A role for a new herpes virus (KSHV) in different forms of Kaposi's sarcoma.

Kaposi's sarcoma (KS) is a previously rare, tumour-like lesion of controversial biological nature. KS has since the early 1980s become frequent in patients with AIDS, particularly in homosexuals. KS is also endemic in Central Africa predominantly in otherwise healthy men but also in women and children. Recently, evidence for the presence of novel, herpes virus DNA sequences in more than 90% of AIDS Kaposi lesions (AKS) was presented. This DNA was identified using representational difference analysis (RDA) generating short, unique sequences with variable homology to several herpes virus, but no intact virus was recovered. If these DNA-sequences are also present in other, non-HIV-associated forms of Kaposi's sarcoma this would strongly suggest a specific, aetiopathological involvement of this putative new herpes virus in the pathogenesis of Kaposi's sarcoma, rather than a contamination of yet another opportunistic virus in immunosuppressed AIDS patients.

PIP: Samples were examined by polymerase chain reaction (PCR) for the presence of the putative Kaposi's sarcoma herpes virus (KSHV). KS DNA from HIV-negative, African, endemic (EKS) samples, and epidemic HIV-positive KS (AKS), and sporadic KS (SKS) samples were tested from Tanzania and Sweden. All of the HIV KS (18 African EKS and 4 Swedish SKS) as well as the HIV-positive AIDS-related KS (16 African and 7 Swedish AKS) biopsies were shown to contain the previously described DNA sequences. KS lesions from children, females, and males in various tissues were analyzed including skin, lymph nodes, gut and oral mucosa. All forms of KS showed a single PCR product of the expected size (233 base pairs). To exclude amplification of other types of herpes virus, virus preparations of Epstein-Barr virus (EBV), herpes simplex virus, cytomegalovirus, vesicular stomatitis, and human herpes virus type 6 (HHV6) were assayed, again by PCR, using the KSHV primers. No PCR products were obtained with any of these virus strains. However, most HIV-positive and HIV-negative KS DNA samples also contained either EBV and/or HHV6 sequences. All biopsies from non-KS tissues (cells) of HIV-positive and HIV-negative individuals were consistently negative for KSHV by PCR. The observation that the same herpes virus-like DNA sequence is present in endemic and sporadic, as well as AIDS-related, Kaposi's sarcoma cases suggests a possible pathogenic association between this putative novel, herpes-like virus and KS. The herpes virus-like DNA sequences described by Y. Chang in 1994 may indeed represent a novel herpes (KSHV), etiopathologically associated with various clinical forms of Kaposi's sarcoma. Its pathogenic importance is indicated by its presence in different KS tissues with various clinical types of KS and its absence from non-KS-involved tissues. Furthermore, the presence of KSHV in KS of children suggests a nonsexual mode of transmission.

Clinical features and predictive markers of disease progression in cynomolgus monkeys experimentally infected with simian immunodeficiency virus.

OBJECTIVE: To study the pathogenicity of simian immunodeficiency virus (SIVsm) in cynomolgus monkeys in order to establish an animal model for human AIDS. METHODS: Thirty-three cynomolgus monkeys were monitored for more than 2 years following experimental infection with SIVsm. RESULTS: All the macaques became SIV-infected, as demonstrated by virus recovery from peripheral blood lymphocytes and by the appearance of viral antibodies. SIVsm was found to be pathogenic, killing 29 out of the 33 monkeys (88%) within 26 months. Clinically, infected monkeys developed lymphadenopathy, splenomegaly, diarrhoea, weight loss, neurological symptoms and a remarkably high incidence (39%) of malignant lymphomas. All lymphomas were high-grade malignant and of B-cell origin. Disease progression was associated with low CD4+ lymphocyte count, involution of initially hyperplastic follicular B-cell areas in lymph nodes, reappearance of viral antigen in serum, loss of anti-Gag antibodies and development of systemic giant cell disease in 55% of the monkeys. CONCLUSIONS: There are many similarities between SIVsm-induced AIDS in cynomolgus monkeys and human AIDS with regard to clinical, virological, immunological and pathological manifestations.

Spindle cell ploidy and proliferation in endemic and epidemic African Kaposi's sarcoma.

Comparative studies of ploidy and proliferative activity of spindle cells in sections of 20 (skin, 17; lymph node, 3) biopsy specimens from African patients, 10 with endemic Kaposi's sarcoma (EKS) and 10 with AIDS-associated Kaposi's sarcoma (AKS) were performed by histopathology, feulgen-based DNA measurement and proliferating cell nuclear antigen (PCNA)/cyclin immunohistochemistry, respectively. All specimens were classified as nodular lesions with basically the same histology. In 17 cases immunostained for cyclin/PCNA, the percentage of proliferating spindle cells range between 2-18, with a higher mean rate in AKS although this was not statistically significant. In situ measurement of DNA showed no significant values greater than the diploid level of control cells indicating that spindle cells in both EKS and AKS have euploid DNA content. Our findings indicate that both EKS and AKS represent the same type of euploid low rate cell proliferations. This corroborates previous suggestions that KS could represent a reactive process to yet undefined stimulus rather than a clonal proliferation, of transformed malignant cells.

A histological and immunohistological study of malarial placentas.

Sections of 18 malaria-infected placentas were stained with haematoxylin and eosin, periodic acid and methenamine silver, and immunohistochemically with monoclonal antibodies against human common leukocyte antigen, CLA (CD 45), B cells (CD 20, L 26), T cells (CD 45RO, UCHL-1) and collagen IV. Parasitized erythrocytes accumulated in the maternal villous spaces, with none in the foetal circulation. These were found in association with inflammatory leukocytes and pigments. Fibrinoid necrosis was more prevalent in the heavily infected placentas. Thickening and reduplication of foetal capillary basement membranes, and a decrease in leukocytes, including B and T cells, were seen in the heavily infected placentas. These findings are in keeping with previously reported depression of cellular and humoral immunity in patients with heavy parasitaemia.

Kaposi's sarcoma before and during a human immunodeficiency virus epidemic in Tanzanian children.

BACKGROUND: With the onset of AIDS increased frequency of Kaposi's sarcoma (KS) has been reported. However, there is no case-based comparison of childhood (<14 years) KS before and during the HIV pandemic in sub-Saharan Africa. Here we report on the Tanzanian cancer registry data of pediatric KS in Tanzania and implications with regard to pathogenic factors. METHODS: One hundred fifty histologically confirmed pediatric KS (PKS) cases registered during 1968 through 1995 (28 years) were analyzed with regard to demographic and clinical characteristics before and during the AIDS epidemic. Statistical analysis was done with the Epi-Info program and chi square test. RESULTS: Of children with PKS 126 (84%) were male and 24 (16%) were female. The gender ratio was 5.1:1 and 5.4:1 during the endemic and epidemic periods, respectively. The highest occurrence of PKS was observed in the 0- to 5-years age group. Overall 73 (4.9/year) of these cases were registered during the pre and 77 (5.9/year) during the AIDS period. Over time a significant increase in anatomically disseminated KS cases was evident during the AIDS epidemic (P = 0.003). CONCLUSIONS: These observations indicate that children younger than 5 years are at high risk for developing KS, possibly reflecting low resistance to human herpesvirus (HHV) 8 infection. It is also likely that an increased susceptibility to HHV8 infection and morbidity is related to progressive immunodeficiency. The increase in AIDS PKS incidence appears to reflect a direct or indirect promoting effect of HIV on the development of KS lesions. Recognition of the high KS risk in small children warrants considerations of possible prevention measures including HIV/HHV8 vaccination and therapeutic options.

Immunological profile of endemic and epidemic Kaposi's sarcoma patients in Dar-es-Salaam, Tanzania.

Kaposi's sarcoma (KS) presents in four clinicopathological types namely classical/sporadic (CKS), endemic African (EKS), iatrogenic (IKS) and that associated with AIDS (AKS). Recently a putative herpes virus (HHV-8) was described and shown to be present in all four types of KS. The immunological status of patients with EKS has been conflicting. In this study total leucocyte counts, total lymphocyte counts and lymphocyte subsets of patients with EKS and AKS were determined by flow cytometry and compared to those of healthy HIV-1 seronegative controls. Results show that 50% of EKS lesions were of nodular type. Patients with EKS had significantly lower levels of CD4+ T- lymphocytes and CD4:CD8 ratio but significantly higher CD8+ T-lymphocytes compared to controls. Patients with AKS had significantly lower levels of CD4+ T-lymphocytes and also CD4:CD8 ratios but significantly higher percentage of CD8+ T-lymphocytes when compared with EKS patients. These findings indicate that in both forms of KS there is a certain degree of immunological disturbance which is more conspicuous in AKS because of HIV infection and suggests that HIV-1 acts synergistically with the aetiological agent (HHV-8) to cause a more aggressive type of KS.

Breast cancer before and during the AIDS epidemic in women and men: a study of Tanzanian Cancer Registry Data 1968 to 1996.

Kaposi's sarcoma and malignant lymphoma are two cancers that are most often associated with human immunodeficiency virus (HIV) infection. Recently, other cancers, including cervical cancer, have been associated with AIDS. The role of HIV in the pathogenesis of these malignancies is not well understood, and few studies have been done to determine any general increase in cancers after the onset of the HIV epidemic. This study compared breast cancer before and during the AIDS period by studying the total Tanzanian Cancer Registry data (1968 to 1996). The mean age among males increased from 50.88 to 52.63 years (p = 0.45) and among females decreased from 44.79 to 43.23 years (p = 0.005) before and during the AIDS epidemic, respectively. A statistically significant decrease in the incidence of breast cancer was observed during the AIDS epidemic period in both males (p = 0.001) and females (p = 0.021). The male-to-female ratio widened significantly from 0.09:1 to 0.03:1 (p = 0.0001). Further studies are needed to determine the incidence and observed changes of different cancers, including breast among patients with HIV/AIDS.

Paediatric malignancies in Tanzania.

Using the Tanzania Cancer Registry, the pattern of childhood malignancies up to the age of 15 years over a period of 22 years (1973-1995) was analysed. Lymphomas were most frequent (38.9%) followed by soft tissue sarcomas (13.1%), retinoblastomas (11.1%) and squamous cell carcinoma (6.7%). The frequency of soft tissue sarcomas is relatively high but appears not to be related to human immunodeficiency virus (HIV) infection pandemic. The high frequency of squamous cell carcinoma also needs further investigation.

Monocyte/macrophage giant cell disease in SIV-infected cynomolgus monkeys.

A non-opportunistic, generalized giant cell disease (GCD) was found in 12 out of 25 (48%) cynomologus monkeys infected with SIVsm. Most organs were affected notably the lymph nodes (LN), spleen, gut, liver, lungs and CNS. The multinucleated GC varied considerably in cell size and in the number and cytoplasmic distribution of the nuclei. Immunohistochemically most GC expressed SIV antigens and markers of mononuclear phagocytes (CD68), CD4 and also occasionally the T-cell markers CD45RO, CD43 and CD2. Monkeys with GCD had more pronounced immunosuppression with lower CD4-cell counts, more often demonstrable SIV antigen in the blood and LN and had been infected for a longer time period, as compared to monkeys without GCD. These findings show that SIV infection in cynomolgus monkeys is frequently associated with extensive formation of multinucleated GC of macrophage origin, which appears to be related to the pathogenesis of the infection and the degree of immunosuppression.

Heterogeneity of spindle cells in Kaposi's sarcoma: comparison of cells in lesions and in culture.

The immunophenotype of spindle cells in epidemic, endemic, and classic (sporadic) Kaposi's sarcoma (KS) lesions was defined by the demonstration of various cell markers and compared with that of KS-derived cell lines. No significant histological or immunophenotypic differences were observed between the three clinical types of KS at comparable stages. The spindle-cell compartment of the different KS types was composed predominantly of a mixture of proliferating CD45+/CD68+ bone-marrow-derived monocytes and TE7+/collagen+ fibroblastic cells with varying expression of EN4/PAL-E/CD31/CD34/CD36 endothelial-associated antigens and/or smooth-muscle-specific alpha-actin (alpha-actin). The latter cells appeared to represent transitional forms of fibroendothelial and fibromyocytic cells. The in vitro cultured KS-derived cell lines (KS-3, KS-6, and KS-8) expressed the fibroblastic antigen TE7 and smooth-muscle-specific alpha-actin but not leukocytic or endothelial-associated antigens consistent with the phenotype of fibromyoid spindle cells of primary lesions. Neither HIV antigen nor provirus DNA was demonstrable in the epidemic KS lesions. The observed heterogeneity of the spindle-cell compartment further substantiates the view that Kaposi's sarcoma, irrespective of clinical setting, expresses salient features more compatible with reactive, tumor-like lesion than clonal sarcoma.

Expression of adhesion molecules in endemic and epidemic Kaposi's sarcoma.

Spindle cells and vascular endothelium in nodular lesions of AIDS associated (epidemic) and endemic Kaposi's sarcoma showed similar immunohistochemical patterns of expression for cell adhesion molecules and extracellular matrix proteins. Spindle cells as well as endothelium also expressed both alpha 5 and alpha V integrin subunits and ICAM-1 suggesting a possible role for inflammatory cytokines in spindle cell formation. The spindle cell compartment was rich in collagen, laminin, fibronectin and tenascin suggesting an important reactive component in the evolution of Kaposi's sarcoma. The lack of thrombospondin expression in the spindle cells favours the contention that they could be transitional, proliferating cells of endothelial origin. Specific expression of tat protein was not seen suggesting minimal if any HIV replication in these lesions. Our findings suggest similar histopathogenetic mechanisms for endemic and epidemic Kaposi's sarcoma. The clinically more malignant features of most AIDS related cases may reflect an important effect of systemic and focal cytokines in HIV patients and possibly other cofactor(s), i.e. tat protein in the induction and growth of the lesions.

Induction of intercellular adhesion molecule-1 (CD54) on isolated mouse pancreatic beta cells by inflammatory cytokines.

Insulin-dependent diabetes mellitus (IDDM) results from a T cell-dependent autoimmune destruction of insulin-producing pancreatic beta cells. In the present study, expression of adhesion molecule ICAM-1 (CD54) on pancreatic beta cells was studied in normal, obese hyperglycemic (ob/ob), and nonobese diabetic (NOD) mice. Freshly isolated pancreatic beta cells from ob/ob mice did not express ICAM-1, but treatment of the cells with IL-1-beta, TNF-alpha, or INF-gamma strongly induced its expression as measured by immunofluorescence flow cytometry. The cytokines acted in a dose- and time-dependent manner. Maximal induction by either cytokine occurred at 24 hr and thereafter expression decreased, except for INF-gamma. Immunoprecipitation from IL-1-beta-treated beta cells demonstrated a cell-surface glycoprotein with an apparent molecular weight of 95 kDa. ICAM-1 expression was undetectable on pancreatic beta cells of normal and ob/ob mice as measured by immunohistochemistry. In NOD mice at different ages (1 to 6 months) ICAM-1 was also undetectable on beta cells, in contrast to the strong expression on infiltrating mononuclear cells. The present study indicates that mouse pancreatic beta cells, under certain conditions, can express ICAM-1.

Malignant lymphomas in cynomolgus monkeys infected with simian immunodeficiency virus.

Malignant lymphomas were observed in 38% (9 of 24) of simian immunodeficiency virus (SIV)-infected cynomolgus monkeys (Macaca fascicularis) 5 to 15 months after inoculation with SIV strain SMM3. Lymphomagenesis in the SIV-infected monkeys was not related directly to the SIV-infectious dose given. All SIV-infected animals developed severe immunodeficiency. No significant difference in immunodeficiency was observed between tumor-bearing and non-tumor-bearing animals. In contrast, no lymphomas were observed in a comparable group of HIV-2-infected monkeys, which did not develop immunodeficiency; nor did the noninfected control monkeys. All 9 SIV-related tumors were high-grade B-cell lymphoblastic or pleomorphic lymphomas with extranodal, disseminated growth. Most tumors showed marked infiltration by monocytes and CD8+ T lymphocytes. Occasional tumor infiltrating cells showed immunohistochemical reaction for SIV. The cells of two tumors were established in vitro and shown to be of B-cell phenotype. The tumor cell cultures showed no reverse transcriptase activity and no evidence of virus infection by electron microscopy. Our observations indicate that SIV-induced immunodeficiency in cynomolgus monkeys also mimics HIV infection and AIDS in humans with regard to increased lymphomagenesis and type of lymphomas.

B-cell lymphomagenesis in SIV-immunosuppressed cynomolgus monkeys.

B-cell lymphomas developed frequently (approx. 40%) in SIVsm (SMM3) immunosuppressed monkeys and were mostly extranodal, aggressive and all associated with an EBV-related simian herpes virus operationally designated herpes virus Macaca fascicularis (HVMF-I). Lymphoma tissues from 21 monkeys were studied by PCR and DNA PAGE for mono/oligoclonality of the VDJ-rearranged IgH genes. Most lymphomas (n = 15) showed a monoclonal and approximately 1/3 (n = 6) an oligoclonal VDJ rearrangement pattern. The time after infection to tumor presentation was significantly shorter for oligoclonal than for monoclonal lymphomas, suggesting that oligoclonal selection frequently precedes the outgrowth of a single malignant clone. Comparison of the VDJ rearrangements in an established lymphoma cell line and the original, oligoclonal lymphoma tissue indicated in vitro selection of one HVMF-infected clone. Longitudinal studies of sequential lymph-node biopsies showed that the malignant lymphoma clone in 3 out of 8 lymphomas could be identified as a predominant clone in lymph nodes 2-12 months after SIV infection and 6-10 months before clinical presentation of the lymphomas. VDJ-rearranged DNA corresponding to that of the lymphomas was also detected in most sera at the time of lymphoma manifestation but not in corresponding PBL preparations. Clearly, the SIVsm AIDS model in cynomolgus monkeys represents a powerful tool for biological and clinical studies of herpes-virus-associated lymphomagenesis in immunosuppressed states.

Thymic immunopathology and progression of SIVsm infection in cynomolgus monkeys.

Thymuses from 22 cynomolgus monkeys infected with simian immunodeficiency virus (SIVsm) developed characteristic cortical and medullary changes including formation of B-cell follicles (8/21) and accumulation of virus immune complexes. Advanced thymic histopathology was correlated with more pronounced immunodeficiency. SIVsm provirus was detected by polymerase chain reaction (PCR) in most (16/18) thymuses and spliced viral env mRNA in 3 (3/7) thymuses with advanced histopathologic changes indicative of thymic SIVsm replication. By combined in situ hybridization (ISH) and immunohistochemistry, viral RNA was localized mainly to the follicular dendritic network, macrophages, multinucleated giant cells, and lymphocytes of the medullary regions. Latent infection by an Epstein-Barr-related herpesvirus (HVMF1) was also found by PCR and by ISH in medullary regions of three (3 of 8) thymuses with B-cell follicles, suggestive of an inductive role for B-cell proliferation in these thymuses. In a control group of HIV-2-infected nonimmunosuppressed monkeys, no comparable thymic changes were observed. Our results indicate that SIV, and probably by analogy HIV, can have direct and diverse pathogenic effects on the thymus that are important in the development of simian (human) AIDS.

[Opportunistic malignant lymphomas in SIV infected primates--a model for Epstein-Barr virus associated lymphomas in AIDS]. / Opportunistische maligne Lymphome bei SIV-infizierten Primaten--Ein Modell für Epstein-Barr Virus assoziierte Lymphome bei AIDS.

In a series of 33 cynomolgus monkeys (Macaca fascicularis) experimentally infected with Simian Immunodeficiency virus (SIV), strain smm3, 13 animals developed malignant Non-Hodgkin lymphomas. These lymphomas presented with unusual primary manifestations like in the orbita, testes, and brain. The morphological features and immunophenotyping identified the tumors as high malignant B-cell lymphomas. In all tumors as well as in tumor-derived cell lines a cynomolgus B-lymphotropic herpes virus (CBLV) with structural homogeneity to the Epstein-Barr virus (EBV) could be demonstrated by Southern blotting with EBV-specific probes. The lymphoma cells also expressed CBLV-associated nuclear antigens involved in B-cell transformation crossreacting with EBNA-specific human sera and monoclonal antibodies. Ig-gene rearrangement studies revealed clonal populations, however, no translocations of the c-myc oncogene could be detected. The lymphomas developing with high frequency in SIV-induced immunodeficiency resemble a major subtype of human EBV-associated AIDS lymphomas. This animal model can therefore be used to further elucidate interactions of HIV and EBV in AIDS-related lymphomagenesis.

Expression of HHV-8 latency-associated T0.7 RNA in spindle cells and endothelial cells of AIDS-associated, classical and African Kaposi's sarcoma.

Analysis by polymerase chain reaction (PCR) and serological studies have demonstrated a close association between the novel human herpes virus, Kaposi's sarcoma-associated herpes virus (KSHV) or human herpes virus-8 (HHV-8) and the development of Kaposi's sarcoma (KS). To clarify the role of HHV-8 in KS pathogenesis, we investigated at the cellular level by in situ hybridization the expression of a recently described 0.7-kb HHV-8-encoded mRNA (T0.7 mRNA) in KS tissues of different epidemiological origin (AIDS-KS, African endemic KS and classical KS). The T0.7 mRNA likely encodes a small membrane protein, supposedly expressed in latently HHV-8-infected cells. Indeed, we detected T0.7 mRNA in virtually all cells of the cell line BCBL-1 established from a body cavity-based lymphoma (BCBL) and latently infected with HHV-8. In all KS biopsies examined, independent of their epidemiological type, the late-stage (nodular) KS tissues showed a high level of T0.7 mRNA expression in typical KS spindle cells but also in endothelial cells lining blood vessels, indicating latent HHV-8 infection of these cells. The presence of T0.7-expressing cells was restricted to KS tumor tissue and therefore appears to indicate an important role of latent HHV-8 infection in KS pathogenesis.