Giovanna Tosato

Activity of Thalidomide in AIDS-Related Kaposi’s Sarcoma

PURPOSE To assess the toxicity and activity of oral thalidomide in Kaposis sarcoma (KS) in a phase II dose-escalation study. PATIENTS AND METHODS Human immunodeficiency virus (HIV)-seropositive patients with biopsy-confirmed KS that progressed over the 2 months before enrollment received an initial dose of 200 mg/d of oral thalidomide in a phase II study. The dose was increased to a maximum of 1,000 mg/d for up to 1 year. Anti-HIV therapy was maintained during the study period. Toxicity, tumor response, immunologic and angiogenic factors, and virologic parameters were assessed. RESULTS Twenty patients aged 29 to 49 years with a median CD4 count of 246 cells/mm(3) (range, 14 to 646 cells/mm(3)) were enrolled. All patients were assessable for toxicity, and 17 for response. Drowsiness in nine and depression in seven patients were the most frequent toxicities observed. Eight (47%; 95% confidence interval [CI], 23% to 72%) of the 17 assessable patients achieved a partial response, and an additional two patients had stable disease. Based on all 20 patients treated, the response rate was 40% (95% CI, 19% to 64%). The median thalidomide dose at the time of response was 500 mg/d (range, 400 to 1,000 mg/d). The median duration of drug treatment was 6.3 months, and the median time to progression was 7.3 months. CONCLUSION Oral thalidomide was tolerated in this population at doses up to 1,000 mg/d for as long as 12 months and was found to induce clinically meaningful anti-KS responses in a sizable subset of the patients. Additional studies of this agent in KS are warranted.

Defective EBV-Specific Suppressor T-Cell Function in Rheumatoid Arthritis

Several lines of evidence, including high antibody titers to Epstein-Barr virus (EBV)-associated antigens and rapid transformation of B cells into lymphoblastoid cells lines, suggest an association between EBV and rheumatoid arthritis. When lymphocytes from normal immune donors were infected with EBV in culture, they produced an exponentially increasing number of immunoglobulin-secreting cells for eight to 10 days. Thereafter, there was a marked late suppression of their response, mediated by immunoregulatory T cells; by 12 days in culture, this suppression averaged 90 per cent. Lymphocytes from 20 EBV-immune patients with rheumatoid arthritis also responded with increasing production of immunoglobulin-secreting cells, but the late suppression expected in immune donors was absent. Tests of several other T-cell functions in these patients gave normal results, suggesting a more restricted defect in suppressor-T cell function relating specifically to EBV. Since EBV persists in host B cells and thus represents a potential stimulus for immunoglobulin production, this persistence, along with a specific regulatory T-cell defect, may contribute to many of the immune abnormalities underlying rheumatoid arthritis.

Parameters affecting the development of non-Hodgkin's lymphoma in patients with severe human immunodeficiency virus infection receiving antiretroviral therapy.

PURPOSE To investigate the occurrence of non-Hodgkins lymphoma (NHL) in human immunodeficiency virus (HIV)-infected patients receiving long-term antiretroviral therapy and factors associated with the development of these lymphomas. PATIENTS AND METHODS The charts of 55 patients with advanced HIV infection receiving zidovudine (formerly known as azidothymidine [AZT])-based therapy and 61 patients receiving dideoxyinosine (ddI) were examined for the occurrence of NHL. Stored samples from the AZT-based treatment cohort were examined retrospectively for parameters predictive of the subsequent development of lymphoma. RESULTS Eight of 55 patients receiving AZT-based therapy developed NHL, yielding an estimated probability of 12% (95% confidence interval [CI], 4.7% to 27.1%) after 24 months, and 29.2% (95% CI, 15.2% to 48.7%) after 36 months. Four of 61 patients receiving ddI developed NHL, yielding a 6.2% (95% CI, 2.1% to 17%) estimated probability after 24 months, and 9.5% (95% CI, 3.6% to 22.8%) after 36 months. The difference between these cohorts was not significant (two-tailed P [P2] = .13). Patients with less than 50 CD4 cells/microL developed NHL at a significantly higher rate (P2 = .0085). This was particularly true for patients who presented with primary CNS lymphoma (PCNSL). For patients receiving AZT-based therapy, pretreatment serum interleukin-6 (IL-6) levels were somewhat higher in those who subsequently developed NHL than in those who did not (P2 = .048). CONCLUSION HIV-infected patients with profound immunodeficiency, especially those with less than 50 CD4 cells/microL, are at substantial risk of developing NHL and particularly PCNSL. Additional studies are needed to define the role of other factors such as IL-6 in the pathogenesis of these opportunistic tumors.

Contribution of the CXC chemokines IP-10 and Mig to the antitumor effects of IL-12.

The mechanisms by which interleukin‐12 (IL‐12) exerts antitumor effects have been difficult to dissect. In this study, we examined the potential contribution of the chemokines interferonγ‐inducible protein‐10 (IP‐10) and Mig to the antitumor effects of IL‐12. Using an athymic mouse model, local inoculations with IL‐12 consistently produced tumor size reductions associated with characteristic tumor necrosis and vascular damage. These effects were indistinguishable from those produced by IP‐10 or Mig injected locally in the same tumor model. Local and systemic treatment with IL‐12 was associated with expression of the interferon‐γ (IFN‐γ), IP‐10, and Mig genes and proteins in the tumor. Levels of IP‐10 and Mig expression in the tumor, the liver, and the kidney were inversely correlated with tumor size. Administration in vivo of neutralizing antibodies to IP‐10 and Mig reduced substantially the antitumor effects of IL‐12 inoculated locally into the tumors. These results support the notion that IP‐10 and Mig contribute to the antitumor effects of IL‐12 through their inhibitory effects on tumor vasculature. J. Leukoc. Biol. 64: 384–392; 1998.

An Interleukin-6-Related Systemic Inflammatory Syndrome in Patients Co-Infected with Kaposi Sarcoma-Associated Herpesvirus and HIV but without Multicentric Castleman Disease

BACKGROUND Kaposi sarcoma-associated herpesvirus (KSHV) is the causal agent for Kaposi sarcoma (KS) and multicentric Castleman disease (MCD) in human immunodeficiency virus (HIV)-infected patients. Patients with KSHV-MCD develop fevers, wasting, hypoalbuminemia, cytopenias, and hyponatremia that are related to overproduction of KSHV-encoded viral interleukin (IL)-6 (vIL-6) and human IL-6 (hIL-6). METHODS We identified 6 HIV-infected patients with KS or serological evidence of KSHV infection who had severe inflammatory MCD-like symptoms but in whom we could not diagnose MCD, and we hypothesized that these symptoms resulted from vIL-6 overproduction. Serum vIL-6 levels were assessed in these 6 patients and compared with levels in 8 control patients with symptomatic KSHV-MCD and 32 control patients with KS. KSHV viral load, serum hIL-6 level, and human IL-10 level were also evaluated. RESULTS Patients with inflammatory MCD-like symptoms but without MCD had elevated vIL-6 levels, comparable with levels in patients with symptomatic KSHV-MCD, and had levels that were significantly greater than those in control patients with KS (P = .003). Elevated hIL-6, IL-10, and KSHV viral loads were also comparable to patients with symptomatic KSHV-MCD and significantly greater than those with KS. CONCLUSIONS A subset of patients with HIV and KSHV co-infection, but without MCD, can develop severe systemic inflammatory symptoms associated with elevated levels of KSHV vIL-6, IL-6, and KSHV viral loads. Excess lytic activation of KSHV, production of the lytic gene product vIL6, and associated immunologic dysregulation may underlie the pathophysiology of these symptoms. This IL-6-related inflammatory syndrome is important to consider in critically ill patients with HIV and KSHV co-infection.

Mast cell–derived angiopoietin-1 plays a critical role in the growth of plasma cell tumors

Multiple myeloma in humans is frequently associated with mast cell infiltration and neovascularization, which correlate directly with disease severity, but the mechanisms underlying this relationship remain unclear. Here, we report that primary murine mast cells express angiopoietin-1 (Ang-1) and low levels of VEGF-A but not Ang-2 and that 2 established murine plasmacytoma cell lines express high levels of VEGF-A but little or no Ang-1 or Ang-2. An in vivo angiogenesis assay using extracellular matrix components shows that mast cells and plasmacytoma cells, together, promote marked neovascularization composed of dilated vessels, which is prevented by neutralization of VEGF-A and Ang-1 but is only partially reduced by neutralization of either VEGF-A or Ang-1. Mast cells within extracellular matrix components express Ang-1, and recombinant Ang-1 together with plasmacytoma cells promotes extracellular matrix neovascularization similar to that induced by mast cells. A transplantation assay shows that primary mast cells accelerate tumor growth by established plasmacytoma cell lines and that neutralization of Ang-1 alone or with VEGF-A reduces significantly the growth of plasmacytomas containing mast cells. These results demonstrate that mast cell-derived Ang-1 promotes the growth of plasmacytomas by stimulating neovascularization and provide further evidence supporting a causal relationship between inflammation and tumor growth.

A role for the interferon-inducible protein 10 in inhibition of angiogenesis by interleukin-12

We have developed a nude mouse model in which tumor regression is reproducibly induced by coinjection with or intratumor inoculation of EBV-immortalized B cells. A wide spectrum of tumor-derived human cell lines can be established as subcutaneous tumors in sublethally irradiated athymic mice. Most of these tumors are induced to regress by human B cells immortalized with EBV. The tumor regression process is characterized by superficial necrosis and scarring that progressively extends itself to involve all or most of the tumor. Microscopically, tumor regression is characterized by tumor tissue necrosis, evidence of vascular damage with intimal thickening and capillary thrombosis, and macrophage, but not lymphocyte or neutrophil, infiltration. Profiles of cytokine expression differed between progressive and regressive Burkitts tumors in that regressive tumors expressed larger levels of TNF-alpha, IL-6, IFN-gamma, IP-10, Mig, and IL-12 p35, but not other chemokines/cytokines. IP-10 and IL-12 were found to act as potent inhibitors of angiogenesis in vivo. IL-12 is an inducer of IFN-gamma and indirectly of IP-10, an IFN-gamma-inducible protein, raising the possibility that the antiangiogenic effect of IL-12 is mediated by IP-10. Previous studies have demonstrated that IL-12 has potent antitumor activity in vivo. Much of this activity was dependent on the presence of IFN-gamma and of immune T cells. The observation, made in our studies, that IP-10 is an inhibitor of angiogenesis raises the possibility that IP-10 might contribute to the antitumor effects of IL-12 by inhibiting angiogenesis. Inhibition of angiogenesis by IP-10 and IL-12 is T-cell independent, suggesting that IL-12 targets at least two compartments, T cells and capillaries, each capable of mediating antitumor effects.

B Cell Differentiation and Immunoregulatory T Cell Function in Human Cord Blood Lymphocytes

The functional maturity of T and B lymphocyte populations from human newborns was evaluated using a reverse hemolytic plaque assay to detect immunoglobulin-secreting cells generated in in vitro cultures stimulated with pokeweed mitogen (PWM), a T cell-dependent polyclonal activator, and the Epstein-Barr virus (EBV), a T cell-independent B cell activator. Cord blood lymphocytes failed to produce immunoglobulin in response to PWM, but did respond with immunoglobulin synthesis to stimulation with EBV. Co-culture experiments demonstrated that cord blood T cells would inhibit immunoglobulin production by adult cells stimulated with PWM, but not with EBV. Cord blood T cells did suppress immunoglobulin production by cord blood B cells when stimulated with a mixture of EBV and PWM, indicating that cord blood, in contrast to adult blood, contains a population of suppressor T cell precursors that are easily activated by PWM. Irradiation of the cord blood T cells with 2,000 rad eliminated the suppressor activity and revealed normal helper function for immunoglobulin (Ig) G, A, and M when these T cells were co-cultured with adult B cells. Cord blood B cells co-cultured with adult T cells or irradiated cord blood T cells did produce immunoglobulin in response to PWM, but the response was significantly lower than that of adult B cells, and only IgM was produced in these cultures. These studies demonstrate that both the T and B cells of the human newborn have significant functional differences compared with the functions of T and B lymphocyte populations in adults.

The Epstein-Barr virus and the immune system

Publisher Summary This chapter focuses on both on the effects of Epstein–Barr virus (EBV) on human B cells and on the immune response to viral infection. The chapter also presents how this information helps understanding the diverse aspects of virus–-host interaction. Epstein–Barr virus is a ubiquitous and potentially oncogenic pathogen for man. Once infected with the virus, the B cell acquires the potential to grow indefinitely. Tumor development, however, rarely occurs in the EBV-infected individual because strict controls assure that the virus reaches a state of equilibrium with its host. Some of these controls are immunologic, and they act at several levels to suppress the growth of virally infected cells. Part of the uniqueness of the interaction between EBV and the immune system arises from the fact that the target for infection is the immune system itself. This imposes certain controls on the infected cells that derive from the multitude of regulatory pathways involving the B cell as a central element of the immune system. The interactions among cells of the immune system help unraveling the defense mechanisms against EBV. During primary infection, nonspecific suppressor and cytotoxic T cells that become activated in vivo regulate and destroy EBV-infected B cells. After primary infection, a few B cells continue to harbor EBV in a latent form, and, as a consequence, are capable of unlimited proliferation. At this time, in conjunction with nonspecific functions, virus-specific suppressor and cytotoxic T cells that become activated continuously act to limit the expansion of EBV-infected cells.

Generation of Epstein‐Barr Virus (EBV)–Immortalized B Cell Lines

Immortalization of B lymphocytes by EBV is an effective procedure for inducing long‐term growth of certain human B lymphocytes. The protocol described in this unit to accomplish this can be divided into three stages: preparation of virus, preparation of target cells to be immortalized, and EBV infection and growth of infected cells.