Claudia D'Agostino

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.

Increased activity of matrix metalloproteinases in the cerebrospinal fluid of patients with HIV-associated neurological diseases

Matrix metalloproteinases (MMPs) have been identified as mediators of brain injury in HIV-associated neurological diseases. The activity of the 72 kDa gelatinase A (MMP-2) and 92 kDa gelatinase B (MMP-9) was detected by zymography in the cerebrospinal fluid (CSF) of 138 HIV-infected patients (40 with AIDS dementia, 83 with brain opportunistic infections and 15 neurologically asymptomatic), 26 HIV-seronegative individuals with inflammatory neurological diseases (IND) and 12 HIV-seronegative subjects with noninflammatory neurological diseases (NIND). MMP-2 was present in all CSF samples from HIV-seropositive and HIV-seronegative individuals, including those of subjects with NIND. On the contrary, MMP-9 was absent in the CSF of NIND controls, whereas the activity of this MMP was found in the 77 - 100% of CSF samples from HIV-infected patients, including those with HIV dementia, central nervous system (CNS) opportunistic infections or neurologically asymptomatic subjects. The highest levels of MMP-9 were found in the CSF of patients with cryptococcosis, cytomegalovirus encephalitis and tuberculous meningitis and were comparable with those found in the CSF of HIV-negative patients with multiple sclerosis or meningitis. A significant correlation between CSF MMP-9 activity and CSF cell count was found only in patients with HIV dementia. The increased CSF activity of MMPs capable to degrade components of the extracellular matrix of blood-brain barrier may contribute to the transendothelial migration of virus-infected cells into the CNS and development of HIV-associated neurologic damage.

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.

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.

Synergistic activity and effectiveness of a double-carbapenem regimen in pandrug-resistant Klebsiella pneumoniae bloodstream infections

Herein, we evaluated through antibiotic kill studies the in vitrosynergistic activity of meropenem plus ertapenem againstpandrug-resistant CP-Kp isolated from three patients with bacter-aemia who were successfully treated with double-carbapenemtherapy.For each patient, informed consent to participate in the studywas obtained.

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.

Antiretroviral therapy inhibits matrix metalloproteinase-9 from blood mononuclear cells of HIV-infected patients

Objective: To investigate whether antiretroviral therapy (ART) influences the release of matrix metalloproteinase (MMP)-9 from peripheral blood mononuclear cells (PBMC) of HIV-infected individuals. Design: Culture supernatants were collected from PBMC isolated from 46 HIV-infected subjects and 19 healthy donors (HD). Among the HIV-infected subjects, 30 were receiving ART therapy, while 16 were naive for any ART treatment. Methods: Zymography of culture supernatants was followed by determination of MMP-9 using computered scanning densitometry. MMP-9 net enzymatic activity was determined by the sensitive fluorescent-activated substrate conversion assay (FASC) to complement the zymography data. Results: MMP-9 was significantly elevated in culture supernatants from PBMC of ART-naive subjects in comparison with HD. In the supernatants from 30 HIV-infected subjects receiving ART therapy, MMP-9 was significantly lower than that in those from ART-naive subjects. Analysis by the reverse transcriptase polymerase chain reaction indicated that MMP-9 expression was increased in ART-naive subjects in comparison with HD but ART induced a decrease of MMP-9 expression to levels comparable with those of HD. FASC used as a functional assay showed conversion of fluorescent gelatine in ART-naive subjects, indicating the presence of active MMP-9. By contrast, in both HD and ART-treated subjects, there was no MMP-9 activity, indicating that MMP-9 was completely blocked by binding to its natural tissue inhibitor TIMP-1. Conclusions: The present findings show for the first time that ART can reduce the capacity of PBMC from HIV-infected patients to secrete increased amounts of MMP-9.

Bactericidal and synergistic activity of double-carbapenem regimen for infections caused by carbapenemase-producing Klebsiella pneumoniae

Available therapeutic options against carbapenem-resistant Klebsiella pneumoniae (CR-Kp) are limited because of the high level of resistance to other antimicrobial classes including polymyxins. The double-carbapenem regimen has been recently considered a possible therapeutic strategy. In the present study, we evaluated the in vitro bactericidal and synergistic activity of a double-carbapenem regimen consisting of ertapenem plus high-dose meropenem in a series of patients with healthcare-associated CR-Kp infections in whom the use of colistin was not indicated because of potential nephrotoxicity and/or resistance. In vitro synergy was evaluated using checkerboard and killing studies. A total of 15 patients were included in the study, with sepsis, severe sepsis and septic shock found in two (13.3%), five (33.3%) and one (6.7%) patients, respectively. Overall, the clinical/microbiological response was 12/15 (80%). Synergy was observed in 11/14 (78.6%) isolates using the checkerboard method whereas in killing studies 12/14 (85.7%) and 14/14 (100%) strains were synergistic and bactericidal at 24 h at concentrations of 1 × MIC MEM+1 × MIC ERT and 2 × MEM+1 × MIC ERT, respectively, with a significant decrease of log CFU/mL compared with other combinations (p <0.0001). The double-carbapenem regimen showed clinical and in vitro effectiveness in patients with CR-Kp infections.

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.

Immune Activation, Immunosenescence, and Osteoprotegerin as Markers of Endothelial Dysfunction in Subclinical HIV-Associated Atherosclerosis

HIV-infected patients have a significantly greater risk of cardiovascular disease. Several markers including osteoprotegerin have been shown to be involved in the development and progression of atherosclerosis. We investigated the relationship between T-cell phenotype, osteoprotegerin, and atherosclerosis evaluated by carotid intima-media thickness (c-IMT) in 94 HIV+ patients on suppressive antiretroviral therapy with Framingham score <10%. As for the control group, 24 HIV-negative subjects were enrolled. c-IMT was assessed by ultrasound. CD4+/CD8+ T-cell activation (CD38+ HLADR+) and senescence (CD57+ CD28−) were measured by flow cytometry. IL-6 and OPG levels were measured by ELISA kit. c-IMT was higher in HIV+ than in controls. Among HIV+ patients, 44.7% had pathological c-IMT (≥0.9 mm). CD8+ T-cell activation and senescence and OPG plasma levels were higher in HIV+ patients than in controls. Subjects with pathological c-IMT exhibited higher CD8+ immune activation and immunosenescence and OPG levels than subjects with normal c-IMT. Multivariate analysis showed that age, CD8+ CD38+ HLADR+, and CD8+ CD28− CD57+ were independently associated with pathological c-IMT. Several factors have been implicated in the pathogenesis of atherosclerosis in HIV patients. Immune activation and immunosenescence of CD8+ T cell together with OPG plasma levels might be associated with the development and progression of early atherosclerosis, even in the case of viral suppression.