Transplantation-associated malignancies: restriction of human herpes virus 8 to Kaposi's sarcoma.

BACKGROUND: The human herpes virus 8 (HHV8) has been detected in all forms of Kaposi's sarcoma. HHV8 was also reported to be present in epithelial skin tumors of patients after renal transplantation, raising the question of the clinical relevance of HHV8 in transplant-related tumors. METHODS: Using a highly sensitive nested polymerase chain reaction assay, we analyzed for the presence of HHV8-DNA in the tumor tissue of renal transplant recipients with Kaposi's sarcoma (n=2) and non-Hodgkin's lymphomas (n=6), and in 32 tumors from 10 patients with multiple epithelial skin tumors. RESULTS: HHV8-DNA was detected in both cases of Kaposi's sarcoma but not in either the non-Hodgkin's lymphomas or the epithelial skin tumors. CONCLUSIONS: Our data confirm the association of HHV8 with Kaposi's sarcoma but not with other transplant-related tumors. Further studies are needed to analyze the risk for transmission of HHV8 by the donor and the possible exclusion of HHV8-positive patients as organ donors.

Detection of herpesvirus-like DNA by nested PCR on archival skin biopsy specimens of various forms of Kaposi sarcoma.

AIMS: To detect herpesvirus-like DNA sequences, defining a new herpesvirus, human herpesvirus 8 (HHV8), in paraffin wax embedded skin biopsy specimens of the various forms of Kaposi sarcoma. METHODS: DNA was extracted from archival skin biopsy specimens of Kaposi sarcoma, other mesenchymal skin tumours and various inflammatory skin lesions of HIV seropositive and negative patients. HHV8 DNA was detected by using a nested PCR assay. Human beta-globin DNA served as an internal control. RESULTS: Twenty two samples of Kaposi sarcoma were analysed, comprising 12 of the endemic type, nine HIV associated and one transplantation related. HHV8 DNA was detected by nested PCR in all forms of Kaposi sarcoma. By contrast, no HHV8 DNA was detected in five mesenchymal skin tumours or nine biopsy specimens of unspecific inflammatory skin lesions of HIV seropositive and negative patients. CONCLUSIONS: Detection of HHV8 DNA in paraffin wax embedded tissue can be used to confirm a diagnosis of Kaposi sarcoma.

Detection and typing of hepatitis C RNA in liver biopsies and its relation to histopathology.

This paper describes the correlation of hepatitis C genotypes detected in liver tissue with histological grading (inflammatory activity) and staging (degree of fibrosis/cirrhosis). The viral genotype was analysed by type-specific polymerase chain reaction (PCR) and correlated with histology and age of patients. In 69 patients with chronic hepatitis C (HCV) infection, genotypes 1a and 1b were detected in 13 (18.8%) and 31 (44.9%) liver biopsies, respectively. Genotypes 2a and 2b were each detected once (1.5%) and 12 (17.4%) tissue samples showed a mixed infection with two genotypes. In 11 (15.9%) biopsies, no genotype could be established. The liver specimens were grouped according to the presence or absence of genotype 1b: group A consisted of specimens infected with genotypes 1a, 2a, and 2b (n = 16), Group B contained biopsies infected with genotype 1b (n = 42), and group C were biopsies with no detectable genotype (n = 11). Activity (grade) of chronic hepatitis was not different in these three groups. However, advanced fibrosis/cirrhosis was observed in 16 (38.1%) biopsies in group B (containing genotype 1b), compared with none in group A (P = 0.01). The mean age of patients in group B was significantly higher than that in group A (P = 0.038), and the mean age of patients with advanced fibrosis was higher than that of patients with low fibrosis scores within these two groups (P = 0.004). Stepwise logistic regression revealed an independent association of age and genotype 1b (group B) with advanced fibrosis/cirrhosis. These data indicate that patients infected with genotype 1b have an higher risk of developing cirrhosis than do patients with other genotypes.

Huntingtin inclusions do not down-regulate specific genes in the R6/2 Huntington's disease mouse.

Transcriptional dysregulation is a central pathogenic mechanism in Huntington's disease (HD); HD and transgenic mouse models of HD demonstrate down-regulation of specific genes at the level of mRNA expression. Furthermore, neuronal intranuclear inclusions (NIIs) have been identified in the brains of R6/2 mice and HD patients. One possibility is that NIIs contribute to transcriptional dysregulation by sequestering transcription factors. We therefore assessed the relationship between NIIs and transcriptional dysregulation in the R6/2 mouse, using double-label in situ hybridization combined with immunohistochemistry, and laser capture microdissection combined with quantitative real-time PCR. There was no difference in transcript levels of specific genes between NII-positive and NII-negative neurons. These results demonstrate that NIIs do not cause decreases in D2, PPE and PSS mRNA levels in R6/2 striatum and therefore are not involved in the down-regulation of these specific genes in this HD model. In addition, these observations argue against the notion that NIIs protect against transcriptional dysregulation in HD.

[Detection and typing of hepatitis C RNA in liver biopsies in comparison with histopathology]. / Nachweis und Typisierung von Hepatitis C RNA im Lebergewebe im Vergleich zur Histopathologie.

Total RNA of 55 frozen liver biopsies were extracted and tested for the presence of HCV RNA and the genotype by RT-PCR using primers of the 5' non-coding region and a type specific primer set for HCV genotyping. In paralell, the activity of chronic hepatitis, the stage of fibrosis as well as chracteristic features of chronic hepatitis C were evaluated by conventional histology. HCV RNA was detected in 49 (89.1%) of 55 liver specimen by either primer set and genotyping was successful in 42 (76.4%) of liver biopsies. The samples were divided in 3 groups: Group A consisted of specimens infected with genotypes 1a, 2a and 2b (n = 13), Group B contained biopsies infected with genotype 1b (n = 24) and Group C were biopsies with two or no detectable genotype. The histology showed a significant higher degree of fibrosis/cirrhosis in Group B (genotype 1b) compared to Group A (11/24 vs. 0/13, p = 0.011). In addition, an advanced fibrosis/cirrhosis was found more often in Group C than B, however, this difference was not significant (5/15 vs. 0/13, p = 0.072). No difference was seen between the three groups with respect to the activity of chronic hepatitis, presence of lymphoid follicles, bile duct lesions or steatosis. We conclude that HCV RNA can readily be detected and typed in frozen liver tissue. Patients infected with HCV genotype 1b have an increased risk developing liver fibrosis and cirrhosis.

[Detection of human herpesvirus 8 (HHV 8) in Kaposi sarcomas of the gastrointestinal tract]. / Nachweis von humanem Herpesvirus 8 (HHV 8) in Kaposi-Sarkomen des Magen-Darm-Traktes.

Involvement of Kaposi's sarcoma in the gastrointestinal tract is common in AIDS patients. The disease is, however, usually asymptomatic and, due to the tumor growth primarily in the submucosa, biopsy diagnosis is possible in under 25%. The recently described human herpes virus 8 (HHV8) is closely associated with all forms of Kaposi's sarcoma. Detection of HHV8 in the tissue samples may therefore improve the diagnosis of gastrointestinal Kaposi's sarcoma. In the present study we analyze autopsy samples of tumor and non-tumor tissue from the gastrointestinal tract in patients with and without Kaposi's sarcoma for the presence of HHV8 DNA using a nested polymerase chain reaction (PCR) assay. HHV8 DNA was present in all 15 tissues with Kaposi's sarcoma. In contrast, HHV8 DNA was present only in 3 (18.8%) of 16 gastrointestinal tissues of patients with Kaposi's sarcoma but without histologically detectable tumor. No HHV8 DNA was present in 15 tissue samples of AIDS patients without Kaposi's sarcoma. Our data show that detection of HHV8 DNA using a nested PCR assay is a highly sensitive and specific diagnostic test for Kaposi's sarcoma in autopsy tissue samples from the gastrointestinal tract. It should therefore be possible to use detection of HHV8 DNA in biopsy material as an assay for the diagnosis of Kaposi's sarcoma.

Distribution of human herpesvirus 8 DNA in tumorous and nontumorous tissue of patients with acquired immunodeficiency syndrome with and without Kaposi's sarcoma.

Human herpesvirus 8 (HHV-8) DNA is present in virtually all Kaposi's sarcomas (KSs). Conflicting results, however, exist with respect to the presence of HHV-8 in nontumorous tissue samples. To define the specificity and predictive value of HHV-8 DNA detection in KS, we analyzed autopsy-derived tissue samples from patients with acquired immunodeficiency syndrome (AIDS) with and without KS for the presence of HHV-8 DNA, using single-step and nested polymerase chain reaction. Semiquantitative analysis of HHV-8 DNA was performed by endpoint dilution assays. HHV-8 DNA was detected in 41 (100%) tumor tissue samples of KSs. According to nested polymerase chain reaction results, HHV-8 DNA was also present in 16 (32%) of 50 nontumorous specimens of patients with AIDS patients with KS and in 3 (2.7%) of 113 specimens of patients with AIDS without KS; it was absent in 26 autopsy tissues and 15 transurethral resected prostatic specimens of patients without AIDS. By use of a second, unrelated primer set, the presence of HHV-8 DNA was confirmed in 12 (63.2%) of 19 nontumorous samples and detected in another 6 (17.7%) of 34 samples tested. Significantly higher titers of HHV-8 DNA were found in tumorous than in nontumorous tissues samples (1.9 x 10(4) vs. 1.2 x 10(2); P < .05). Specificity and positive predictive values for the diagnosis of KS by detecting HHV-8 DNA in a given tissue sample were 56 and 65.1% in patients with manifest KS and 97.4 and 100% in patients without previously known KS. An increased specificity and a positive predictive value were observed when the presence of KS anywhere in a given patient was considered (92.9 and 77.8%, respectively). In conclusion, the detection of HHV-8 DNA is a sensitive test for the diagnosis of KS. Its specificity, however, might be lower because HHV-8 can be detected in histologically unaffected tissue of patients with KS.

Detection of hepatitis B and C viruses in liver tissue with hepatocellular carcinoma.

Polymerase chain reaction was used to investigate the presence of the hepatitis B and C viruses in liver tissue from Taiwanese patients with hepatocellular carcinoma by examining paired samples (tumor and non-tumor) from 38 cases. We used a DNA-polymerase chain reaction protocol with primers spanning the regions of the hepatitis B virus genome corresponding to HBs, HBc, and HBx genes and RNA-polymerase chain reaction protocol with primers spanning the 5' untranslated region of the hepatitis C virus. Co-infection with hepatitis B and hepatitis C viruses was seen in nine patients (23%), only three of whom had anti-hepatitis C virus in serum. One of these three was HBsAg-negative in serum while the other two and four of the other six from this group were HBsAg-positive. One of the patients with anti-HCV and no HBsAg in serum had no hepatitis C virus-RNA in liver tissue, while hepatitis B virus-DNA was detectable by using the HBc and HBx specific primers. We detected hepatitis C virus as a single agent in the liver in only one patient. This patient was anti-HCV positive and HBsAg-negative. The remaining 27 patients (71%) had infection with hepatitis B virus only. Twenty-five of 27 patients had HBsAg in their sera. HBs-specific primers detected hepatitis B virus-DNA in non-tumor tissue from 23 patients and in tumor tissue from 25 patients. HBc-specific primers detected hepatitis B virus-DNA in non-tumor tissue from 24 patients and in tumor tissue from 20 patients. Finally, HBx-specific primers detected hepatitis B virus-DNA in non-tumor tissue from 24 patients and in tumor tissue from 25 patients. These data indicate that in a hyperendemic area, hepatitis B virus is closely associated with the development of hepatocellular carcinoma but that infection with hepatitis C virus may play a secondary role.

Hepatitis B X-gene expression in hepatocellular carcinoma.

A RNA-PCR method and different sets of primers were used to investigate the expression of different hepatitis B (HB) virus genes at the RNA level. We tested paired samples (tumor and non-tumor) from the liver tissues of 48 Taiwanese patients with primary hepatocellular carcinoma (HCC). By using a set of primers which spanned the sequences of the S-gene, we found expression in only 2 patients. In one HBs-RNA was only detected in the tumor tissue and in the other only in the surrounding non-tumor tissue. Using primers covering the C-gene, expression was found in 7 patients. In 2 of these RNA was detected in both the tumor and the surrounding tissue, in 2 in the tumor tissue, and in 3 in the surrounding tissue only. Finally, when primers spanning the X-gene sequences were used, RNA was detected in 40/48 patients. In 33 of these cases HBx-RNA was detected in both tumor and non-tumor tissue, in 3 patients in tumor tissue only, and in 4 in the surrounding tissue. Among the cases in which HBc and HBs-RNA was expressed, all showed HBx expression also. These data indicate that the expression of the HBx gene in HCC may play an important role in hepatocarcinogenesis.

Detection of herpesvirus-like DNA in the bronchoalveolar lavage fluid of patients with pulmonary Kaposi's sarcoma.

Pulmonary involvement is a clinically important form of visceral Kaposi's sarcoma in immunocompromised patients. Recently, herpesvirus-like deoxyribonucleic acid (DNA) sequences, defining a new herpesvirus termed "human herpesvirus 8" (HHV8) or "Kaposi's sarcoma-associated herpesvirus" (KSHV), were detected in Kaposi's sarcoma of acquired immune deficiency syndrome (AIDS) and non-AIDS patients. We describe the successful detection of HHV8 DNA in the bronchoalveolar lavage (BAL) fluid of patients with pulmonary Kaposi's sarcoma. Three immunocompromised patients, two HIV seropositive and one after kidney transplantation, suffered from respiratory symptoms and showed pulmonary infiltrates on chest radiography after development of biopsy proven Kaposi's sarcoma of the skin. Bronchoscopy revealed the typical Kaposi like livid endobronchial lesions. BAL fluid was analysed for the presence of HHV8 DNA using a nested polymerase chain reaction (PCR) assay. HHV8 DNA was detected in the BAL fluid of all three patients. In addition, HHV8 DNA could be detected in the skin biopsy tissue, lymph node, and peripheral blood mononuclear cells of these patients. Our data show that human herpesvirus 8 deoxyribonucleic acid can be detected in the bronchoalveolar lavage fluid of patients with pulmonary Kaposi's sarcoma. If further studies reveal a high specificity for human herpesvirus 8 deoxyribonucleic acid detection, this test will improve the tools for the diagnosis of pulmonary Kaposi's sarcoma without further need of biopsies.

Diagnosis of pulmonary Kaposi's sarcoma by detection of human herpes virus 8 in bronchoalveolar lavage.

Human herpes virus 8 (HHV8) DNA has recently been detected in sarcoma tissue of patients with Kaposi's sarcoma. HHV8 DNA could also be found in bronchoalveolar lavage (BAL) fluid of patients with tracheobronchial Kaposi's sarcoma. To determine the specificity, sensitivity and predictive values of HHV8 DNA detection in the BAL for the diagnosis of pulmonary Kaposi's sarcoma, 100 consecutive BAL were prospectively analyzed for the presence of HHV8 DNA using a nested PCR assay. In addition, 19 BAL samples of 14 AIDS patients with cutaneous or visceral Kaposi's sarcoma were retrospectively investigated. The prospective group consisted of 79 BAL performed in immunocompromised and of 21 BAL in nonimmunocompromised patients. Four patients of the prospectively analyzed group undergoing six BAL showed tracheobronchial Kaposi's sarcoma at five bronchoscopies. All of the five BAL samples performed in these patients with endoscopically visible Kaposi's sarcoma were positive for HHV8 DNA. Following chemotherapy and antiretroviral treatment tracheobronchial Kaposi's sarcoma was no longer detectable at a subsequent bronchoscopy and HHV8 DNA in BAL became negative in one patient. One BAL sample of a HIV-positive patient with no evidence of Kaposi's sarcoma was HHV8 DNA-positive. The sensitivity, specificity, positive and negative predictive values of HHV8 detection for the diagnosis of tracheobronchial Kaposi's sarcoma were 100%, 98.9%, 83.3%, and 100%, respectively. Twelve of 19 BAL samples of the retrospective group were HHV8 DNA-positive. In this group, 10 patients undergoing a total of 14 BAL suffered from pulmonary Kaposi's sarcoma. HHV8 DNA was documented in 10 of these 14 BAL samples. In three BAL of this group HHV8 DNA was positive, but pulmonary Kaposi's sarcoma was diagnosed at a later stage. In conclusion, the detection of HHV8 DNA in BAL is restricted to patients with Kaposi's sarcoma and is highly sensitive and specific for pulmonary involvement of Kaposi's sarcoma.

[Detection of herpes virus-like DNA in various forms of Kaposi's sarcoma but not in other mesenchymal tumors or inflammatory changes in skin]. / Nachweis von Herpes-Virus ähnlicher DNA in den verschiedenen Formen des Kaposi Sarkoms aber nicht in anderen mesenchymalen Tumoren oder entzündlichen Veränderungen der Haut.

Recently, herpes-virus like DNA sequences defining a new herpes virus termed human herpes virus 8 (HHV8), were detected in Kaposi's sarcoma of AIDS and non-AIDS patients. We describe the successful detection of HHV8 DNA in archival skin biopsies of the various forms of Kaposi's sarcoma. DNA was extracted from archival skin biopsies of Kaposi's sarcoma, other mesenchymal skin tumors and various inflammatory skin lesion of HIV seropositive and negative patients. The extracted DNA was analyzed for the presence of HHV8 DNA using a nested PCR assay. All samples were tested for the presence of appropriate DNA using a internal cellular control PCR-reaction. A total of 23 Kaposi's sarcoma were analyzed, including 12 of the endemic type, 9 HIV-associated and 2 transplant related. HHV8 DNA was detected by nested PCR in all forms of Kaposi's sarcoma. In contrast, no HHV8 DNA could be found in 17 mesenchymal, especially vascular skin tumors or in 7 biopsies with unspecific inflammatory skin lesions of HIV seropositive and negative patients. HHV8 DNA was present in all forms of Kaposi's sarcoma tested but not in other mesenchymal tumors or unspecific inflammatory lesions of the skin. This data support the idea of a strong association of HHV8 and Kaposi's sarcoma.

Binding of isolated 3T3 surface membranes to growing 3T3 cells and their effect on cell growth.

We have quantitated by autoradiography the binding of [125I]labeled 3T3 plasma membrane fragments to 3T3 cells growing on the surface of plastic dishes; ie, the same conditions in which these membranes specifically arrest the growth of 3T3 cells early in the G1 phase of the cell cycle. We have been able to demonstrate that binding of membranes to cells is coincidental with the expression of the growth inhibitory activity of protein(s) present in the membrane fragments. Treatments that reduce binding (heat denaturation of the membranes or culture in the presence of high serum) also reduce growth inhibitory activity. [125I]labeled membranes bound to cells are located primarily on the cell surface (as determined by electron microscope autoradiography) and are exchangeable with unlabeled membranes. We conclude that binding of membranes to cells is necessary but may not be sufficient for the expression of the growth inhibitory activity of these membranes. This approach provides information not only on the average level of binding of membranes to cells, but also provides a quantitative assessment of the variation of the level of membrane to cell binding between different cells in the population.

A serine protease activity in C3H/10T1/2 cells that is inhibited by anticarcinogenic protease inhibitors.

Several different protease inhibitors have the ability to suppress transformation in vitro and carcinogenesis in vivo. The mechanism(s) by which protease inhibitors suppress carcinogenesis, however, is not fully understood. Presumably, these agents inhibit one or more intracellular proteases whose functions are essential for the induction and/or expression of the transformed phenotype. We have isolated an endopeptidase activity capable of hydrolyzing the substrate Boc-Val-Pro-Arg-MCA (Boc = butoxycarbonyl; MCA = 7-amino-4-methylcoumarin) from C3H/10T1/2 mouse embryo fibroblast cells. This intracellular protease was inhibited by the soybean-derived Bowman-Birk inhibitor (BBI), chymostatin, and L-1-tosylamido-2-phenylethyl chloromethyl ketone, all of which have anticarcinogenic activity, but was unaffected by soybean trypsin inhibitor, which lacks anticarcinogenic activity. Other protease inhibitors affected the proteolytic activity to an extent that correlates with their relative ability to suppress transformation in vitro. The enzyme has a mass of about 70 kDa, contains a single subunit, and exhibits maximal activity at pH 7.0. Diisopropyl fluorophosphate covalently binds to this enzyme and blocks its activity, indicating that the enzyme is a serine protease. We have previously demonstrated that several protease inhibitors are effective suppressors of radiation-induced transformation of C3H/10T1/2 cells. Since these agents reduce the Boc-Val-Pro-Arg-MCA-hydrolyzing activity to an extent that correlates with their ability to inhibit malignant transformation in vitro, this endopeptidase activity may be a cellular target of the anticarcinogenic protease inhibitors.