Fabio Mengoni

Improvement in neutrophil and monocyte function during highly active antiretroviral treatment of HIV-1-infected patients

OBJECTIVE To investigate the effect of highly active antiretroviral treatment (HAART) on neutrophil and monocyte function in patients with moderately advanced HIV-1 infection. DESIGN Eighteen HIV-1-infected patients with CD4 T cell counts below 350/microl, no concomitant active infection, and no previous use of protease inhibitors were treated with indinavir or ritonavir and two reverse-transcriptase inhibitors and were followed up for 9 months. Ten age- and sex-matched healthy subjects were included as controls. METHODS The functional activity of neutrophils and monocytes was measured by assessing chemotaxis towards a bacterial peptide, killing activity against Candida albicans, and oxidative burst as measured by chemiluminescence production. RESULTS Neutrophils and monocytes from the treatment group exhibited a significantly diminished baseline chemotactic and fungicidal activity compared with healthy controls (P < 0.001). After starting HAART, there was a significant improvement in chemotaxis and fungicidal activity of phagocytic cells (P < 0.001). Values of chemotaxis reached normal ranges in 13 out of 18 patients (72%) for neutrophils and eight out of 18 (44%) for monocytes, whereas phagocyte killing was rarely restored to normal values (3/18 cases for monocytes and 0/18 for neutrophils). The administration of HAART was also associated with significantly increased phagocyte chemiluminescence production in response to phorbol-12-myristate 13-acetate or opsonized C. albicans (P < 0.01). CONCLUSION The functional improvement of two critical components of innate antimicrobial immunity, such as neutrophils and monocytes, may contribute to the improved cell-mediated immune responses against opportunistic infections in HAART-treated patients.

Interleukin-15 in HIV infection: immunological and virological interactions in antiretroviral-naive and -treated patients.

Objective To investigate the immunological and virological interactions between interleukin (IL)-15 and HIV in antiretroviral-naive and highly active antiretroviral therapy (HAART)-treated patients. Design Three groups of HIV-infected patients were studied: 20 untreated patients with advanced disease; eight patients with viral suppression and immunological response to HAART; and 10 patients with virological and immunological treatment failure. Eleven healthy blood donors were included as controls. Methods The following parameters were evaluated: the production of IL-15 by peripheral blood mononuclear cells (PBMC) stimulated with lipopolysaccharide, Candida albicans and Mycobacterium avium complex; the ability of IL-15 to induce the secretion of IL-8 and monocyte chemotactic protein-1 (MCP-1) from HIV-positive monocytes; and the virological effect of IL-15 and IL-2 on HIV replication in mononuclear cells. Results IL-15 production by PBMC was significantly decreased in antiretroviral-naive patients and in those with treatment failure. On the contrary, in patients with response to HAART IL-15 production was comparable to that of healthy donors. IL-15 was able to stimulate HIV-positive monocytes to produce chemokines, such as IL-8 and MCP-1, that specifically attract neutrophils and monocytes to the site of inflammation thus possibly improving immune response to pathogens during HIV infection. Finally, IL-15 had no major effect on HIV replication in vitro, while only simultaneous administration with IL-2 may induce high levels of HIV production. Conclusions This in vitro study provides new insights in the area of IL-15–HIV interactions and suggests that IL-15 may represent a potential candidate for cytokine treatment in combination with HAART during HIV infection.

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.

Chemokine profiles in the cerebrospinal fluid (CSF) during the course of pyogenic and tuberculous meningitis

The concentrations of the chemokines IL‐8, monocyte chemotactic protein‐1 (MCP‐1) and macrophage inflammatory protein‐1α (MIP‐1α) were measured in 120 CSF samples from 23 patients with pyogenic meningitis and from 11 patients with tuberculous meningitis (TBM) and in 10 CSF from subjects with non‐infectious neurological diseases. The chemokine concentrations in patients with meningitis were significantly higher than in control subjects (P < 0.0001). The highest CSF levels were found for IL‐8 (median 2917 pg/ml) and MCP‐1 (median 2557 pg/ml), whereas those of MIP‐1α were less significantly elevated (median 24 pg/ml) (P < 0.0001). Patients with pyogenic meningitis had higher levels of IL‐8 and MCP‐1 than those with TBM (P < 0.0001). In serial samples from patients with pyogenic meningitis IL‐8 levels declined before MCP‐1 and MIP‐α. In the case of TBM, IL‐8, MCP‐1 and MIP‐1α decreased more gradually during treatment and were detectable in the CSF for several weeks, without any characteristic time course of elimination. These data indicate that patients with pyogenic meningitis and TBM show different chemokine profiles in CSF. The distinct chemokine pattern could be responsible for a differential attraction and activation of leucocytes in the CSF which is reflected in differences in the inflammatory response and clinical course of pyogenic meningitis and TBM.

In vitro effect of anti-human immunodeficiency virus CCR5 antagonist maraviroc on chemotactic activity of monocytes, macrophages and dendritic cells

Compounds targeting the chemokine receptor CCR5 have recently been approved for treatment of human immunodeficiency virus (HIV) infection. Given the central role of CCR5 in inflammation and recruitment of antigen‐presenting cells (APC), it is important to investigate the immunological consequences of pharmacological inhibition of CCR5. We evaluated the in vitro effect of different concentrations of CCR5 antagonist maraviroc (MVC) on cell migration of monocytes, macrophages (MO) and monocyte‐derived dendritic cells (MDC) towards peptide formyl‐methionyl‐leucyl‐phenylalanine (fMLP) and chemokines regulated upon activation normal T cell expressed and secreted (RANTES) and CCL4/macrophage inflammatory protein‐1 (MIP‐1β) and CCL2/monocyte chemotactic protein‐1 (MCP‐1). Results of flow cytometric analysis showed that monocytes treated in vitro with MVC exhibited a significant dose‐dependent reduction of chemotaxis towards MIP‐1β and MCP‐1. fMLP‐induced chemotactic activity decreased only at higher concentration (1 µM and 10 µM of MVC). In addition, all concentrations of MVC (0·1, 1 and 10 µM) induced in vitro a significant inhibition of chemotaxis of MO and MDC in response to all tested chemoattractants. No change in phenotype (CD1a and CD14) and CCR1, CCR4, CCR5 and formyl peptide receptor (FPR) expression was seen after in vitro treatment with MVC. These findings suggest that CCR5 antagonist MVC may have the in vitro ability of inhibiting the migration of innate immune cells by mechanism which could be independent from the pure anti‐HIV effect. The drug might have a potential role in the down‐regulation of HIV‐associated chronic inflammation by blocking the recirculation and trafficking of MO and MDC.

Circulating dendritic cells and interferon‐α production in patients with tuberculosis: correlation with clinical outcome and treatment response

Dendritic cells (DC) have been characterized recently as having an important role in the initiation and control of immunological response to Mycobacterium tuberculosis infection. Blood DC have been subdivided into myeloid (mDC) and plasmacytoid (pDC) subsets, on the basis of differences in phenotype markers and function. Little is known about the enumeration and functional evaluation of circulating DC in patients with tuberculosis and their correlation with clinical outcome during the course of anti‐tuberculous treatment. We assessed circulating mDC and pDC counts measured by a newly developed single‐platform flow cytometric assay based on TruCOUNT, as well as the production of interferon (IFN)‐α after in vitro stimulation by herpes simplex virus (HSV‐1) in 24 patients with active tuberculosis (TB) and 37 healthy donors. Absolute numbers of both DC subsets were decreased significantly in patients with active TB compared to controls. Similarly, the production of IFN‐α was highly impaired. In 13 patients these parameters were assessed longitudinally, before and after the specific anti‐microbial treatment. Most interestingly, in all nine patients with successful anti‐tuberculous therapy there was a significant and marked increase of pDC counts and IFN‐α production. In contrast, no significant longitudinal variations in DC counts and IFN‐α production were observed in four patients with lack of response to specific treatment. In conclusion, active TB is associated with a defect in blood DC numbers and IFN‐α production that is restored after bacterial clearance and clinical improvement, as a result of effective anti‐tuberculous treatment.

Circulating levels of interleukin-7 in antiretroviral-naïve and highly active antiretroviral therapy-treated HIV-infected patients

Abstract Interleukin (IL)-7 is a critical cytokine regulating T-lymphocyte development, regeneration, and function. Purpose: This study analyzes the endogenous IL-7 production in HIV-infected patients receiving highly active antiretroviral treatment (HAART). Method: Plasma levels of IL-7 were measured by enzyme-linked immunosorbent assay (ELISA) in 11 patients with untreated advanced HIV disease, in 8 patients who successfully responded to HAART, and in 9 individuals with virological and immunological treatment failure. Results: We found that in the patients with advanced HIV disease and no treatment IL-7 concentrations were elevated and were inversely related to both CD4 + and CD8 + T-cell counts. When IL-7 was assessed in treated patients, this cytokine was below the detection limit of the assay in all participants who responded to HAART. On the contrary, patients with evidence of HAART failure had increased concentrations of IL-7 that were comparable to those found in the untreated group with progressive disease. Conclusion: These data suggest that IL-7 may play a role in the immune reconstitution of T-cells during HIV infection, especially in the context of potent antiretroviral treatments.

Neuroprotection by melatonin on astrocytoma cell death.

Glial cells play an active role in the homeostatic regulation of the central nervous system (CNS). Astrocytes, the most abundant glial cell types in the brain, provide mechanical and metabolic support for neurons. The regulation of astrocyte apoptosis, therefore, is important for physiological and pathological processes in the CNS. Melatonin is a neurohormone that regulates target cells via binding to specific high‐affinity plasma membrane receptors, MT1/MT2. In addition to regulating circadian rhythms, melatonin has recently attracted much interest for its potential regulation of cell apoptosis. We recently showed that melatonin antagonizes apoptosis on U937 cells via intersecting signal transduction events involving binding to MT1/MT2 and activation of lipoxygenase. Here we describe the neuroprotective potential of melatonin, showing that melatonin significantly reduces damage‐induced apoptosis in astrocytoma cells. The mechanism of protection is different from that shown in U937 cells, because it does not involve MT1/MT2 or lipoxygenase; likewise, Ca2+ influx is not involved. Intriguingly, inhibition of phospholipase C (PLC) with neomycin reverses melatonin protection, suggesting that a PLC‐dependent signal transduction, different from that triggered by MT1/MT2, is involved in the antiapoptotic pathway of melatonin.

Anti-apoptotic effect of HIV protease inhibitors via direct inhibition of calpain

Treatment with drugs designed to inhibit the HIV protease ameliorates immune functions in AIDS patients, reducing cell deletion by apoptosis even in the absence of inhibition of viral spread. This suggests that they interact with the intrinsic apoptotic signaling. We found that caspases, the main executioner of the apoptotic process, are not directly inhibited. In search for the mechanism responsible for their anti-apoptotic effect, we have found that indinavir and ritonavir are able to inhibit apoptosis only in those cell systems where apoptosis involves the activation of calpains. They directly inhibit a calpain-like activity expressed in lysates from apoptotic cells, to the same extent as commercially available calpain inhibitor 1. In in vitro assays with purified calpains, indinavir and ritonavir strongly inhibit m-calpain, and moderately mu-calpain. These results have great therapeutic implications, going beyond AIDS treatment, since many degenerative disorders involve abnormal calpain activation, indicating calpain as an ideal pharmacological target. Indinavir and ritonavir, potent m-calpain inhibitors, largely used since several years on humans without important negative side effects, may become powerful tools against those pathologies.

Ex vivo and in vitro effect of human immunodeficiency virus protease inhibitors on neutrophil apoptosis

Polymorphonuclear leukocytes (PMNL) from human immunodeficiency virus (HIV)-infected patients exhibit accelerated apoptosis and impaired functional activity. HIV protease inhibitor-based therapy produces improvements in both acquired and innate immune responses. Ex vivo and in vitro effects of HIV protease inhibitors on apoptosis and chemotaxis of PMNL were evaluated. After therapy, there was a rapid and significant decrease of PMNL apoptosis, which correlated with increased chemotactic function. These findings were found both in patients with immunological and virological response and in control subjects who showed an increase in CD4(+) T cell counts but no concomitant decline in HIV load. After in vitro treatment with ritonavir or indinavir, apoptosis of both HIV-infected and -uninfected PMNL markedly decreased and correlated with significant enhancement of chemotaxis. These results suggest that HIV protease inhibitors may improve the PMNL function by reducing the apoptosis rate and that this effect may, at least in part, be independent of their antiviral activity.