Marcello Mazzetti

Infection of peripheral mononuclear blood cells by hepatitis C virus

We investigated the infection of peripheral blood mononuclear cells (PBMNC) by hepatitis C virus (HCV) in 5 patients with HCV-related chronic hepatitis. The presence of HCV-RNA-positive and -negative strands was tested with the polymerase chain reaction (PCR) method. In all subjects, HCV-RNA was shown in PBMNC. In 3 cases, HCV-RNA was shown in the T- and B-cell populations, with viral RNA also present in the monocyte-macrophage fraction of two of these. HCV-RNA-negative stranded molecules, indicative of the viral multiplication, were significantly increased in cells maintained in cultures with PHA/PMA stimulation. The results indicate that HCV infect blood mononuclear cells, thus suggesting that this cellular tropism may play a role in HCV infection.

Detection by Flow Cytometry of ESAT-6- and PPD-Specific Circulating CD4+ T Lymphocytes as a Diagnostic Tool for Tuberculosis

Background: The identification of mycobacteria represents the gold standard in the diagnosis of tuberculosis (TB), but it is not applicable in all patients, and immunological tests, such as the tuberculin skin test (TST), are not specific and sensitive enough. Methods: By flow cytometry, we measured the CD4 T-cell response to purified protein derivative (PPD) and early secretory antigenic target-6 (ESAT-6) protein using the intracellular cytokine staining technique on whole blood samples obtained from active TB (n = 16), latent TB (n = 17), Bacille Calmette-Guérin (BCG)-vaccinated (n = 11) and healthy (n = 10) donors. All the patients were also tested with conventional TST. Results: The identification by flow cytometry of PPD-specific T lymphocytes upon antigen stimulation of whole blood enables the discrimination of active TB, latent TB and BCG-vaccinated subjects from healthy individuals, whereas the ESAT-6 response discriminated active TB from healthy and BCG-vaccinated individuals. Moreover, this test enables identification of active TB patients who were negative on TST and to distinguish between TB and non-typical mycobacteria TB infections. Conclusions: The identification by flow cytometry of antigen-specific T lymphocytes upon antigen stimulation of whole blood has a better positive predictive value than TST, and could represent a further tool in the diagnosis of TB infection.

T cells specific for Candida albicans antigens and producing type 2 cytokines in lesional mucosa of untreated HIV-infected patients with pseudomembranous oropharyngeal candidiasis

Factors influencing the susceptibility to mucosal candidiasis in HIV-infected patients are not clearly understood. Since in animal models of candidiasis the T helper (Th)1- or Th2-responses are protective or non-protective, respectively, this study was aimed to evaluate the cytokine profile of T-cell response to Candida albicans in the blood and lesional tissues of human immunodeficiency virus (HIV)-infected individuals, suffering, or not, from pseudomembranous oropharyngeal candidiasis (POPC), of HIV-negative women suffering from recurrent vaginal candidiasis (RVC) and of healthy controls. Peripheral blood mononuclear cells from HIV-infected and RVC patients proliferated to C. albicans antigen more than controls. Upon antigen activation, T cells from HIV-infected patients produced low interferon (IFN)-gamma, while only T cells from patients with POPC displayed high interleukin (IL)-4 and IL-5 production. POPC-positive patients also showed higher serum IgE levels than POPC-negative patients. T-cell clones generated from the oral mucosa of one HIV-infected patient with POPC produced IL-4, but not IFN-gamma (Th2 phenotype), whereas clones obtained from vaginal mucosa from one RVC patient or one healthy donor showed a Th1 profile. These findings, showing a non-protective Th0/Th2 response to C. albicans antigen in the blood and lesional mucosa of HIV-infected patients with POPC, may explain the high susceptibility of candidiasis in these subjects.

Enhanced expression of the CXCR4 co-receptor in HIV-1-infected individuals correlates with the emergence of syncytia-inducing strains

The aim of this study was to investigate the mechanisms responsible for the emergence in some HIV-1-infected individuals of highly aggressive, syncytia-inducing (SI) HIV-1 strains, which have been shown to use CXCR4 as co-receptor to enter target cells. To this end, the percentages of circulating CXCR4+CD4+ T cells were evaluated by flow cytometry in 39 untreated and 61 highly active antiretroviral therapy (HAART)-treated HIV-1-infected individuals in comparison with 35 HIV-1 seronegative subjects. Plasma viremia was also measured, and HIV primary isolates, from both untreated and HAART-treated HIV-1-infected subjects, were tested for the presence of SI strains. The results of this study showed enhanced proportions of CXCR4+CD4+ T cells in untreated patients in comparison with HAART-treated and healthy subjects. Furthermore, the results of a 12-month longitudinal study in a cohort of 11 patients undergoing HAART showed a significant reduction of CXCR4 expression after successful therapy. Finally, a significant positive correlation among the proportions of circulating CXCR4-expressing CD4+ T cells, plasma viremia, and the probability to isolate SI strains was found. These in vivo data are in keeping with previous in vitro results suggesting a bidirectional link between HIV-1 and CXCR4 expression on CD4+ T cells, and provide some clues to understanding the mechanisms exerting a selective pressure toward the emergence of SI strains.

Different reactivity of activated human B cells to B-cell growth factor and interleukin 2 in the costimulation assay with anti-IgM antibody and in the preactivation assay with Staphylococcus bacteria

The two main assay systems which have been developed for the study of lymphokine-mediated human B-cell proliferation, i.e., the costimulation assay with anti-mu antibody and the preactivation assay with Staphylococcus aureus Cowan I (SAC) bacteria, were compared. Purified interleukin 2 (IL-2), obtained by the recombinant DNA technology (r-IL-2), enhanced the proliferative response of anti-mu-stimulated human B cells in the costimulation assay with anti-mu antibody and maintained the B-cell proliferation induced by preactivation with SAC bacteria. Although the majority of T-cell clones, established from normal peripheral blood T lymphocytes, showed production of both IL-2 and B-cell growth factor (BCGF) following phytohemagglutinin (PHA)-stimulation, some T-cell clones were found whose supernatants (PHA-SN), apparently free of IL-2, manifested strong BCGF activity in the costimulation assay with anti-mu antibody. However, the same clonal, IL-2-free, T-cell SN displayed no BCGF activity in the preactivation assay with SAC bacteria. When B cells were activated for 3 days with anti-mu antibody, followed by the addition of r-IL-2 or clonal T-cell SN containing BCGF for an additional 3 days, r-IL-2 showed the ability to maintain B-cell proliferation, whereas clonal SN containing BCGF had virtually no effect. These data indicate that the costimulation assay with anti-mu antibody explores the reactivity of normal human B cells to both BCGF and IL-2, whereas the preactivation assay with SAC bacteria, due to a shorter reactivity to BCGF of activated human B cells, essentially represents a probe for the study of IL-2-promoted B-cell proliferation.

Increased production of IgE protein and IgE antibodies specific for fungal antigens in patients with the acquired immunodeficiency syndrome

SummaryLevels of IgE protein and IgE antibodies specific for 8 different allergenic extracts were measured in the serum of a large series of patients infected by the human immunodeficiency virus (HIV) and in HIV-seronegative subjects belonging to the same risk groups (intravenous drug-users, homosexual men and hemophiliacs). The proportion of subjects showing elevated IgE levels was higher among HIV-infected patients with group IV disease than among HIV-infected patients with group II–III diseases or seronegative individuals. In addition, many HIV-infected patients with elevated IgE levels showed the presence in their serum of IgE antibodies specific for fungal antigens.

Screening and confirmatory tests for antibody to HTLV-III/LAV retrovirus in individuals at risk for AIDS

SummaryThe prevalence of specific antibodies to HTLV-III/LAV retrovirus was investigated during 1985 in a large group of subjects at risk for AIDS living in the Florence area. Two hundred and thirty-two of 774 (29.9%) intravenous drug users (IDU), 40 of 164 homosexuals (24.3%), 43 of 147 hemophiliacs (29.2%), 7 of 10 children born from IDU mothers and 4 of 88 heterosexual partners of IDU were found to be seropositive using different ELISA kits. The positivity in all serum samples showing low antibody titres and/or discordant results with different sandwich ELISA kits was confirmed by a competition ELISA assay and the Western blot technique. Serum samples from 4 of 210 hemodialyzed and from one of 17 polytransfused patients also showed positive reactions in the assays based on the sandwich principle, but gave negative results in both the competition ELISA assay and the Western blot technique. In addition, all these sera showed positive reactions using immunofluorescence and ELISA procedures that control for reactivity with H9 human cell line material used for culturing the HTLV-III/LAV retrovirus. These data demonstrate that exposure to HTLV-III/LAV retrovirus is widespread in groups of subjects at risk for AIDS living in the Florence area. Furthermore, the results of the present study suggest that, in addition to the Western blot technique, the competition ELISA assay as well as assays that control reactivity with H9 human cell line material may be of value for detecting false positive reactions due to antibody cross-reactive with human cellular components.

In vitro Infection with hiv of antigen-specific t cell clones derived from hiv-seronegative individuals. effects on cytokine production and helper function

SummaryThree human T cell clones (TCC) specific for purified protein derivative of Mycobacterium tuberculosis were incubated in the presence of polybrene and phytohemagglutinin with irradiated mononuclear cells from one individual exhibiting seropositivity for human immunodeficiency virus (HIV) and high levels of circulating p24 antigen. After three weeks, TCC showed HIV integration in their DNA, as shown by polymerase chain reaction analysis and Southern blot technique. All the three HIV-infected TCC maintained their ability to recognize the specific antigen, even if their proliferative ability was reduced. The ability of the HIV-infected TCC to produce IL-2, IL-4 and IFN-y in response to phorbol myristate acetate plus anti-CD3 monoclonal antibody was decreased, whereas their ability to produce TNF-α was unaffected or even enhanced. Two our. of the three HIV-infected TCC showed the ability to provide helper function for polyclonal immunoglobulin production when cocukured with autologous B cells in the absence of any stimulant. These data suggest that in vitro infection of normal human TCC may provide a useful model for the study of immunological alterations induced by HIV.