Dimitra Bourboulia

Kaposi sarcoma herpesvirus–induced cellular reprogramming contributes to the lymphatic endothelial gene expression in Kaposi sarcoma

The biology of Kaposi sarcoma is poorly understood because the dominant cell type in Kaposi sarcoma lesions is not known. We show by gene expression microarrays that neoplastic cells of Kaposi sarcoma are closely related to lymphatic endothelial cells (LECs) and that Kaposi sarcoma herpesvirus (KSHV) infects both LECs and blood vascular endothelial cells (BECs) in vitro. The gene expression microarray profiles of infected LECs and BECs show that KSHV induces transcriptional reprogramming of both cell types. The lymphangiogenic molecules VEGF-D and angiopoietin-2 were elevated in the plasma of individuals with acquired immune deficiency syndrome and Kaposi sarcoma. These data show that the gene expression profile of Kaposi sarcoma resembles that of LECs, that KSHV induces a transcriptional drift in both LECs and BECs and that lymphangiogenic molecules are involved in the pathogenesis of Kaposi sarcoma.

Transformation of human mesenchymal stem cells increases their dependency on oxidative phosphorylation for energy production

An increased dependency on glycolysis for ATP production is considered to be a hallmark of tumor cells. Whether this increase in glycolytic activity is due mainly to inherent metabolic alterations or to the hypoxic microenvironment remains controversial. Here we have transformed human adult mesenchymal stem cells (MSC) using genetic alterations as described for differentiated cells. Our data suggest that MSC require disruption of the same pathways as have been shown for differentiated cells to confer a fully transformed phenotype. Furthermore, we found that MSC are more glycolytic than primary human fibroblasts and, in contrast to differentiated cells, do not depend on increased aerobic glycolysis for ATP production during transformation. These data indicate that aerobic glycolysis (the Warburg effect) is not an intrinsic component of the transformation of adult stem cells, and that oncogenic adaptation to bioenergetic requirements, in some circumstances, may also rely on increases in oxidative phosphorylation. We did find, however, a reversible increase in the transcription of glycolytic enzymes in tumors generated by transformed MSC, indicating this is a secondary phenomenon resulting from adaptation of the tumor to its microenvironment.

Serologic Evidence of Human Herpesvirus 8 Transmission by Homosexual but Not Heterosexual Sex

Epidemiologic studies link Kaposis sarcoma with a sexually transmitted agent. Human herpesvirus 8 (HHV-8) is likely to be that agent, but routes of transmission are poorly described. A seroepidemiologic study was conducted to determine whether HHV-8 is transmitted sexually between heterosexuals. Sera from 2718 patients attending a sexually transmitted disease (STD) clinic were tested for antibodies to HHV-8 and herpes simplex virus type 2 (HSV-2). Information on sex partners in the previous 12 months and past STDs were obtained by questionnaire. Relationships between possible risk factors and HHV-8 infection were assessed by logistic regression. Overall, seroprevalence of HHV-8 was 7.3%. Independent risk factors for HHV-8 in the whole group were homo/bisexuality and birth in Africa and, among homo/bisexual men, a history of syphilis and HSV-2 and human immunodeficiency virus seropositivity. Among heterosexuals there was no evidence for sexual transmission; the only independent risk factor for HHV-8 seropositivity was birth in Africa.

Prospective study of the effects of antiretroviral therapy on Kaposi sarcoma--associated herpesvirus infection in patients with and without Kaposi sarcoma.

Objective: To investigate the effect of highly active antiretroviral therapy (HAART) on circulating Kaposi sarcoma (KS)‐associated herpesvirus (KSHV) load in HIV‐infected individuals with and without KS. Design: Twenty‐nine HIV‐infected subjects (21 with KS and 8 without KS) were recruited for the study; they were prospectively studied before and at regular intervals during HAART. Methods: Patients with KS were clinically assessed using Adult Clinical Trials Group (ACTG) criteria, and sequential blood samples were obtained from all patients for determination of plasma HIV‐1 load, CD4 cell count, titer of antibody to KSHV, and KSHV load. Results: Ten of 21 patients with KS had a favorable KS response (complete response, 6; partial response, 4) with HAART alone. Of the 20 subjects with detectable KSHV viremia prior to HAART, over one half (12 [60%]) had an undetectable KSHV load with antiretroviral therapy. There was no significant difference between subjects receiving protease inhibitor‐ or nonnucleoside reverse transcriptase inhibitor‐based treatment combinations. Achieving undetectable KSHV viremia is associated with a better clinical outcome in patients with KS. Conclusions: To our knowledge, we demonstrate for the first time that both protease inhibitor‐based and nonnucleoside reverse transcriptase inhibitor‐based antiretroviral treatment combinations may lead to an undetectable KSHV load and confirm that an undetectable KSHV load is associated with KS regression. There was no clear association between CD4 cell count response and KS response to HAART, but there was a significant relationship between HIV load response to HAART and clinical improvement of KS.

Short- and long-term effects of highly active antiretroviral therapy on Kaposi sarcoma-associated herpesvirus immune responses and viraemia.

Objective: To investigate the effect of highly active antiretroviral therapy (HAART) on Kaposi sarcoma-associated herpesvirus (KSHV) DNA load, anti-KSHV antibody responses and KSHV-specific CD8 T cell responses in HIV-infected individuals over a 2 year period. Design: Prospective study of 27 HIV-infected antiretroviral therapy-naive individuals, with (n = 4) and without KS (n = 23), before HAART and at 3-month intervals, during treatment with HAART. Methods: Sequential blood samples were collected for anti-KSHV antibody detection, KSHV DNA load in peripheral blood mononuclear cells (PBMC) and plasma, HIV Gag-specific and KSHV-specific CD8 T cell responses, HIV-1 plasma RNA load and CD4 and CD8 T cell counts. Results: KSHV DNA in PBMC and plasma became less detectable over time during HAART, in particular after 12 months. KSHV DNA was undetectable in plasma after 24 months on HAART. Anti-KSHV lytic, but not latent, antibody levels increased within 12 months of treatment. KSHV-specific CD8 T cell responses were absent prior to HAART but became detectable in some patients within 6 months of starting treatment, and continued to increase thereafter. Conclusions: HAART (both protease inhibitor-based and non-nucleoside reverse transcriptase inhibitor-based antiretroviral combinations) is associated with immune reconstitution to KSHV and with undetectable KSHV viraemia. However, this restoration is apparent (in particular) only after a relatively long (> 24 months) period of treatment. These immune responses could contribute to the decreased incidence of KS during HAART, but it is unlikely to be a complete explanation for the often rapid resolution of KS when HAART is started.

Identification of Kaposi's Sarcoma-Associated Herpesvirus (KSHV)-Specific Cytotoxic T-Lymphocyte Epitopes and Evaluation of Reconstitution of KSHV-Specific Responses in Human Immunodeficiency Virus Type 1-Infected Patients Receiving Highly Active Antiretroviral Therapy

ABSTRACT Following the introduction of highly active antiretroviral therapy (HAART), the incidence of Kaposis sarcoma (KS) has significantly declined in human immunodeficiency virus type 1 (HIV-1)-positive (HIV-1+) individuals and clinical remission is often observed. We hypothesize that these effects are partly due to anti-KS-associated herpesvirus (KSHV) immune restoration. Here, 15-mer overlapping peptides from proteins K12 and K8.1 were used to identify novel KSHV-specific cytotoxic T-lymphocyte epitopes. Three immunogenic peptides, two lytic and one latent, were subsequently used to monitor the anti-KSHV CD8+ T-cell responses in a cohort of 19 HIV-1+ KSHV+/− KS+/− individuals during 52 weeks of HAART. KSHV and HIV-1 loads, KSHV antibody titers, and both CD4+ and CD8+ T-lymphocyte counts were enumerated. Prior to HAART, the total number of spot-forming cells (SFC) for all three peptides correlated with both CD4+ and CD8+ T-lymphocyte counts (P ≤ 0.05) in the KSHV-positive KS-positive cohort (n = 11). Following 52 weeks of HAART, significant decreases in HIV-1 and KSHV loads were associated with significant increases in CD4+ T-lymphocyte counts and number of SFC for the three KSHV-specific peptides. Although these increases were modest in comparison to the number of SFC observed with the HIV-1 gag peptide SLYNTVATL, they represented a fourfold increase from the baseline, continuing an upward trend to week 52.

Kaposi's Sarcoma–Associated Herpesvirus-Encoded Interleukin-6 and G-Protein–Coupled Receptor Regulate Angiopoietin-2 Expression in Lymphatic Endothelial Cells

Kaposis sarcoma (KS) is caused by Kaposis sarcoma-associated herpesvirus (KSHV) and consists of proliferating spindle cells, which are related to lymphatic endothelial cells (LEC). Angiopoietin-2 (Ang2) is a secreted proangiogenic and lymphangiogenic molecule. Here, we show the expression of Ang2 protein in KS and confirm that KSHV infection up-regulates Ang2 in LEC. We show that a paracrine mechanism contributes to this up-regulation. A lentiviral library of individual KSHV-encoding genes, comprising the majority of known latent genes and a selection of lytic viral genes, was constructed to investigate the underlying mechanism of this up-regulation. Two lytic genes, viral interleukin-6 (vIL6) and viral G-protein-coupled receptor (vGPCR), up-regulated Ang2 expression in LEC. Both vIL6 and vGPCR are expressed in KSHV-infected LEC and caused up-regulation of Ang2 in a paracrine manner. KSHV, vIL6, and vGPCR up-regulated Ang2 through the mitogen-activated protein kinase (MAPK) pathway. Gene expression microarray analysis identified several other angiogenic molecules affected by KSHV, including the vascular endothelial growth factor (VEGF)/VEGF receptor (VEGFR) axis, which is also affected by vIL6 and vGPCR in LEC, and matrix metalloproteinases, which could act in concert with Ang2 to contribute to KS development. These findings support the paracrine and autocrine roles of the lytic KSHV-encoded proteins, vIL6 and vGPCR, in KS pathogenesis and identify Ang2 as a potential therapeutic target for this neoplasm.

Kaposi's Sarcoma-Associated Herpesvirus Cytotoxic T Lymphocytes Recognize and Target Darwinian Positively Selected Autologous K1 Epitopes

ABSTRACT Kaposis sarcoma-associated herpesvirus (KSHV) is the infectious cause of Kaposis sarcoma (KS) and certain lymphoproliferations particularly in the context of human immunodeficiency virus (HIV) type 1-induced immunosuppression. The introduction of effective therapies to treat HIV has led to a decline in the incidence of KS, suggesting that immune responses may play a role in controlling KSHV infection and pathogenesis. Cytotoxic-T-lymphocyte (CTL) activity against KSHV proteins has been demonstrated; however, the identification of KSHV CTL epitopes remains elusive and problematic. Although the herpesvirus genomic layout is generally conserved, KSHV encodes a unique hypervariable protein, K1, with intense biological selection pressure at specific amino acid sites. To investigate whether this variability is partly driven by cellular immunity, we designed K1 peptides that match only the unique viral sequence for every individual studied here (autologous peptides). We identified functional CTL epitopes within K1s most variable areas, and we show that a given individual responds only to autologous peptides and not to peptides from other individuals. Furthermore, these epitopes are highly conserved sequences within KSHV isolates from a specific strain but are not conserved between different strains. We conclude that CTL recognition contributes to K1, and therefore to KSHV, evolution.

Viral Coinfections among African Children Infected with Human Immunodeficiency Virus Type 1

City-dwelling children from Kenya who were infected with human immunodeficiency virus type 1 (HIV-1) were tested for coinfection with cytomegalovirus (CMV), human T cell lymphotropic viruses 1 and 2, Kaposi sarcoma-associated herpesvirus (KSHV), or hepatitis B, C, and G viruses. All children were found to be coinfected with CMV, whereas 5% had hepatitis G virus coinfection and 15% had KSHV coinfection. A protective role for hepatitis G virus cannot be excluded but likely affects only a minority of HIV-1-infected African children.

Seroprevalence of human herpesvirus 8 and its association with Kaposi sarcoma in Brazil

Background: Epidemiologic studies have indicated that human herpesvirus 8 is implicated in the development of Kaposi sarcoma in different geographic areas worldwide. Goal: To provide information on the prevalence of human herpesvirus 8 in Brazil and its association with Kaposi sarcoma. Study Design: An immunofluorescence assay was performed to test 1,044 serum samples from 747 blood donors, 73 patients presenting to casualty departments, and 224 patients attending sexually transmitted disease/AIDS clinics. The sexually transmitted disease group was composed of 88 patients with HIV (40 Kaposi sarcoma positive, 48 Kaposi sarcoma negative) and 136 patients without HIV. Results: Antibodies to human herpesvirus 8‐latent nuclear antigens were found in 34 blood donors (4.6%), and in seven casualty patients (9.6%). The highest frequency of human herpesvirus 8 antibodies was found in the sexually transmitted disease group: 32 HIV‐positive patients with Kaposi sarcoma (80%) and seven patients without Kaposi sarcoma (14.6%). Conclusion: The presence of human herpesvirus 8 in patients with HIV was strongly associated with Kaposi sarcoma (odds ratio, 23.4; 95% CI, 7.7‐71.4), male gender, homosexual or bisexual orientation, and hepatitis B virus infection, but not with the other sexually transmitted diseases that were investigated.