 14 which contained the HLA-A and -B determinants.     

荧光原位杂交检测 EWSR1基因易位在尤因肉瘤家族肿瘤疑难病例诊断中的应用

目的：探讨荧光原位杂交( fluorescence in situ hybridization, FISH)法在尤因肉瘤家族肿瘤( Ewing family tumor, EFT)石蜡包埋组织中检测EWSR1基因易位的可行性及其在临床病理学中的应用价值。方法收集4例EFT疑难病例,观察临床病理学特点,并采用EWSR1双色分离型探针检测EWSR1基因是否断裂。另以15例其他类型的软组织肿瘤作阴性对照。结果4例EFT 疑难病例的EWSR1基因均出现易位,对临床特点、病理学形态或免疫表型不典型的EFT 病例具有辅助诊断价值。结论 FISH 检测EWSR1基因易位可作为EFT 诊断的重要辅助依据,诊断时仍需结合病理形态学和免疫表型综合判断。