Raffaella Rossi

In Vivo and In Vitro Effects of Antituberculosis Treatment on Mycobacterial Interferon-γ T Cell Response

Background In recent years, the impact of antituberculous treatment on interferon (IFN)-γ response to Mycobacterium tuberculosis antigens has been widely investigated, but the results have been controversial. The objective of the present study was: i) to evaluate longitudinal changes of IFN-γ response to M. tuberculosis-specific antigens in TB patients during antituberculous treatment by using the QuantiFERON-TB Gold (QFT-G) assay; ii) to compare the differences in T-cell response after a short or prolonged period of stimulation with mycobacterial antigens; iii) to assess the CD4+ and CD8+ T cells with effector/memory and central/memory phenotype; iv) to investigate the direct in vitro effects of antituberculous drugs on the secretion of IFN-γ. Principal Findings 38 TB patients was evaluated at baseline and at month 2 and 4 of treatment and at month 6 (treatment completion). 27 (71%) patients had a QFT-G reversion (positive to negative) at the end of therapy, while 11 (29%) TB patients remained QFT-G positive at the end of therapy. Among the 11 patients with persistent positive QFT-G results, six had a complete response to the treatment, while the remaining 5 patients did not have a resolution of the disease. All 27 patients who became QFT-G negative had a complete clinical and microbiological recovery of the TB disease. In these patients the release of IFN-γ is absent even after a prolonged 6-day incubation with both ESAT-6 and CFP-10 antigens and the percentage of effector/memory T-cells phenotype was markedly lower than subjects with persistent positive QFT-G results. The in vitro study showed that antituberculous drugs did not exert any inhibitory effect on IFN-γ production within the range of therapeutically achievable concentrations. Conclusions The present study suggests that the decrease in the M. tuberculosis-specific T cells responses following successful anti-TB therapy may have a clinical value as a supplemental tool for the monitoring of the efficacy of pharmacologic intervention for active TB. In addition, the antituberculous drugs do not have any direct down-regulatory effect on the specific IFN-γ response.

Influence of previous tuberculin skin test on serial IFN-γ release assays

Tuberculin skin test (TST) and interferon-γ release assays (IGRAs) have been proposed for serial testing in tuberculosis. In the present study, we assessed the effect of TST on subsequent QuantiFERON-TB Gold In-Tube (QFT-GIT) results by monitoring the evolution of responses during a follow-up period of 6 weeks. One hundred and two subjects were initially tested with QFT-GIT and subsequently with TST; then the QFT-GIT was performed serially 1, 2, 4, and 6 weeks after the TST. A subgroup of 40 subjects was also assessed by older version of the QuantiFERON-TB Gold (QFT-G) assay. The results showed no significant variation in IFN-γ response over time in the tested patients, although two TST-positive subjects showed evidence of possible boosting effect. In addition, a direct comparison between the QFT-G and QFT-GIT test showed no significant differences at any time point with excellent agreement between two tests. No significant differences were seen in IFN-γ responses between BCG-unvaccinated and BCG-vaccinated patients at each time point. In conclusion, our findings indicate that TST does not influence the outcome of subsequent IGRAs testing in individuals with negative TST results, but it can boost the IFN-γ response in subjects sensitized to TB antigens and not detected by IGRA.

Plasmacytoid Dendritic Cells Count in Antiretroviral-Treated Patients is Predictive of HIV Load Control Independent of CD4+ T-Cell Count

The depletion in circulating dendritic cells (DCs) and inverse correlation with viral load have been described in human immunodeficiency virus (HIV)-infected patients. The aim of this study was to investigate whether the DC blood count in antiretroviral-treated patients might be predictive of viral load control independent of CD4+ T cell count. Plasmacytoid DCs (pDCs) and myeloid DCs (mDCs) were enumerated using a newly developed flow cytometric assay based on TruCOUNT. A significant reduction of circulating pDCs and mDCs was detected both in untreated and -treated subjects. The probability of experiencing viral load increase according to pDC, and CD4 count at baseline was evaluated in 39 treated patients. Individuals with lower baseline pDCs were more likely to have an increase of HIV-RNA during the 30 month follow-up in comparison with patients with high pDCs (p <0.001). In particular, the pDC measurement may be useful in the context of a high CD4 count, to distinguish the patients who have virological failure despite high CD4 counts. These findings suggest that in treated patients the enumeration of circulating DCs, especially pDC count, can augment the predictive value of CD4 measurement in predicting virologic failure.

Severe and Persistent Depletion of Circulating Plasmacytoid Dendritic Cells in Patients with 2009 Pandemic H1N1 Infection

Background Dysregulation of host immune responses plays a critical role in the pathogenesis of severe 2009 pandemic H1N1 infection. Whether H1N1 virus could escape innate immune defense in vivo remains to be investigated. The aim of this study was to evaluate the pattern of innate immune response during human 2009 H1N1 infection. We performed the enumeration of circulating myeloid dendritic cells (mDC) and plasmacytoid DC (pDC) in blood from patients with H1N1 pneumonia shortly after the onset of symptoms and during follow-up at different intervals of time. The analysis of CD4 and CD8 count, CD38 T-cell activation marker and serum cytokine/chemokine plasma levels was also done. Methodology/Principal Findings Blood samples were collected from 13 hospitalized patients with confirmed H1N1-related pneumonia at time of admission and at weeks 1, 4, and 16 of follow-up. 13 healthy donors were enrolled as controls. In the acute phase of the disease, H1N1-infected patients exhibited a significant depletion in both circulating pDC and mDC in conjunction with a decrease of CD4 and CD8 T cell count. In addition, we found plasmatic hyperproduction of IP-10 and RANTES, whereas increase in T-cell immune activation was found at all time points. When we assessed the changes in DC count over time, we observed a progressive normalization of mDC number. On the contrary, H1N1-infected patients did not achieve a complete recovery of pDC count as values remained lower than healthy controls even after 16 weeks of follow-up. Conclusions H1N1 disease is associated with a profound depletion of DC subsets. The persistence of pDC deficit for several weeks after disease recovery could be due to H1N1 virus itself or to a preexisting impairment of innate immunity.

HIV protease inhibitor therapy reverses neutrophil apoptosis in AIDS patients by direct calpain inhibition

The reduction of neutrophils apoptosis is one of the main non-virological effects of protease inhibitor (PI) therapy. We explore here whether this may be due to the cross-inhibition of calpain, an important non-virological target of PI in vitro. We found that the high basal level of neutrophils apoptosis in AIDS patients is strictly related to an increased intracellular calpain activity. Both alterations disappear after PI treatment, with apoptosis and calpain going back to normal levels after 3 months of PI therapy, independently of a proficient antiviral effect. PI drugs exerted a similar antiapoptotic and anticalpain effects on neutrophils in ex vivo experiments: strikingly, the effects were mimicked by commercially available calpain inhibitors. This study shows, for the first time, that apoptosis of neutrophils in AIDS patients is mediated by calpain, and that neutrophil survival in PI treated AIDS patients is a non virological effect due to calpain inhibition.

QuantiFERON-TB Gold and selected region of difference 1 peptide-based assays for the detection of Mycobacterium tuberculosis infection in a cohort of patients enrolled with suspected tuberculosis

Recently, a simple whole blood test, QuantiFERON-TB Gold (QFT-Gold), has been adopted for immunologic diagnosis of Mycobacterium tuberculosis infection. A total of 369 individuals with suspected tuberculosis (TB) were tested with the QFT-Gold, whereas a subgroup of 62 patients was tested simultaneously with QFT-Gold test and an assay based on selected peptides from early secreted antigenic target 6 and culture filtrate protein 10 proteins. The sensitivity of the QFT-Gold assay for active TB was 72.9%, and the agreement between this test and tuberculin skin test was 75%. Using selected peptides, we found a positive response in the majority of active TB patients, with a sensitivity of 84% and a specificity of 89%. Our study suggests the usefulness of QFT-Gold in routine clinical practice on unselected patients with suspected TB. Moreover, the assay based on selected peptides is a useful tool to discriminate between active and latent TB and shows a high predictive value.

Active HCV infection is associated with increased circulating levels of interferon-gamma (IFN-γ)-inducible protein-10 (IP-10), soluble CD163 and inflammatory monocytes regardless of liver fibrosis and HIV coinfection

BACKGROUND AND OBJECTIVE Interferon-gamma (IFN-γ)-inducible protein-10 (IP-10), soluble (s) CD163 and sCD14 play an important role in the pathogenesis of HCV and HIV infection and are involved in inflammation and liver fibrosis. The aim of the present study was to evaluate at a single time point, plasma soluble biomarkers and inflammatory monocytes subsets in different groups of subjects: (i) HIV monoinfected patients on suppressive ART; (ii) HIV/HCV coinfected patients on ART, with undetectable HIV viremia (including either subjects who had active HCV replication or those who cleared HCV); (iii) HCV monoinfected individual with active viral replication. METHODS Hundred and twenty-nine plasma samples were analyzed including HCV and HIV monoinfected patients, HIV/HCV coinfected patients, with active HCV infection (AHI) or with HCV viral clearance (VHC) and healthy donors (HD). Levels of IP-10, sCD163 and sCD14 were measured by ELISA. Absolute cell counts of monocyte subpopulations were enumerated in whole blood by using flow cytometric analyses. RESULTS IP-10 and sCD163 plasma levels were higher in HCV monoinfected and in AHI coinfected pts compared to HIV monoinfected and HD, whereas sCD14 levels were higher only in HIV monoinfected patients. Considering the degree of fibrosis, sCD163 and sCD14 levels positively correlated with kPa values (as assessed by fibroscan) and FIB-4 in HCV monoinfected group. On the other hand, IP-10 did not correlate with the fibrosis stage and it was found increased also in patients with low fibrosis. Moreover, we found an increase of the inflammatory NCM subset, in non-cirrhotic HCV subjects, while no alterations were observed in HIV, AHI and VHC. CONCLUSIONS Our study suggests a scenario in which active HCV infection is associated with a strong pro-inflammatory state, even in the initial stage of liver fibrosis, regardless the presence of HIV coinfection, thus underlying the need of an early anti-HCV treatment.

Blood Myeloid Dendritic Cells and slanDC in Antiretroviral Therapy- Suppressed HIV-Infected Patients.

Myeloid dendritic cells (mDCs) play a complex role in HIV infection regardless of viral replication. The aim of our study was to analyse mDCs in long term antiretroviral therapy (ART)- suppressed HIV-infected patients. We evaluated the numbers of mDCs and slanDC in the context of different degree of CD4+ T cell recovery, persistent T cell activation (as HLA-DR+/CD3+ expression) and monocyte-macrophage activation assessed in terms of circulating levels of both sCD14 and sCD163. We enrolled 72 aviremic patients under effective ART and 34 healthy donors (HD). Patients were divided into two groups on the bases of ΔCD4, indicating the difference between the value of CD4 at the time of sampling and CD4 nadir. Higher levels of mDCs and slanDC were found in patients with ΔCD4>200/mmc in comparison to HD. In those patients also an increased level of sCD14 was found, whereas sCD163 seemed to be at normal levels. An augmentation of activated CD4 T lymphocytes was found, although less pronounced in patients with ΔCD4<200/mmc. In conclusion, our findings showed an expansion of mDCs with a shift to inflammatory slanDC that could sustain both microbial translocation and HIV latency in CD4 T cells.