Insulin-like growth factor-I receptor activity is essential for Kaposi's sarcoma growth and survival.

Kaposi's sarcoma (KS) is a highly vascular tumour and is the most common neoplasm associated with human immunodeficiency virus (HIV-1) infection. Growth factors, in particular vascular endothelial growth factor (VEGF), have been shown to play an important role in its development. The role of insulin-like growth factors (IGFs) in the pathophysiology of different tumours led us to evaluate the role of IGF system in KS. The IGF-I receptors (IGF-IR) were identified by immunohistochemistry in biopsies taken from patients with different AIDS/HIV-related KS stages and on KSIMM cells (an established KS-derived cell line). Insulin-like growth factor-I is a growth factor for KSIMM cells with a maximum increase of 3H-thymidine incorporation of 130 +/- 27.6% (P < 0.05) similar to that induced by VEGF and with which it is additive (281 +/- 13%) (P < 0.05). Moreover, specific blockade of the receptor (either by alpha IR3 antibody or by picropodophyllin, a recently described selective IGF-IR tyrosine phosphorylation inhibitor) induced KSIMM apoptosis, suggesting that IGF-IR agonists (IGF-I and -II) mediate antiapoptotic signals for these cells. We were able to identify an autocrine loop essential for KSIMM cell survival in which IGF-II is the IGF-IR agonist secreted by the cells. In conclusion, IGF-I pathway inhibition is a promising therapeutical approach for KS tumours.

Dendritic cells in patients with non-progressive B-chronic lymphocytic leukaemia have a normal functional capability but abnormal cytokine pattern.

Dendritic cells (DC) are attractive candidates for use in vaccine-based immunotherapy. We have analysed the functional capability of DC generated in vitro from blood CD14(+) cells of chronic lymphocytic leukaemia (CLL) patients and healthy donors by culturing for 10 d with granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin 4 (IL-4) and tumour necrosis factor-alpha (TNF-alpha). Two distinct DC populations were identified in patients as well as in controls. The majority of DC expressed CD11c and a minority also CD123. Most of the DC generated from both patients and controls exhibited a mature phenotype indicated by CD83 and major histocompatibility complex (MHC) class II expression, as well as by a characteristic morphology. Less than 1% of DC exhibited CD14. CLL DC had a similar expression of accessory molecules (CD54, CD80 and CD86) as control DC. The mean fluorescence intensity of CD80 and MHC class I molecules was significantly higher on CLL DC than on control DC (P < 0.05). At the gene level (real-time polymerase chain reaction) the expression of IL-10 was higher in CLL (P = 0.028) than in control DC. IL-1 beta and IL-12p(35) transcripts were also more abundant in CLL than in control DC but did not reach statistical significance. The expression of IL-4 and TNF-alpha was similar to that of control DC. The interferon gamma (IFN-gamma) gene expression level in CLL DC was decreased compared with control DC. DC of CLL patients had a similar capacity to stimulate in mixed leucocyte reaction as well as to present a recall antigen (PPD) as control DC. Thus, DC of CLL patients seem to have a normal function and may serve as antigen preserving cells for presentation of tumour antigens in a therapeutic vaccination approach. The mechanisms behind the observed increase in some surface molecules and the abnormal cytokine profile of CLL DC is not clear but might indicate pre-activation of DC in vivo, which may have a regulatory role in the pathobiology of CLL.

Immunohistochemical expression of extracellular matrix proteins and adhesion molecules in pancreatic carcinoma.

BACKGROUND/AIMS: Carcinoma invasion and metastasis in general involve multiple steps including dynamic changes in the composition and structure of extracellular matrix proteins and cell surface receptors. In the present study, the usually highly invasive carcinoma of the pancreas was investigated regarding the expression of various extracellular matrix proteins and their corresponding integrin receptors, as well as E-cadherin. METHODOLOGY: Phenotypic expression of various markers was investigated immunohistochemically in frozen sections of 16 pancreatic carcinomas and normal pancreatic tissue. RESULTS: An irregular and discontinuous deposition of type IV collagen and laminin in the basement membrane was found in cancer tissue and a pronounced desmoplastic reaction with deposition of type I, type III, and type IV collagen in the tumor stroma. In contrast, the noninvolved pancreas showed an intact basement membrane and a sparse stroma. The collagen type IV and laminin receptors alpha 2, alpha 3, and beta 1 integrin subunits were expressed on pancreatic cancer cells but not the alpha 6 integrin subunit normally present on epithelial cells, suggesting anchorage independence of the carcinoma cells. An increased capacity for cancer cell motility was suggested by the abundant expression of the "antiadhesive" extracellular matrix proteins, tenascin and vitronectin close to the cancer cells, and the expression of cell surface receptors such as alpha v (vitronectin-binding). Expression of the alpha 4 integrin subunit was also increased on cancer cells. CONCLUSIONS: The distribution of extracellular matrix proteins and the cell surface immune phenotype differed in pancreatic carcinoma as compared to normal pancreatic tissue. The present findings substantiate the notion that disseminated growth of highly malignant carcinomas of the pancreas reflects an invasive interaction of the tumor cells with extracellular matrix proteins of a well-established stroma. Similar findings were observed regardless of tumor histology and patient survival time.

Recombinant Semliki Forest virus vaccine vectors: the route of injection determines the localization of vector RNA and subsequent T cell response.

Vectors based on Semliki Forest virus (SFV) have been widely used in vitro and in vivo to express heterologous genes in animal cells. In particular, the ability of recombinant SFV (rSFV) to elicit specific, protective immune responses in animal models suggests that rSFV may be used as a vaccine vehicle. In this study, we examined the distribution of rSFV in vivo by immunohistochemistry and RT-PCR after intravenous, intramuscular and subcutaneous injection of rSFV particles and related this to the degree of cytotoxic T lymphocyte (CTL) responses and frequency of specific T cells detected by MHC-I tetramers. We found that after i.v. injection, rSFV-RNA was distributed to a variety of different tissues, whereas it was confined locally after i.m. and s.c. injections. The persistence of the rSFV vector was transient, and no viral RNA could be detected 10 days after inoculation. All tested routes of immunization generated significant levels of antigen-specific CTL responses and increased numbers of specific CD8+ T cells, as detected by tetramer binding. The distribution of antigen-specific CTLs correlated with the in vivo distribution pattern of rSFV, with a highest frequency in the spleen or local lymph node, depending on the injection route.

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.

Proliferation and apoptosis in the evolution of endemic and acquired immunodeficiency syndrome-related Kaposi's sarcoma.

Kaposi's sarcoma (KS) is a multifocal lesion that occurs predominantly in the skin, most frequently in people infected with HIV-1, and that evolves through early stages (patch and plaque) to a tumor-like late stage (nodular). Both, endemic African (EKS) and AIDS-associated (AKS) KS expressed human herpesvirus 8 (HHV-8) as shown by PCR. By immunohistochemistry the expression of cellular Bcl-2 and c-myc was confined in early stages of both EKS and AKS to relatively few endothelial cells (EC) whereas in nodular KS most of spindle cells (SC) strongly expressed both genes. CD40 was usually strongly expressed in SC at all KS stages as well as in EC of non-involved tissue whereas CD40L (CD154) was not demonstrable. Fas (CD95) was moderately to weakly expressed by SC whereas p53 and Waf-1 were found in less than 5% of the SC. In both AKS and EKS at nodular stage almost no apoptotic SC were detected. In most AKS and EKS low levels of cell proliferation were seen but AKS showed consistently higher values compared to EKS. All clinical types and stages of KS showed a diploid cellular DNA content by flow cytometric analysis of microselected lesions. Thus, we conclude that KS during evolution represents diploid, probably reactive, cell proliferation, which progressively increases the expression of strong cellular and also viral (HHV-8) antiapoptotic factors.

Antibodies to human herpesvirus 8 latent and lytic antigens in blood donors and potential high-risk groups in Sweden: variable frequencies found in a multicenter serological study.

Human herpesvirus 8 (HHV-8) is a herpesvirus associated with Kaposi's sarcoma (KS). An immunofluorescence assay was used for detection of IgG, IgM, and IgA antibodies against lytic and latent HHV-8 antigens to analyse samples from KS patients (n = 8), healthy blood donors (n = 162), individuals with a high risk sexual behaviour (n = 114), and bone marrow transplant patients (with high risk for bloodborne infections) (n = 34) in Sweden. Of the KS patients, 88% had IgG antibodies to both lytic and latent antigens by immunofluorescence. In all other groups, antilatent antibodies were rare (0-2.6%). IgG antibodies to the lytic antigens were found, by immunofluorescence, in 20% of the blood donors, 31% of the high risk patients, and in 24 and 29% of the bone marrow transplant patients (pre- and post-transplant samples, respectively). For verification of the specificity of the anti-lytic antibodies, 170 of the samples were also tested blindly at different laboratories world-wide with five other assays shown previously to detect HHV-8 antibodies in most KS patients. By using two recombinant HHV-8 proteins (ORF65/vp17 and K8.1/gp 35-37) in ELISA, a whole-virion ELISA and two immunofluorescence assays confirmation of the reactivity against lytic viral antigens was sought. The comparison of the different methods suggested the K8.1 ELISA to be highly specific and also showed a good agreement between two of the immunofluorescence assays. However, generally there was a poor correlation for positive results, indicating the need of further methodological development.

The apoptotic v-cyclin-CDK6 complex phosphorylates and inactivates Bcl-2.

v-cyclin encoded by Kaposi's sarcoma herpesvirus/human herpesvirus 8 (KSHV or HHV8) associates with cellular cyclin-dependent kinase 6 (CDK6) to form a kinase complex that promotes cell-cycle progression, but can also induce apoptosis in cells with high levels of CDK6. Here we show that whereas HHV8-encoded v-Bcl-2 protects against this apoptosis, cellular Bcl-2 has lost its anti-apoptotic potential as a result of an inactivating phosphorylation in its unstructured loop region. Moreover, we identify Bcl-2 as a new substrate for v-cyclin-CDK6 in vitro, and show that it is present in a complex with CDK6 in cell lysates. A Bcl-2 mutant with a S70A S87A double substitution in the loop region is not phosphorylated and provides resistance to apoptosis, indicating that inactivation of Bcl-2 by v-cyclin-CDK6 may be required for the observed apoptosis. Furthermore, the identification of phosphorylated Bcl-2 in HHV8-positive Kaposi's sarcoma indicates that HHV8-mediated interference with host apoptotic signalling pathways may encourage the development of Kaposi's sarcoma.

Sequence analysis and variation of EBNA-1 in Epstein-Barr virus-related herpesvirus of cynomolgus monkey.

OBJECTIVES: The Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA-1) is an important protein for immortalization and tumorigenesis of infected cells. EBNA-1 gene variants may play a role in tumorigenesis. We determined the nucleotide and amino acid (aa) sequences of EBNA-1 in EBV-related herpesviruses from cynomolgus monkeys (cynomolgus-EBV) which induced malignant lymphomas in its natural host and in rabbits, and compared them with sequences of EBV and other lymphocryptoviruses (LCVs). METHODS: Polymerase chain reaction and direct sequencing methods were performed using extracted DNA from cynomolgus-EBV-infected cell lines. RESULTS: The amino acid sequences of cynomolgus-EBV EBNA-1 from two cell lines (Si-IIA: 588 aa; Ts-B6: 619 aa) which are antigenically cross-reactive to human EBV EBNA-1 showed homology with human EBV (Si-IIA: 53%; Ts-B6: 58%) and other LCVs from baboons (54 and 52%) and rhesus monkeys (60 and 58%), especially in the C-terminal unique domain. Homology of the EBNA-1 sequence between Si-IIA and Ts-B6 was 92%. The sequence difference between EBV and the related LCVs was manifested mainly in the length of the internal repeat 3-corresponding region, which contains serine in the glycine/alanine repeat region of nonhuman LCVs. CONCLUSION: Sequence variation of cynomolgus-EBV EBNA-1 from different cell lines was observed. However, their sequences show a relatively high homology with human EBV and share the common features of EBNA-1 of EBV and other LCVs.

Combined analysis of DNA ploidy, proliferation, and apoptosis in paraffin-embedded cell material by flow cytometry.

A flow cytometric assay was developed for correlated measurement of DNA content and apoptotic DNA strand breaks in cell nuclei of formalin-fixed, paraffin-embedded tissues. The assay allows a combined analysis of cell ploidy, proliferation, and apoptosis in sections of fixed paraffin-embedded archival or fresh tissue/cell specimens. It is based on (a) proteolytic release of cell nuclei from deparaffinized and rehydrated 90-microm thick sections of the fixed embedded specimen, (b) the inactivation of the protease, (c) FITC-labeling of DNA strand breaks by the terminal deoxynucleotidyl transferase (TdT)-mediated FITC-dUTP nick end-labeling (TUNEL) reaction, and (d) DNA staining with 4'6-diamidino-2-phenyleindole. The fluorescence was recorded with a double-beam flow cytometer equipped with a mercury arc lamp and an argon ion laser. Cytograms obtained with this assay correlated closely with those produced using nonembedded material from the same specimen. Furthermore, a significant correlation was found between flow cytometric analysis of apoptosis in cell nuclei released from paraffin blocks and conventional evaluation of TUNEL on (corresponding) sections (p < 0.001). Since necrotic cells can stain positively by TUNEL, the possibility to microscopically select nonnecrotic tumor regions for flow cytometric analysis is an important advantage of the assay.

Antibody spectrum against the viral transactivator protein in patients with human immunodeficiency virus type 1 infection and Kaposi's sarcoma.

We analyzed patterns of antibody response to recombinant transactivator protein (human Immunodeficiency virus type 1 [HIV-1] tat) in serum samples from HIV-1-negative subjects (n = 60), HIV-1-infected asymptomatic patients (n = 20), HIV-1-infected patients with Kaposi's sarcoma (n = 25), and patients with Kaposi's sarcoma without HIV-1 infection. None of the healthy subjects possessed anti-tat immunoglobulin G (IgG) in their serum. All asymptomatic patients with HIV-1 infection were anti-tat IgG-positive. Epitope mapping revealed that these sera had anti-tat IgG to all the functional domains of tat protein. Histochemical studies on lymph nodes from five asymptomatic HIV-1-infected patients showed that, in all cases, tat-positive cells were present within the germinal center at the stage of follicular fragmentation containing immunoblasts and small lymphocytes. Of the 25 HIV-1-infected patients with Kaposi's sarcoma, 4 were anti-tat IgG-positive; however, the epitope analysis revealed that IgG to functional domains of tat protein--in particular to transactivating response element (TAR)-binding site--were absent. All patients with Kaposi's sarcoma without HIV-1 infection were anti-tat IgG-negative. Presence or absence of anti-tat IgG and a prevalence of different antibody profiles in different groups of patients indicated the pathophysiologic role of tat protein. Thus, a passive immunization with anti-tat IgG could be a useful strategy to influence the pathophysiologic state of the disease.

Anti-CD3 induced thymocyte apoptosis in vivo require the antibody Fc domain.

We have earlier found that explanted thymic epithelial cells (TEC) can produce glucocorticoid (GC) activity in vitro and that the GC receptor (GR) antagonist RU486 partially inhibit thymic apoptosis induced by the anti-CD3 monoclonal antibodies (MoAb) 2C11, both in vivo and in new-born thymic organ cultures. To explain the inhibitory effect of RU486 in this system we have now investigated the importance of the 2C11 Fc as this MoAb bind with high affinity to cellular FcR. We have found both that whole 2C11 MoAb can bind to explanted TEC in vitro and that F(ab)'2 fragments from this MoAb loose this ability, in addition with the capacity to induce thymic apoptosis in vivo. We interpret our results to indicate that the injected 2C11 MoAb may establish a close contact between GC producing, FcR positive TEC cells and CD3 positive thymocytes and thereby subject the later to high paracrine GC concentrations and subsequent induction of apoptosis.

Recruitment of monocyte derived dendritic cells ex vivo from SIV infected and non-infected cynomolgus monkeys.

This study shows that characteristic dendritic, antigen presenting cells, can be generated from adherent peripheral blood mononuclear cells (PBMC)/monocytes of uninfected and SIVsm-infected cynomolgus monkeys after stimulation in vitro with granulocyte macrophage colony stimulating factor (GM-CSF) and interleukin (IL)-4. The recruitment of monocyte derived dendritic cells (MDDC) was usually possible irrespective of the level of immunodeficiency (CD4-level) and viremia. The cynomolgus MDDC closely resembled their human counterpart (immature MDDC) with regard to capacity to upregulate CD1a, CD40, CD86 and human leukocyte antigen (HLA)-DR and develop dendrites and veiled processes. Such MDDC also increased their capacity for antigen uptake (dextran endocytoses/macropinocytosis) and for induction of T-cell proliferation in mixed leukocyte reaction (MLR) assays. However, although no clear difference with regard to phenotype and morphology was seen between MDDC from SIV-infected and uninfected monkeys, a reduction in MLR responsiveness in MDDC from SIV infected monkeys was consistently detected within each experiment.

Epstein-Barr virus expression in Hodgkin's disease in relation to patient characteristics, serum factors and blood lymphocyte function.

Epstein-Barr virus (EBV) expression was investigated by immunohistochemistry (latent membrane protein 1 [LMP-1]) and in situ hybridization (EBV encoded RNA [EBER]) in biopsies from 95 patients with untreated Hodgkin's disease (HD). Tumour EBV status was related to EBV antibody titres, spontaneous and concanavalin A induced blood lymphocyte DNA synthesis, serum levels of soluble (s) CD4, sCD8, sCD25, sCD30, sCD54, beta2-microglobulin, thymidine-kinase, routine chemistry, patient characteristics, complete remission and survival. The median follow-up time was 145 months (range 60-257). Tumour EBV-positive (n = 30; 33%) and negative (n = 62; 67%) patients did not differ with regard to sex, age, stage, presence of bulky disease or B-symptoms, remission rate or survival. The proportion of EBV+ cases was significantly higher among patients with mixed cellularity histopathology (58%) as compared to the nodular sclerosis subtype (18%; P < 0.001). The total white blood cell (WBC) counts were significantly lower in EBV+ patients (P < 0.01), who also had significantly higher levels of sCD54 (P < 0.02) and a tendency towards lower levels of sCD30 (P = 0.056). Patients in the tumour EBV+ group had significantly higher IgG antibody titres to restricted early antigen (EA-R) (P < 0.02). Hence, clinical features and outcome were not related to tumour EBV status. However, HD patients with EBV+ tumours had elevated sCD54 levels, higher antibody titres to EA-R and decreased total WBC counts. A potential causal relationship between EBV tumour status and these findings needs to be further explored.

Kaposi's sarcoma-associated herpesvirus-encoded v-cyclin triggers apoptosis in cells with high levels of cyclin-dependent kinase 6.

Kaposi's sarcoma-associated herpesvirus (KSHV) has a key etiological role in development of Kaposi's sarcoma (KS). v-Cyclin is a KSHV-encoded homologue to D-type cyclins that associates with cellular cyclin-dependent kinase 6 (CDK6). v-Cyclin promotes S-phase entry of quiescent cells and has been suggested to execute functions of both D- and E-type cyclins. In this study, expression of v-cyclin in cells with elevated levels of CDK6 led to apoptotic cell death after the cells entered S phase. The cell death required the kinase activity of CDK6 because cells expressing a kinase-deficient form of CDK6 did not undergo apoptosis upon v-cyclin expression. Studies on the mechanisms involved in this caspase-3-mediated apoptosis indicated that it was independent of cellular p53 or pRb status, and it was not suppressed by Bcl-2. In contrast, the KSHV-encoded v-Bcl-2 efficiently suppressed v-cyclin-/CDK6-induced apoptosis, demonstrating a marked difference in the antiapoptotic properties of c-Bcl-2 and v-Bcl-2. In KS lesions, high CDK6 expression was confined to a subset of cells, some of which displayed signs of apoptosis. These results suggest that v-cyclin may exert both growth-promoting and apoptotic functions in KS, depending on factors regulating CDK6 and v-Bcl-2 levels.

The inhibitor of death receptor signaling, FLICE-inhibitory protein defines a new class of tumor progression factors.

Death receptor-mediated apoptosis can be modulated by several antiapoptotic proteins, such as the FLICE (FADD [Fas-associated death domain]-like IL-1beta-converting enzyme)-inhibitory proteins (FLIPs). The FLIP family includes both cellular and viral members. The Kaposi's sarcoma-associated herpesvirus protein (KSHV)-FLIP is expressed by human herpesvirus 8 (HHV-8), which is associated with malignancies such as Kaposi's sarcoma and certain lymphomas. In this paper, we demonstrate that KSHV-FLIP protects cells from Fas-mediated apoptosis by inhibiting caspase activation and permits clonal growth in the presence of death stimuli in vitro. Furthermore, we show that KSHV-FLIP can act as a tumor progression factor by promoting tumor establishment and growth in vivo. When injected into immunocompetent recipient mouse strains, murine B lymphoma cells (A20) transduced with KSHV-FLIP rapidly develop into aggressive tumors showing a high rate of survival and growth. The tumor-progressive activity of KSHV-FLIP is mediated by prevention of death receptor-induced apoptosis triggered by conventional T cells. Consequently, inhibitors of death receptor signaling can be regarded as a new class of tumor progression factors, and HHV-8-associated tumors may represent naturally occurring examples of the tumorigenic effect of such inhibitors.

Expression of K13/v-FLIP gene of human herpesvirus 8 and apoptosis in Kaposi's sarcoma spindle cells.

BACKGROUND: Human herpesvirus 8 (HHV8) infection is associated with all forms of Kaposi's sarcoma (KS). The HHV8 genome locus ORFK13-72-73 (ORF = open reading frame) encodes proteins that may be important in HHV8-mediated pathogenesis, i.e., the latency-associated nuclear antigen (encoded by ORF73), viral-cyc-D (v-cyc-D), a viral homologue of cellular cyclin D (encoded by ORF72), and viral-FLIP (v-FLIP), a homologue of the cellular FLICE (Fas-associated death domain-like interleukin 1 beta-converting enzyme) inhibitory protein (encoded by ORFK13; is an inhibitor of apoptosis [programmed cell death]). Through differential splicing events, this locus expresses individual RNA transcripts that encode all three proteins (tricistronic transcripts) or just two of them (v-FLIP and v-cyc-D; bicistronic transcripts). We examined expression of these transcripts in KS tissues. METHODS: We collected tissues from patients with KS of different stages. By use of an optimized in situ hybridization procedure, we examined different ORFK13-72-73 locus transcripts in HHV8-infected cells in skin lesions and in one adjacent lymph node. Apoptosis in KS lesions was determined by use of an in situ assay. RESULTS AND CONCLUSIONS: Our results indicate the following: 1) Transcripts from the ORFK13-72-73 locus appear to be spliced differentially in latently infected KS cells in skin lesions and in HHV8-infected cells in lymph nodes; specifically, ORFK13-ORF72 bicistronic transcripts were expressed abundantly in KS cells, whereas ORFK13-ORF72-ORF73 tricistronic transcripts were detected only in lymph node cells. 2) Sequences encoding the antiapoptotic protein v-FLIP are expressed at very low levels in early KS lesions, but expression increases dramatically in late-stage lesions. 3) The increase in expression of v-FLIP-encoding transcripts is associated with a reduction in apoptosis in KS lesions. IMPLICATIONS: These data suggest that functional v-FLIP is produced in vivo and that antiapoptotic mechanisms may be involved in the rapid growth of KS lesions, where only a few cells undergoing mitosis are generally observed.

A cytotoxic, apoptotic, low-molecular weight factor from pineal gland.

Previous studies suggest that the pineal gland may play a role in tumour growth inhibition. In this respect, melatonin, as the major hormone of this gland, has been extensively studied. However, there is growing evidence for the existence of other yet unknown pineal factors that may have tumour growth inhibiting properties. Here we describe the partial purification of a highly cytotoxic low molecular weight (<400 Da) hydrophilic fraction (designated F2M3R), starting from a porcine pineal extract (PE), via methanol precipitation followed by reverse-phase HPLC. F2M3R is cytotoxic for a highly apoptosis-resistant human erythroleukemia cell line (K562) at a concentration as low as 30 microg/ml. The viability of the cells was not influenced by an identical prepared porcine pituitary extract or by melatonin. PE induces apoptosis in K562 cells as indicated by three different criteria: morphology, in situ TUNEL assay and bi-parametric FACS analysis with annexin V and propidium iodide, but does not influence the viability of stimulated peripheral blood mononuclear cells. These observations warrant further purification and validation of the cytotoxicity in a panel of different human tumour and non-malignant cells.

Lymphoid disorders associated with HHV-8/KSHV infection: facts and contentions.

Following the demonstration in 1994, that Kaposi's sarcoma (KS) was associated with a novel virus (KSHV or HHV-8) belonging to the lymphotropic herpes family, this virus was also found in certain lymphoid neoplasias of immunodeficient (HIV+) and immune competent hosts. The association of HHV-8/KSHV infection is now well established with primary effusion lymphoma (PEL) or body cavity based lymphoma (BCBL) and multicentric Castleman's disease (MCD) of the plasma cell type. A possible pathogenic role of HHV-8/KSHV in other lymphoid tumours including primary central nervous system lymphoma (PCNSL) and multiple myeloma (MM) as well as some atypical lymphoproliferations and sarcoidosis has also been suggested, but this is at present a controversial matter, or not confirmed. Several HHV-8/KSHV genes, including potential oncogenes, genes homologous to various cellular genes and growth factors have been incriminated in the pathogenesis of KS and PEL/BCBL, but a common pathogenic mechanism for the clearly diverse proliferations represented by PEL, MCD and KS is at present not evident.