Barbara Mantelli

The Binding Subunit of Pertussis Toxin Inhibits HIV Replication in Human Macrophages and Virus Expression in Chronically Infected Promonocytic U1 Cells

We have recently shown that the binding subunit of pertussis toxin (PTX-B) inhibits the entry and replication of macrophage-tropic (R5) HIV-1 strains in activated primary T lymphocytes. Furthermore, PTX-B suppressed the replication of T cell-tropic (X4) viruses at a postentry level in the same cells. In this study we demonstrate that PTX-B profoundly impairs entry and replication of the HIV-1ADA (R5), as well as of HIV pseudotyped with either murine leukemia virus or vesicular stomatitis virus envelopes, in primary monocyte-derived macrophages. In addition, PTX-B strongly inhibited X4 HIV-1 replication in U937 promonocytic cells and virus expression in the U937-derived chronically infected U1 cell line stimulated with cytokines such as TNF-α and IL-6. Of interest, TNF-α-mediated activation of the cellular transcription factor NF-κB was unaffected by PTX-B. Therefore, PTX-B may represent a novel and potent inhibitor of HIV-1 replication to be tested for efficacy in infected individuals. In support of this proposition, a genetically modified mutant of PTX (PT-9K/129G), which is safely administered for prevention of Bordetella pertussis infection, showed an in vitro anti-HIV profile superimposable to that of PTX-B.

Dual role of TNF‐α in NK / LAK cell‐mediated lysis of chronically HIV‐infected U1 cells. Concomitant enhancement of HIV expression and sensitization of cell‐mediated lysis

The U937‐derived chronically HIV‐infected U1 cell line and uninfected U937 cell clones were efficiently lysed by both unstimulated (NK) and IL‐2‐stimulated (lymphokine‐activated killer; LAK) peripheral blood mononuclear cells (PBMC) of healthy HIV‐seronegative donors. Pretreatment of target cells with IFN‐γ down‐modulated killing of both U1 cells and two U937 cell clones, and up‐regulated MHC class I expression. In contrast, TNF‐α enhanced the sensitivity of infected U1 cells, but not of U937 cell clones to NK / LAK cell lysis. Co‐cultivation of IL‐2‐stimulated PBMC with U1 cells triggered expression and replication of HIV by cell‐cell contact, and this effect was inhibited by anti‐TNF‐α antibodies (Ab); virus production was partially inhibited by zidovudine. Of interest, anti‐TNF‐α Ab protected U1 cells from LAK cell activity. Thus, TNF‐α can induce HIV expression from chronically infected U1 cells, but also plays an important role in sensitizing these cells to lysis.

A simplified flow cytometry method of CD4 and CD8 cell counting based on thermoresistant reagents: implications for large scale monitoring of HIV-infected patients in resource-limited settings.

To validate a simplified flow cytometry assay for CD4 and CD8 T cell counting based on monoclonal antibodies which are made resistant to high temperatures (simplified thermoresistant assay (STRA)).

Double-edged effect of Vγ9/Vδ2 T lymphocytes on viral expression in an in vitro model of HIV-1/mycobacteria co-infection

A reciprocal influence exists between mycobacteria and HIV: HIV‐infected individuals are more susceptible to mycobacterial infections and, on the other hand, mycobacterial infection results inacceleration of HIV disease progression. Vγ9/Vδ2 T lymphocytes are known to participate in the defense against intracellular pathogens, including Mycobacterium tuberculosis. Indeed, they kill mycobacteria‐infected macrophages and, upon recognition of mycobacterial Ag, release TNF‐α and IFN‐γ, which are also up‐regulators of HIV expression. To assess whether mycobacteria‐activated γ δ T lymphocytes contribute to the enhancement of HIV replication, we established an in vitro model mimicking HIV and mycobacteria co‐infection with the latently HIV‐infected promonocytic U1 cell line and Vγ9/Vδ2 peripheral lymphocytes stimulated with mycobacterial Ag. γ δ T cell activation determined two distinct, but connected effects, namely U1cell death and HIV expression. Both effects were mainly mediated by release of TNF‐α and IFN‐γ from activated γ δ lymphocytes, although Fas‐FasL interaction also contributed to U1 apoptosis. The final outcome on U1 survival, and thus, on HIV expression, highly depended on mycobacterial Ag concentration coupled to the differential secretory potency of γ δ cells. In particular, the induction of viral expression prevailed at low Ag concentration and with lower cytokine production by mycobacteria‐activated γ δ cells. Notably, during the course of HIV infection, Vγ9/Vδ2 lymphocytes are reported to be functionally impaired and may thus indirectly influence the progression of HIV disease. In addition, a predominant inhibition of viral replication was encountered when mycobacteria‐activated γ δ T cells were co‐cultured with primary HIV‐infected macrophages. Thus, we suggest that specific recognition of mycobacterial Ag by γ δ T lymphocytes in co‐infected individuals may modulate viral replication through the complex array of soluble factors released.

CD30 ligation differentially affects CXCR4-dependent HIV-1 replication and soluble CD30 secretion in non-Hodgkin cell lines and in γ δ T lymphocytes

We studied whether signaling through CD30, a member of the TNF receptor family, affected acute infection with HIV‐1, encompassing its entire replicative cycle. Several non‐Hodgkin cell lines, targets of CXCR4‐dependent (X4) HIV‐1 infection, were positive for CD30 expression. CD30 ligation induced up‐regulation of viral replication only in certain CD30+ cell lines. Enhancement ofX4 virus replication by CD30 engagement inversely correlated with both CD30 surface density and constitutive NF‐κB activation. Conversely, expression of CD30, but not of other members of the TNF receptor family, was proportional to constitutive NF‐κB binding. Concomitantly, secretion of soluble (s) CD30 increased in all cell lines by CD30 ligation. sCD30 release was enhanced by engagement of CD30 alone and, to a greater extent, by co‐engagement of CD3 also in primary γ δ T lymphocytes, along with complementary modulations of their surface CD30 expression. sCD30‐containing supernatant specifically inhibited HIV‐1 expression induced by CD30 engagement in chronically infected ACH‐2 T cells; thus sCD30 may act as a negative feed‐back molecule. In conclusion, we have delineated novel features of CD30 biology and underline the peculiar link of CD30 expression to constitutive NF‐κB activation which is pivotal to both HIV replication and cell survival.