Carla Montesano

Mycobacterium tuberculosis—Induced Apoptosis in Monocytes/Macrophages: Early Membrane Modifications and Intracellular Mycobacterial Viability

Apoptosis has been observed in monocytes/macrophages in the course of in vivo and in vitro Mycobacterium tuberculosis (MTB) infection. In order to define the early events of MTB-induced apoptosis, membrane CD14 expression and the exposure of Annexin V-binding sites in MTB-infected monocytes/macrophages have been monitored. Moreover, the role of MTB-induced apoptosis was further analyzed in vitro in terms of mycobacterial viability. Results show that monocyte/macrophage apoptosis is a very early event that is strictly dependent on the MTB amount, and this apoptosis is associated with a selective down-regulation of surface CD14 expression. Furthermore, no statistically significant decrease in mycobacterial viability was observed, which indicates that the apoptotic pathway triggered by high doses of MTB is associated with parasite survival rather than with killing of the parasite.

Infection of Human Monocytes with Mycobacterium tuberculosis Enhances Human Immunodeficiency Virus Type 1 Replication and Transmission to T Cells

Mycobacterium tuberculosis and human immunodeficiency virus type 1 (HIV-1) are virulent intracellular pathogens that invade and multiply within macrophages. The effect of M. tuberculosis on HIV-1 infection and replication was analyzed in vitro using human monocyte-derived macrophages (MDM) isolated from peripheral blood mononuclear cells by countercurrent centrifugal elutriation. Preinfection of MDM with M. tuberculosis followed by HIV-1 infection resulted in an increase in p24 release, reverse transcriptase activity, and infective virus production. In contrast, no increase in HIV-1 production was observed when MDM were infected with Mycobacterium avium complex or heat-killed M. tuberculosis. Coinfected MDM were potent stimulators of T cell proliferation, while HIV-1-infected MDM failed to present exogenous tuberculin to T cells. Furthermore, coinfected MDM showed an increased capacity to transmit HIV-1 to activated T cells. These results suggest that M. tuberculosis infection can both up-regulate HIV-1 infection and replication within MDM and increase the efficiency of virus transmission from infected MDM to T cells.

Expansion of pre-terminally differentiated CD8 T cells in chronic HIV-positive patients presenting a rapid viral rebound during structured treatment interruption.

Objective: The influence of structured treatment interruption on effector/memory CD8 T cell dynamics was analysed in chronic HIV-infected patients showing a rapid or delayed viral rebound. Design: Structured treatment interruption consisted of at least one month of discontinuation, followed by highly active antiretroviral therapy (HAART) resumption. Two groups of HIV structured treatment interruption patients were selected on the basis of plasma viral HIV-RNA value (> 30 000 copies/ml, branched DNA): group A (n = 14), patients with a rapid viral rebound (within one month) and group B (n = 6), patients with a delayed or no viral rebound (after a minimum of 4 months). Methods: A clinical and immunological follow-up was performed at HAART suspension (t 0), one month from suspension (t 1), at HAART resumption (t 2), and 30 days from resumption (t 3). Results: A sustained viral rebound was observed in group A patients, showing a rapid expansion of circulating CD8 T lymphocytes. In this group, the frequencies of CD8 T cells releasing IFN-γ after mitogen-induced or Gag-specific stimulation were highly increased after HAART discontinuation. Nevertheless, these CD8 T lymphocytes were mainly composed of pre-terminally differentiated cytotoxic T lymphocytes (CTL) expressing a CCR7− CD27+/− CD45RA− phenotype and a reduced amount of perforin. In contrast, group B patients showed no significant changes in immunological parameters during a prolonged drug-free period. Conclusion: These data indicate that monitoring CD8 T cell dynamics during structured treatment interruption could be clinically relevant, and new therapeutic strategies should aim qualitatively to restore CTL effector functions.

Antioxidant and anti-inflammatory activities of extracts from Cassia alata, Eleusine indica, Eremomastax speciosa, Carica papaya and Polyscias fulva medicinal plants collected in Cameroon

Background The vast majority of the population around the world has always used medicinal plants as first source of health care to fight infectious and non infectious diseases. Most of these medicinal plants may have scientific evidence to be considered in general practice. Objective The aim of this work was to investigate the antioxidant capacities and anti-inflammatory activities of ethanol extracts of leaves of Cassia alata, Eleusine indica, Carica papaya, Eremomastax speciosa and the stem bark of Polyscias fulva, collected in Cameroon. Methods Chemiluminescence was used to analyze the antioxidant activities of plant extracts against hydrogen peroxide or superoxide anion. Comet assays were used to analyze the protection against antioxidant-induced DNA damage induced in white blood cells after treating with hydrogen peroxide. Flow cytometry was used to measure γδ T cells proliferation and anti-inflammatory activity of γδ T cells and of immature dendritic cells (imDC) in the presence of different concentrations of plant extracts. Results Ethanol extracts showed strong antioxidant properties against both hydrogen peroxide and superoxide anion. Cassia alata showed the highest antioxidant activity. The effect of plant extracts on γδ T cells and imDC was evidenced by the dose dependent reduction in TNF-α production in the presence of Cassia alata, Carica papaya, Eremomastax speciosa Eleusine indica, and Polyscias fulva. γδ T cells proliferation was affected to the greatest extent by Polyscias fulva. Conclusion These results clearly show the antioxidant capacity and anti-inflammatory activities of plant extracts collected in Cameroon. These properties of leaves and stem bark extracts may contribute to the value for these plants in traditional medicine and in general medical practice.

Levels of Interleukin-15 in Plasma May Predict a Favorable Outcome of Structured Treatment Interruption in Patients with Chronic Human Immunodeficiency Virus Infection

Structured treatment interruption (STI) may help to alleviate the problems associated with long-term antiretroviral therapy (ART) in human immunodeficiency virus (HIV)-infected patients. We analyzed the role that baseline levels of cytokines in plasma play as markers of a favorable outcome of STI. Two groups of patients were defined: STI responders and STI nonresponders. STI responders showed a higher baseline concentration of interleukin (IL)-15 in plasma than did STI nonresponders and showed lower levels of tumor necrosis factor (TNF)-alpha during STI. No differences were observed in levels of IL-2, IL-7, or interferon-alpha in plasma. Our data show that (1) levels of TNF-alpha in plasma correlate with HIV viremia and (2) monitoring baseline levels of IL-15 in plasma allows for the identification of a favorable outcome of STI.

Therapeutic DNA Vaccination of Vertically HIV-Infected Children: Report of the First Pediatric Randomised Trial (PEDVAC)

Subjects Twenty vertically HIV-infected children, 6–16 years of age, with stable viral load control and CD4+ values above 400 cells/mm3. Intervention Ten subjects continued their ongoing antiretroviral treatment (ART, Group A) and 10 were immunized with a HIV-DNA vaccine in addition to their previous therapy (ART and vaccine, Group B). The genetic vaccine represented HIV-1 subtypes A, B and C, encoded Env, Rev, Gag and RT and had no additional adjuvant. Immunizations took place at weeks 0, 4 and 12, with a boosting dose at week 36. Monitoring was performed until week 60 and extended to week 96. Results Safety data showed good tolerance of the vaccine. Adherence to ART remained high and persistent during the study and did not differ significantly between controls and vaccinees. Neither group experienced either virological failure or a decline of CD4+ counts from baseline. Higher HIV-specific cellular immune responses were noted transiently to Gag but not to other components of the vaccine. Lymphoproliferative responses to a virion antigen HIV-1 MN were higher in the vaccinees than in the controls (p = 0.047), whereas differences in reactivity to clade-specific Gag p24, RT or Env did not reach significance. Compared to baseline, the percentage of HIV-specific CD8+ lymphocytes releasing perforin in the Group B was higher after the vaccination schedule had been completed (p = 0.031). No increased CD8+ perforin levels were observed in control Group A. Conclusions The present study demonstrates the feasibility, safety and moderate immunogenicity of genetic vaccination in vertically HIV-infected children, paving the way for amplified immunotherapeutic approaches in the pediatric population. Trial registration clinicaltrialsregister.eu _2007-002359-18 IT

Aloe-emodin as antiproliferative and differentiating agent on human U937 monoblastic leukemia cells

AIMS Aloe-emodin (AE), a plant derived anthraquinone, has been shown to have anticancer activity in various human cancer cell lines. We have recently reported that AE possesses a differentiative potential on melanoma cells. The purpose of this study was to investigate the possible modulation of defined markers of monocytic differentiation of AE on human U937 cell line. MAIN METHODS U937 cells differentiation has been confirmed unequivocally by Griess and nitroblue tetrazolium reduction assays, protoporphyrin IX accumulation, expression of CD14 and CD11b surface antigens, phagocytic activity, migration and attachment ability. The effect on polyamine metabolism, apoptosis and cytokine production was also investigated. KEY FINDINGS We showed that AE-treated U937 cells exhibit a noticeably rise in transglutaminase activity. This enhanced enzyme activity correlates with AE-induced growth arrest and differentiation to functionally mature monocytes. SIGNIFICANCE Taken together, the results reported here show that AE can promote the macrophage differentiation of U937 cells, suggesting that this anthraquinone could be a potential candidate as a differentiation-inducing selective agent for therapeutic treatment of leukemia.

Asymptomatic infection and unrecognised Ebola virus disease in Ebola-affected households in Sierra Leone: a cross-sectional study using a new non-invasive assay for antibodies to Ebola virus

BACKGROUND The frequency of asymptomatic infection with Ebola virus is unclear: previous estimates vary and there is no standard test. Asymptomatic infection with Ebola virus could contribute to population immunity, reducing spread. If people with asymptomatic infection are infectious it could explain re-emergences of Ebola virus disease (EVD) without known contact. METHODS We validated a new oral fluid anti-glycoprotein IgG capture assay among survivors from Kerry Town Ebola Treatment Centre and controls from communities unaffected by EVD in Sierra Leone. We then assessed the seroprevalence of antibodies to Ebola virus in a cross-sectional study of household contacts of the survivors. All household members were interviewed. Two reactive tests were required for a positive result, with a third test to resolve any discrepancies. FINDINGS The assay had a specificity of 100% (95% CI 98·9-100; 339 of 339 controls tested negative) and sensitivity of 95·9% (89·8-98·9; 93 of 97 PCR-confirmed survivors tested positive). Of household contacts not diagnosed with EVD, 47·6% (229 of 481) had high level exposure (direct contact with a corpse, body fluids, or a case with diarrhoea, vomiting, or bleeding). Among the contacts, 12·0% (95% CI 6·1-20·4; 11 of 92) with symptoms at the time other household members had EVD, and 2·6% (1·2-4·7; 10 of 388) with no symptoms tested positive. Among asymptomatic contacts, seropositivity was weakly correlated with exposure level. INTERPRETATION This new highly specific and sensitive assay showed asymptomatic infection with Ebola virus was uncommon despite high exposure. The low prevalence suggests asymptomatic infection contributes little to herd immunity in Ebola, and even if infectious, would account for few transmissions. FUNDING Wellcome Trust ERAES Programme, Save the Children.

Delayed early antiretroviral treatment is associated with an HIV-specific long-term cellular response in HIV-1 vertically infected infants

Antiviral T-cell immune responses appear to be crucial to control HIV replication. Infants treated before the third month of life with highly active antiretroviral treatment (HAART) did not develop a persistent HIV-specific immune response. We evaluated how delayed initiation of HAART after 3 months of age influences the development of HIV-1-specific T-cell responses during long-term follow-up in 9 HIV-1 vertically infected infants. These data suggest that a longer antigenic stimulation, due to a larger window for therapeutic intervention with HAART, is associated with the establishment of a persistent specific HIV immune response resulting in a long-term viral control of vertically infected infants.

In HIV-positive patients, myeloid-derived suppressor cells induce T-cell anergy by suppressing CD3ζ expression through ELF-1 inhibition.

Objective:During HIV infection, a down-modulation of CD3&zgr; was found on T cells, contributing to T-cell anergy. In this work, we studied the correlation between myeloid-derived suppressor cells (MDSC) frequency and T-cell CD3&zgr; expression. Moreover, we investigated the mechanisms of CD3&zgr; decrease exploited by MDSC. Design and method:CD3&zgr; expression and MDSC frequency were evaluated by flow cytometry on peripheral blood mononuclear cells from 105 HIV-positive (HIV+) patients. The role of MDSC in the modulation of the HIV-specific T-cell response was evaluated. The level of CD3&zgr; mRNA and ELF-1 protein were analysed by real-time–PCR and western blot, respectively. Results:We found that granulocytic-MDSC (Gr-MDSC) were expanded in HIV+ patients compared with healthy donors; in particular, in cART-treated individuals a higher Gr-MDSC frequency was observed in patients with a CD4+ T-cell count below 400 cells/&mgr;l. We found an inverse correlation between the percentage of Gr-MDSC and CD3&zgr; level. Moreover, in-vitro MDSC depletion induced the up-regulation of CD3&zgr; in T cells, restoring the functionality of &agr;&bgr;, but not &ggr;&dgr; T cells. The in-vitro effect of isolated MDSC on CD3&zgr; expression was found cell contact-dependent, and was not mediated by previously described molecules. CD3&zgr; down-modulation corresponds to the decrease of its mRNA induced by silencing the transcription factor ELF-1. Conclusion:Our data provide new knowledge on mechanisms used by Gr-MDSC in immune-modulation and on their role in the immune reconstitution during antiviral treatments.