Search

We report the results of an external quality control program, including 17 Italian centers involved in the care of patients infected by HIV, to evaluate CD4 T cell count proficiency and reproducibility. The centers received two commercial stabilized blood preparations, one with “normal” and one with “low” CD4 T cell content. The centers were asked to process the samples two times, 1 week apart, with the same procedure used for samples from HIV patients. Most centers showed a good performance of CD4 frequency and absolute count determinations. In particular, the “low” sample was correctly analyzed by all centers; only two underestimated the “normal” sample CD4 frequency, and only one underestimated the CD4 absolute count by >100 CD4 cells/μl. Overall, our data suggest that most Italian laboratories provide reliable and reproducible results in evaluating CD4 T cells in HIV+ samples.

Several abnormalities of lipid metabolism, including hypo-beta-lipoproteinemia and liver steatosis are associated with infection by hepatitis C virus (HCV). The aim of this study was to determine whether circulating lipoproteins of patients with HCV infection could directly cause alterations of lipid cellular metabolism. To this end the metabolic response of human monocyte-derived macrophages (HMDM) to very low-density lipoprotein (VLDL) and low-density lipoprotein (LDL), measuring the cholesteryl ester (CE) and triglyceride (TG) production was analyzed. Lipoproteins were isolated from 18 patients infected with hepatitis C virus (HCV-VLDL and HCV-LDL) and from normal healthy donors (ct-VLDL and ct-LDL). In comparison to ct-lipoproteins, HCV-lipoproteins induced significant differences in HMDM CE and TG production. HCV-VLDL decreased CE and TG production; while HCV-LDL induced an increased TG synthesis. The present findings suggest that HCV infection modifies VLDL and LDL molecular composition, affecting cellular lipid metabolism, thus promoting intracellular lipid accumulation and hypo-beta-lipoproteinemia.

This study aimed to define clinical and virological parameters associated with spontaneous control of HIV replication in patients having initiated HAART during primary HIV infection, who underwent structured therapy interruption by two protocols with either fixed or HIV viremia-guided scheme. At the end of the protocol all patients were changed to viremia-guided scheme and observed for 12 months (follow-up). Patients maintaining HIV viremia below the indications for resumption of HAART during the follow-up, were defined controllers, those who had to resume HAART were defined non-controllers. The following parameters were examined: pre-interruption therapy duration, CD4+, HIV RNA, proviral DNA, evolution of viral quasispecies. No specific advantage was conferred by either interruption of structured therapy in the proportion of controllers and non-controllers. Pre-HAART and zenith CD4+, pre-therapy interruption, HAART duration, but not pre-HAART HIV RNA, were significantly higher in controllers as compared to non-controllers. HIV RNA levels after the first interruption cycle of therapy were significantly lower in controllers than in non-controllers. Proviral DNA levels were also lower in controllers at this time point. HIV RNA and proviral DNA levels associated with the last interruption of therapy cycle were not different from those associated with the first cycle, and, in spite of multiple waves of virus rebound, very few gag quasispecies variants emerged in each patient. The data suggest that pre-treatment clinical parameters and virological events associated with the first viral rebound are crucial factors in determining the ability to control viral replication after multiple cycles of interruption of treatment. J. Med. Virol. 79: 1047–1054, 2007. © 2007 Wiley-Liss, Inc.

Body piercing is a social phenomenon on the rise especially among young people. This procedure may be complicated by serious bacterial and viral infections. We report a case of Staphylococcus aureus infective endocarditis and meningitis arising from the site of a nape piercing, after its removal. A 21-year-old Italian female was admitted to hospital with neurological impairment and sepsis. A diagnosis of endocarditis associated with meningitis by S. aureus, complicated by septic emboli in the brain, retina, skin and kidney, was formulated on the basis of modified Duke's criteria. The likely port-of-entry was the site of a nape piercing, removed two months before. In view of the widespread practice of body piercing, provision of correct and timely information concerning the associated serious risks is now imperative. Such information should emphasise the option for antibiotic prophylaxis, and the importance of careful local hygiene, even after piercing removal.

The impact of pre-existing variants in hepatitis C virus (HCV) quasispecies, carrying resistance-associated mutations (RAMs), on the outcome of treatment with direct acting antiviral agents (DAA) is debated and it is complicated by the lack of knowledge of quasispecies distribution between the viral reservoir (liver) and the circulating compartment.To evaluate NS3 protease heterogeneity and presence of RAMs on baseline plasma and liver biopsy samples. Plasma dynamics were also analyzed during therapy and after its suspension. Study design Ultra-deep pyrosequencing (UDPS) was performed in two HCV genotype 1a patients who received telaprevir (TVR)-based therapy and developed treatment failure due to TVR-resistance.In both patients the baseline diversity of NS3 quasispecies in plasma was higher than in liver (183.6×10(-4) vs 47.8×10(-4) and 246.0×10(-4) vs 55.0×10(-4) nt substitution/site, respectively, p0.0001), but phylogenetic trees did not evidence compartmentalization between the two compartments. At baseline RAMs (i.e. V36A, T54A) were detected very low levels (range: 0.31-0.52%) in both specimen types. However, phylogenetic analyses revealed that the viral variants carrying these mutations at baseline were different from those that became fixed at breakthrough, when combined V36M+R155K, conferring high-level resistance to TVR, were observed. The frequency of resistance-associated variants declined after withdrawal of drug selective pressure.UDPS allowed extensive evaluation of quasispecies compartmentalization and of their dynamics after withdrawal of TVR. Plasma and liver NS3 quasispecies, including low level RAMs, do not show significant difference.

Direct-acting antiviral drugs (DAA) regimen improve the SVR rate. However, adverse effects often lead to therapy interruption. This underlines the importance to find some predictive parameters of response in order to consider the possibility of a shorter time of antiviral treatment in the appearance of adverse effects without affecting the success of the therapy.We aimed to examine the HCVAg kinetics in the early phase of treatment and its predictive value of SVR in patients undergoing TPV/Peg-IFN/RBV treatment.Twenty-three patients infected by HCV genotype 1 (1a n=11; 1b n=12) were included in this prospective study.At baseline the median Log of HCVAg concentration in RVR and EVR patients were 3.15 fmol/L and 3.45 fmol/L, respectively with no significant differences. The baseline median HCV-RNA to HCVAg ratio was 233.77, this ratio was significantly lower when measured on day 1 (27.52) and on day 6 (24.84) (p0.001). The two-tailed Fisher's exact test indicated that the SVR response is statistically significantly different in patients with detected HCVAg at week1 compared to patients with no detectable HCVAg (p=0.05). The sensitivity, specificity, and negative and positive predictive values (NPV, PPV) were 53.8, 87.5, 53.8 and 87.5%, respectively. The area under the ROC curve was 0.71 at day T6, the best cut-off of 3 fmol/L when evaluated with the HCVAg plasma concentration at day T6.Undetectable HCVAg in the early phase of TPV/Peg-IFN/RBV treatment could represent an important parameter for predicting SVR.

Background: Direct-acting antiviral drugs (DAA) regimen improve the SVR rate. However, adverse effects often lead to therapy interruption. This underlines the importance to find some predictive parameters of response in order to consider the possibility of a shorter time of antiviral treatment in the appearance of adverse effects without affecting the success of the therapy. Objectives: We aimed to examine the HCVAg kinetics in the early phase of treatment and its predictive value of SVR in patients undergoing TPV/Peg-IFN/RBV treatment. Study design: Twenty-three patients infected by HCV genotype 1 (1a n = 11; 1b n = 12) were included in this prospective study. Results: At baseline the median Log of HCVAg concentration in RVR and EVR patients were 3.15 fmol/L and 3.45 fmol/L, respectively with no significant differences. The baseline median HCV-RNA to HCVAg ratio was 233.77, this ratio was significantly lower when measured on day 1 (27.52) and on day 6 (24.84) (p < 0.001). The two-tailed Fisher's exact test indicated that the SVR response is statistically significantly different in patients with detected HCVAg at week1 compared to patients with no detectable HCVAg (p = 0.05). The sensitivity, specificity, and negative and positive predictive values (NPV, PPV) were 53.8, 87.5, 53.8 and 87.5%, respectively. The area under the ROC curve was 0.71 at day T6, the best cut-off of 3 fmol/L when evaluated with the HCVAg plasma concentration at dayT6. Conclusion: Undetectable HCVAg in the early phase of TPV/Peg-IFN/RBV treatment could represent an important parameter for predicting SVR.

Gut-associated immune system has been identified as a major battlefield during the early phases of HIV infection. γδ T-cells, deeply affected in number and function after HIV infection, are able to act as a first line of defence against invading pathogens by producing antiviral soluble factors and by killing infected cells. Despite the relevant role in mucosal immunity, few data are available on gut-associated γδ T-cells during HIV infection. Aim of this work was to evaluate how primary (P-HIV) and chronic (C-HIV) HIV infection affects differentiation profile and functionality of circulating and gut-associated Vδ1 and Vδ2 T-cells. In particular, circulating and mucosal cells were isolated from respectively whole blood and residual gut samples from HIV-infected subjects with primary and chronic infection and from healthy donors (HD).Differentiation profile and functionality were analyzed by multiparametric flow cytometry. P-HIV and C-HIV were characterized by an increase in the frequency of effector Vδ1-T cells both in circulating and mucosal compartments. Moreover, during PHIV mucosal Vδ1 T-cells expressed high levels of CD107a, suggesting a good effector cytotoxic capability of these cells in the early phase of infection that was lost in C-HIV. P-HIV induced an increase in circulating effector Vδ2 T-cells in comparison to C-HIV and HD. Notably, P-HIV as well as HD were characterized by the ability of mucosal Vδ2 T-cells to spontaneously produce IFN-γ that was lost in C-HIV. Altogether, our data showed for the first time a functional capability of mucosal Vδ1 and Vδ2 T-cells during P-HIV that was lost in C-HIV, suggesting exhaustion mechanisms induced by persistent stimulation. Copyright

Background/Aims Hepatitis C virus (HCV) causes a chronic infection that can lead to fibrosis and carcinoma. Immune responses mediated by cytotoxic T lymphocytes (CTLs) could be involved in viral clearance or persistence, and therefore in determining the course of the disease. Methods Intrahepatic and peripheral blood CD8+T cells were obtained from 32 HCV-chronically infected patients and analysed by flow-cytometry for surface markers of differentiation, IFNγ and TNFα production, degranulation capacity and perforin content, after CD3 triggering. Results were compared with those obtained from 13 patients with a non-viral liver disease. Results Intrahepatic CD8+T cells of HCV-infected patients, despite their phenotype of pre-terminally and terminally differentiated effectors (CCR7–CD45RA−/+), are poorly responsive to T cell receptor (TCR)-mediated stimulation compared with those obtained from uninfected subjects. This defect correlates with the severity of fibrosis, is more pronounced in patients with ALT 1.5×NU/ml, and is not evident after mitogen stimulation. Conclusions The present study describes the accumulation of hypo-responsive CD8+T cells in the liver of patients with chronic HCV infection. Understanding the mechanisms underlying this impairment may be helpful in the design of innovative strategies for HCV treatment.

Transaminase elevation is frequently seen in hepatitis C virus (HCV)-HIV-coinfected patients receiving antiretroviral therapy (ART), representing an increase in the immune response against HCV and being one of the mechanisms proposed to be involved. There is a report claiming that HCV genotype 3 is an independent risk factor. Our objectives were to assess the incidence of liver toxicity in an HIV-HCV-coinfected population with relatively preserved cellular immunity, and the role of HCV genotypes in the elevation of liver enzymes, both at baseline and after initiating ART. All HIV(+) patients with positive anti-HCV serology and CD4(+) cell counts above 100/mm(3) who began triple ART were identified, and their HCV-RNA levels and HCV genotype were determined. Liver enzymes were determined at baseline and bimonthly during follow-up. Of anti-HCV patients 147 were included, 128 (87.1%) of whom had detectable plasma HCV-RNA. HCV-1 and HCV-4 genotypes were found to confer an increased probability of having at baseline transaminases within normal limits over the other genotypes. Severe transaminase elevations (grades 3 and 4) occurred in 5/124 patients (4.0%), all with high pre-HAART ALT and positive HCV-RNA levels. Multivariate analysis showed that patients with genotype HCV-3 had a 3.27 times higher risk of developing HAART-related transaminase elevations of any grade. In conclusion, subjects with the HCV-1 genotype more often had transaminases within normal limits at baseline. The incidence of severe transaminase elevation after initiating ART was very low (4%) in this HIV(+) population with relatively preserved cellular immunity. HCV genotype 3 was identified as a risk factor for the development of transaminase elevation of any grade.

Objectives Integrase drug resistance monitoring deserves attention because of the increasing number of patients being treated with integrase strand-transfer inhibitors. Therefore, we evaluated the integrase genotyping success rate at low-level viraemia (LLV, 51–1000 copies/mL) and resistance in raltegravir-failing patients. Methods An integrase genotypic resistance test (GRT) was performed on 1734 HIV-1 samples collected during 2006–13. Genotyping success rate was determined according to the following viraemia levels: 51–500, 501–1000, 1001–10 000, 10 001–100 000 and >100 000 copies/mL. The reproducibility of integrase GRT was evaluated in 41 plasma samples processed in duplicate in two reference centres. The relationship between LLV and resistance prevalence was evaluated in a subset of 120 raltegravir-failing patients. Results Overall, the integrase genotyping success rate was 95.7%. For viraemia levels 51–500 and 501–1000 copies/mL, the rate of success was 82.1% and 94.0%, respectively. GRT was reproducible, producing sequences with a high similarity and an equal resistance profile regardless of the sequencing centre or viraemia level. Resistance was detected both at LLV and at viraemia >1000 copies/mL (51–500 copies/mL = 18.2%; 501–1000 = 37.5%; 1001–10 000 = 53.7%; 10 001–100 000 = 30.0%; and >100 000 = 30.8%). At viraemia ≤500 copies/mL, Q148H/K/R and N155H had the same prevalence (9.1%), while the Y143C/H/R was completely absent. At early genotyping (within 3 months of raltegravir treatment), Q148H/K/R and N155H mutations were detected regardless of the viraemia level, while Y143C/H/R was observed only in samples with viraemia >1000 copies/mL. Conclusions Our findings prove the reliability of HIV-1 integrase genotyping and reinforce the concept that this assay may be useful in the management of failures even at LLV.

Hepatitis C virus (HCV) has evolved complex strategies to evade host immune responses and establish chronic infection. Since human Vgamma9Vdelta2 T lymphocytes play a critical role in the immune response against viruses, we analyzed their antiviral functions on Huh7 hepatoma cells carrying the subgenomic HCV replicon (Rep60 cells). In a transwell culture system, Rep60 cells were co-cultured with either PBMCs or highly purified gammadelta T cells stimulated by non-peptidic antigens. Vgamma9Vdelta2 T cell activation was associated with a dramatic reduction of HCV RNA levels. Neutralizing antibodies targeting IFN-gamma revealed a critical role for this cytokine in the inhibition of HCV replication. Interestingly, drugs already in clinical use, such as Phosphostim and Zoledronate, known to activate gammadelta T cells, were shown to induce the inhibition of HCV replication mediated by Vgamma9Vdelta2 T cells of HCV patients. Our data suggest that the therapeutic activation of Vgamma9Vdelta2 T lymphocytes may represent an additional strategy to inhibit HCV replication and to restore a Th1-oriented immune response in HCV-infected patients.

Alterations in NK cell numbers and function have been repeatedly shown during HIV infection. In this study, NK cell number and MHC class I expression on CD4+ T cells were studied in HIV patients at different stages of disease progression. An increased expression of HLA-E was seen on CD4+ T cells. In parallel, a reduced number of CD94+ NK cells was observed in advanced disease stages. Moreover, a decline in CD94 expression on NK cells was observed at the HIV replication peak in patients undergoing antiretroviral treatment interruption, suggesting a role of viral replication on NK cells alterations. In vitro HIV infection induced a rapid down-regulation of HLA-A,B,C expression, paralleled by an increased expression of HLA-E surface molecules, the formal ligands of CD94 NK receptors. HIV-infected HLA-E expressing cells were able to inhibit NK cell cytotoxicity through HLA-E expression, since cytotoxicity was restored by antibody masking experiments. These data indicate that the CD94/HLA-E interaction may contribute to NK cell dysfunction in HIV infection, suggesting a role of HIV replication in this process.

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.

G.M. Ettorrea, *, G. Vennareccia, R. Santoroa, P. Lepianea, A. Laurenzia, M. Colasantia, L. Carpaneseb, R. Sciutoc, M. Antoninid, G. Doffizie a General Surgery and Transplantation, San Camillo Hospital, Rome, Italy b Interventional Radiology, National Cancer Institute “Regina Elena”, Rome, Italy c Nuclear Medicine, National Cancer Institute “Regina Elena”, Rome, Italy d ICU, National Institute of Infectious Disease “L. Spallanzani”, Rome, Italy e Infectiology and Hepatology Division, National Institute of Infectious Disease “L. Spallanzani”, Rome, Italy

To develop recommendations for the management of acute hepatitis B by the Italian Society for the Study of Infectious and Tropical Diseases.Development of the recommendations divided into three levels of evidence according to the GRADE system: A (high), B (medium) and C (low experts opinion), together with three recommendation levels: 1 (strong), 2 (medium), 3 (weak).The treatment with antivirals is in selected cases the mainstay of management of severe acute hepatitis, and should be started as a matter of urgency in order to prevent death.These recommendations are meant to provide the rationale and practical indications for the management of acute hepatitis B (AHB).

Background We evaluated efficacy and safety of sofosbuvir and daclatasvir ± ribavirin in liver transplant recipients with severe recurrent hepatitis C. Methods Patients included in an international compassionate use programme for treatment with sofosbuvir and daclatasvir ± ribavirin for 24 weeks were prospectively studied. Serum hepatitis C virus RNA was measured at treatment weeks 4, 12, and 24 and during follow-up at weeks 4, 8, and 12. Results Twelve patients (3 with fibrosing cholestatic hepatitis and 9 with cirrhosis; median model for end-stage liver disease score 20) received sofosbuvir 400 mg/day + daclatasvir 60 mg/day, and 6 patients (50%) also received ribavirin 200–800 mg/day. Nine patients completed 24 weeks of treatment (75%), and all had undetectable hepatitis C virus RNA at week 24; 3 patients died (25%, liver failure, gastrointestinal bleeding and sepsis); 4 patients experienced severe liver disease-related adverse events. Post-treatment hepatitis C virus RNA was available for 5 patients (week 8, n = 2; week 4, n = 3) and was undetectable in all cases. Mean Child–Pugh score and albumin level improved significantly at week 24. No changes in immunosuppressant doses were needed. Conclusion All-oral sofosbuvir plus daclatasvir combination shows high virological efficacy in liver transplant recipients and does not interact with immunosuppressants. All adverse events were unrelated to study drugs. These data strongly suggest that this combination must be initiated before decompensation.

UNLABELLED Our objective was to describe morphologic and metabolic disorders in patients treated with highly active antiretroviral therapy (HAART) since primary HIV infection (PHI). Our method was prospective evaluation of patients with PHI initiating HAART at the time of diagnosis. Outcome measures were: development of hyperglycemia, hypercholesterolemia, hypertriglyceridemia, and of body shape abnormalities indicative of lipodystrophy, assessed through self-reported questionnaires and physical examination. RESULTS From May 1997 to April 2001, 41 patients (35 males) with PHI presented at the National Institute for Infectious Diseases "Lazzaro Spallanzani" in Rome, Italy. A protease inhibitor-including regimen was started in 30 patients, and a nonnucleoside reverse transcriptase-inhibitor in 11. Median interval between enrollment and treatment initiation was 30 days (mean 39, range 10-150). Median HAART duration was 19 months (mean 21.2, range 3-47). Thirty-eight patients had undetectable (less than 80 cp/mL) HIV RNA after a median of 3 months (mean 4.1, range 1-15). Mean CD4 cells count increased from 632/mmc at baseline to 936/mmc at the last follow up. No cases of hyperglycemia (glucose level greater than 110 mg/dL) were observed. After a median of 6 months on HAART, 10 patients developed beyond grade 2 (greater than 240 mg/dL) hypercholesterolemia, 5 developed beyond grade 2 (greater than 400 mg/dL) hypertrygliceridemia, and two developed both. Body mass index did not change significantly. Five patients (12.2%) developed lipodystrophy after a median of 14.5 months (mean 15.3, range 2-30), with an incidence of 7.3 per 100 patient-years. CONCLUSIONS Dyslipidemia and lipodystrophy can occur in patients treated with HAART since PHI. This risk of should be taken into account when considering this early antiretroviral treatment of HIV infection.

HIV and hepatis C virus (HCV) coinfection is frequently associated with rapid progression of HCV-related disease, resulting in a higher risk of cirrhosis. Data suggest that natural T cells expressing the Vdelta1 T cell receptor rearrangement are recruited in the liver of chronically HCV-infected patients and are increased in the peripheral blood of HIV-infected persons. We studied gammadelta T cell distribution in the peripheral blood and liver of HCV-infected and HIV/HCV-coinfected patients in the presence and absence of antiretroviral therapy. We observed that Vdelta1+ T cells releasing helper T cell type 1 cytokines are compartmentalized not only in the liver of HCV+ patients, but also of HIV/HCV-coinfected persons. HIV/HCV patients showed an increased frequency of both peripheral and intrahepatic Vdelta1 natural T lymphocytes, resulting in a higher degree of hepatic inflammation when compared with patients with other liver diseases. Finally, highly active antiretroviral therapy (HAART) was unable to restore Vdelta1T cell circulation to normal levels in chronically HIV-infected persons. We conclude that gammadelta T lymphocytes released from tissue to the bloodstream circulation under the influence of chronic HIV infection may contribute to intrahepatic Vdelta1 compartmentalization and progression of liver disease, independently of HAART.

SUMMARYThe functional recovery of the immune system in HIV-infected persons receiving HAART and the role of adjuvant immune therapy are still matters of intensive investigation. We analysed the effects of HAART combined with cytokines in 22 naive asymptomatic individuals, randomized to receive HAART (n = 6), HAART plus a low dose (1000 000 U/daily) of rIL-2 (n = 8), and HAART plus rIL-2 after previous administration of granulocyte colony-stimulating factor (n = 8). After 3 months of therapy, increased CD4+ T cell counts and diminished viral loads were observed in all patients, independently of cytokine addition. A decreased expression of CD95 (Apo 1/Fas) was evident in all groups when compared with values before therapy. The percentages of peripheral blood mononuclear cells (PBMC) expressing CD95 after therapy decreased by 15%, 22% and 18% in the three treatment groups, respectively (P

In chronic hepatitis C virus (HCV) infection, treatment failure and defective host immune response highly demand improved therapy strategies. Vγ9Vδ2 T-cells represent a good target for HCV immunotherapy, since phosphoantigen (PhAg)-activated Vγ9Vδ2 T-lymphocytes are able to inhibit subgenomic HCV replication by interferon (IFN)-γ release. A profound impairment of IFN-γ production by Vγ9Vδ2 T-cells during chronic HCV infection was previously shown. Interestingly, in vitro IFN-α partially restored Vγ9Vδ2 T-cells responsiveness to PhAg, by stabilizing IFN-γ-mRNA. To verify how in vivo IFN-α/ribavirin (RBV) treatment could affect Vγ9Vδ2 T-cells phenotype and responsiveness to PhAg in HCV-infected patients, 10 subjects underwent a longitudinal study before and after treatment. IFN-α/RBV therapy did not significantly modify Vγ9Vδ2 T-cell numbers and differentiation profile. Interestingly, Vγ9Vδ2 T-cell responsiveness remained unmodified until 3 weeks of therapy, but dropped after 1 month, suggesting that repeated in vivo IFN-α administration in the absence of T-cell receptor (TCR)-mediated signals results in Vγ9Vδ2 T-cell anergy. The present work defines the window of possible application of combined strategies targeting Vγ9Vδ2 T-cells during chronic HCV infection; specifically, the first 3 weeks from the beginning of treatment may represent the optimal time to target Vγ9Vδ2 T-cells in vivo, since their function in terms of IFN-γ production is preserved.

Objectives Identifying the predictive factors of Sustained Virological Response (SVR) represents an important challenge in new interferon-based DAA therapies. Here, we analyzed the kinetics of antiviral response associated with a triple drug regimen, and the association between negative residual viral load at different time points during treatment. Methods Twenty-three HCV genotype 1 (GT 1a n = 11; GT1b n = 12) infected patients were included in the study. Linear Discriminant Analysis (LDA) was used to establish possible association between HCV RNA values at days 1 and 4 from start of therapy and SVR. Principal compo- nent analysis (PCA) was applied to analyze the correlation between HCV RNA slope and SVR. A ultrasensitive (US) method was established to measure the residual HCV viral load in those samples which resulted "detected

SUMMARYIn vitro lymphoproliferative responses to HIV-1 recombinant antigens (gp160, p24, and Rev protein) were studied in 83 patients with asymptomatic HIV-1 infection (CDC groups 11 and III) and circulating CD4 lymphocyte numbers > 400/mm3. Significant response to at least one of the three antigens was detected in 52.4% of the subjects, but the responses were weak, and concordance of the response to the three antigens was rare, the frequency of individuals responding to each antigen not exceeding 22.4%. Increasing frequencies of response were observed when recall antigens (tetanus toxoid and Candida albicans glycomannoprotein) (65.5%) and anti-CD3 MoAb (76.6%) were used as stimuli. Although a significant association between lymphocyte response to p24, but not gp160, and steadiness of CD4 lymphocyte numbers before the assay was observed, no predictive value for lack of CD4 cell decrease was confirmed for either antigen, and fluctuation of the responses to HIV antigens was seen during subsequent follow up. The panel of T cell assays used could be regarded as appropriate for monitoring both HIV-specific responses and T lymphocyte function during immunotherapy with soluble HIV antigens.

HCV genome variability is related to both disease progression and treatment response. De novo high-throughput pyrosequencing was used to obtain full length HCV genome characterization directly from clinical samples.

In chronic HCV infection, treatment failure and defective host immune response highly demand improved therapy strategies. Vγ9Vδ2 T-cells may inhibit HCV replication in vitro through IFN-γ release after Phosphoantigen (PhAg) stimulation. The aim of our work was to analyze Vγ9Vδ2 T-cell functionality during chronic HCV infection, studying the role of IFN-α on their function capability. IFN-γ production by Vγ9Vδ2 T-cells was analyzed in vitro in 24 HCV-infected patients and 35 healthy donors (HD) after PhAg stimulation with or without IFN-α. The effect of in vivo PhAg/IFN-α administration on plasma IFN-γ levels was analyzed in M. fascicularis monkeys. A quantitative analysis of IFN-γ mRNA level and stability in Vγ9Vδ2 T-cells was also evaluated. During chronic HCV infection, Vγ9Vδ2 T-cells showed an effector/activated phenotype and were significantly impaired in IFN-γ production. Interestingly, IFN-α was able to improve their IFN-γ response to PhAg both in vitro in HD and HCV-infected patients, and in vivo in Macaca fascicularis primates. Finally, IFN-α increased IFN-γ-mRNA transcription and stability in PhAg-activated Vγ9Vδ2 T-cells. Altogether our results show a functional impairment of Vγ9Vδ2 T-cells during chronic HCV infection that can be partially restored by using IFN-α. A study aimed to evaluate the antiviral impact of PhAg/IFN-α combination may provide new insight in designing possible combined strategies to improve HCV infection treatment outcome. © 2012 Cimini et al.

We analyzed a cohort of intravenous drug abusers with HIV-1 disease attending our institute in the years 1985–1993. We focused particular attention on the epidemiological analysis of patients, and their clinical, immunological and infectious conditions. The significance of biological markers, particulary CD4+ lymphocyte count and the occurence of other viral infections such as Cytomegalovirus, Epstein-Barr virus, Herpes Simplex virus and Hepatitis B and C viruses in the progress of HIV-1 disease were evaluated. At least two-thirds of the patients at different stages of HIV-1 were treated with antiretroviral drugs: zidovudine (AZT) from 1987 up to the present and more recently didanosine (DDI) alone or in combination with AZT. Psychological and behavioral aspects of our HIV-1 infected drug abusers, in particular needle exchange and condom use, were analyzed.

Despite a long-lasting global effort, the Holy Grail quest for a protective vaccine, able to confer prevention to HIV infection, did not reach the hoped for results, nor seems able to do so in the near future. Since mucosal surfaces of the host serve as the main entry point for HIV, it seems now logical to switch from a systemic to a localized view of events, in order to reveal critical steps useful in designing new and different vaccination strategies. In this context, the recent description of the very early phases of infection, from the eclipse to the viremia peak phase, seems to define a point-of-no-return threshold after which the main HIV infection steps, i.e. the massive destruction of the CD4+CCR5+ cell pool, the destruction of the mucosal physical barrier, and the establishment of reservoir sanctuaries, have already been accomplished. Nevertheless, the underlying mechanisms, the timing, and the consequences of evasion mechanisms exploited by HIV are still under scrutiny. Innate immunity, as part of a rapid lymphoid stress surveillance system, is known to play a central role in host responses to many infectious agents. In particular, Vγ9Vδ2 T-cells are able to quickly respond to danger signals without the need for classical major histocompatibility complex presentation, and may act as a bridge between innate and acquired arms of immune response, being able to kill infected/transformed cells, release antimicrobial soluble factors, and increase the deployment of other innate and acquired responses. Many experimental evidences suggest a direct role of circulating Vγ9Vδ2 T-cells during HIV disease. They may exert a direct anti-HIV role by secreting chemokines competing for HIV entry coreceptors as well as other soluble antiviral factors, and by killing infected cells by cytotoxic natural killer-like mechanisms. Moreover, they were found progressively depleted and anergic in advanced stages of HIV disease, this effect being directly linked to uncontrolled HIV replication. Scarce evidences are available on the involvement of mucosal gamma/delta T-cells during the early phases of HIV infection. In particular, the relative cause/effect links between HIV infection, destruction of the mucosal physical barrier, nonspecific activation of the immune system, and mucosal innate cell activation and effector functions, are still not completely defined. In order to attain an effective manipulation of innate immune cells, aiming at the induction of an effective adaptive immunity against HIV, any information on the role of mucosal antiviral factors and innate immune cells will be very important. The aim of this review is to summarize the information on the role of gamma/delta T-cells during HIV infection, from the general circulating population to mucosal sites, in order to better describe areas deserving increased attention. In particular, strategies enhancing gamma/delta T-cell functions may open the possibility to formulate new immunotherapeutic regimens, which could impact the improvement of immune control of HIV disease.

Background When to initiate antiretroviral therapy in HIV infected patients is a diffcult clinical decision. Actually, it is still a matter of discussion whether early highly active antiretroviral therapy (HAART) during primary HIV infection may influence the dynamics of the viral rebound, in case of therapy interruption, and overall the main disease course. Methods In this article we use a computational model and clinical data to identify the role of HAART timing on the residual capability to control HIV rebound after treatment suspension. Analyses of clinical data from three groups of patients initiating HAART respectively before seroconversion (very early), during the acute phase (early) and in the chronic phase (late), evidence differences arising from the very early events of the viral infection. Results The computational model allows a fine grain assessment of the impact of HAART timing on the disease outcome, from acute to chronic HIV-1 infection. Both patients' data and computer simulations reveal that HAART timing may indeed affect the HIV control capability after treatment discontinuation. In particular, we find a median time to viral rebound that is significantly longer in very early than in late patients. Conclusions A timing threshold is identified, corresponding to approximately three weeks post-infection, after which the capability to control HIV replication is lost. Conversely, HAART initiation occurring within three weeks from the infection could allow to preserve a significant control capability. This time could be related to the global triggering of uncontrolled immune activation, affecting residual immune competence preservation and HIV reservoir establishment.

Objectives: HIV-1 V3 quasispecies was analyzed by ultra-deep pyrosequencing, in early HIV-infected patients, to assess possible correlations between quasispecies diversity, frequency of variants predicted to use CXCR4 and need for early antiretroviral treatment. Methods: Twenty patients were retrospectively enrolled: 10 patients (group A) required HAART within 6 months from seroconversion and 10 (group B) remained free of therapy during this period. V3 quasispecies was assessed on plasma viral RNA and in peripheral blood mononuclear cell-associated proviral DNA. Prediction of coreceptor usage was performed by position-specific score matrix analysis. Results: Variants predicted to use CXCR4 were detected (frequency >0.3%) in the plasma of 50% of early infected patients (60% from group A and 40% from group B). Intrapatient frequency of these variants was highly variable (0.3-56.3%). A positive correlation was observed between the proportion of X4 variants and intrapatient quasispecies diversity. Quasispecies diversity and absolute numbers of X4 variants were significantly higher in patients from group A. The analysis of proviral DNA quasispecies, performed in a subgroup of five patients, showed that X4 variants were not detected in patients with RNA frequency below 0.3%, and detected at 3.6% in the patient with 56.3% of X4 plasma variants. Conclusion: Our findings show that X4 variants may be frequently found, at variable intrapatient frequency, in early infected patients, and that quasispecies diversity and absolute numbers of X4 variants are significantly higher in patients undergoing early antiretroviral treatment. Further studies are mandatory to explore the clinical relevance of X4 variants present during early infection with respect to clinical progression and possible therapeutic implications.

Background/aims The life cycle of hepatitis C virus (HCV) is intimately linked to the lipid metabolism of the host. In particular, HCV exploits the metabolic machinery of the lipoproteins in several steps of its life cycle such as circulation in the bloodstream, cell attachment and entry, assembly and release of viral particles. However, the details of how HCV interacts with and influences the metabolism of the host lipoproteins are not well understood. A study was undertaken to investigate whether HCV directly affects the protein composition of host circulating lipoproteins. Methods A proteomic analysis of circulating very low-, low- and high-density lipoproteins (VLDL, LDL and HDL), isolated from either in-treatment naive HCV-infected patients or healthy donors (HD), was performed using two-dimensional gel electrophoresis and tandem mass spectrometry (MALDI-TOF/TOF). The results obtained were further investigated using in vitro models of HCV infection and replication. Results A decreased level of apolipoprotein A-I (apoA-I) was found in the LDL fractions of HCV-infected patients. This result was confirmed by western blot and ELISA analysis. HCV cellular models (JFH1 HCV cell culture system (HCVcc) and HCV subgenomic replicons) showed that the decreased apoA-I/LDL association originates from hepatic biogenesis rather than lipoprotein catabolism occurring in the circulation, and is not due to a downregulation of the apoA-I protein concentration. The sole non-structural viral proteins were sufficient to impair the apoA-I/LDL association. Functional evidence was obtained for involvement of apoA-I in the viral life cycle such as RNA replication and virion production. The specific siRNA-mediated downregulation of apoA-I led to a reduction in both HCV RNA and viral particle levels in culture. Conclusions This study shows that HCV induces lipoprotein structural modification and that its replication and production are linked to the host lipoprotein metabolism, suggesting apoA-I as a new possible target for antiviral therapy.

It was estimated that up to 70% of hepatitis C virus (HCV)-infected subjects became chronically infected, and these patients have an increased risk of developing cirrhosis and hepatocellular carcinoma.1 The standard treatment for HCV-infected patients is pegylated interferon α (IFNα) in combination with ribavirin; however, data on the correlation between treatment outcome and the activation state of components of the immune system are missing. A very interesting paper by Mengshol et al was published in Gut regarding the effect of anti-HCV treatment on the activation state of circulating dendritic cells (DCs).2 The authors showed that levels of CXCR3 and CXCR4 on plasmacytoid DC (pDCs) were higher at baseline compared with controls, and decreased with treatment. Pretreatment levels of the co-stimulatory marker CD40 and the maturation marker CD83 were higher in pDCs of patients chronically infected with HCV compared with healthy controls; moreover, levels of both markers dropped significantly with treatment in the sustained virological responder (SVR) group only. Finally, baseline pDCs chemotaxis to CXCL12 …

In this work we evaluated plasmacytoid (pDC) and myeloid dendritic (mDC) cells activation before and during anti-HCV treatment in HCV+/HIV+ individuals.HCV+/HIV+ patients received Peg-IFN-α2b subcutaneously for 28. days, followed by oral weight-based ribavirin. DCs activation was evaluated by flow cytometry.Baseline pDC CD80 and CD86 expression was correlated with HIV, but not with HCV viral load. A transient decrease of HIV RNA was found not associated with DC activation. When patients were grouped according to early/sustained virological response (EVR/SVR) to anti-HCV treatment, baseline pDC CD80 and CD86 expression was higher in non-EVR and non-SVR compared to EVR and SVR. Moreover, in responder patients CD80 and CD86 were upregulated by IFN-α. Our data suggest a correlation between DCs activation and response to therapy. These findings could be helpful to better understand the mediators of IFN-α action in HCV+/HIV+ patients and to explore possible exploitation of this knowledge to improve therapeutic response. © 2010 Elsevier Inc.

Background The optimal stage for initiating antiretroviral therapies in HIV-1 bearing patients is still a matter of debate. Methods We present computer simulations of HIV-1 infection aimed at identifying the pro et contra of immediate as compared to deferred Highly Active Antiretroviral Therapy (HAART). Results Our simulations highlight that a prompt specific CD8+ cytotoxic T lymphocytes response is detected when therapy is delayed. Compared to very early initiation of HAART, in deferred treated patients CD8+ T cells manage to mediate the decline of viremia in a shorter time and, at interruption of therapy, the virus experiences a stronger immune pressure. We also observe, however, that the immunological effects of the therapy fade with time in both therapeutic regimens. Thus, within one year from discontinuation, viral burden recovers to the value at which it would level off in the absence of therapy. In summary, simulations show that immediate therapy does not prolong the disease-free period and does not confer a survival benefit when compared to treatment started during the chronic infection phase. Conclusion Our conclusion is that, since there is no therapy to date that guarantees life-long protection, deferral of therapy should be preferred in order to minimize the risk of adverse effects, the occurrence of drug resistances and the costs of treatment.

Objective gammadelta T cells bearing the Vgamma9Vdelta2 T-cell receptor exert many antiviral effector functions in humans, including release of anti-HIV factors and direct cytotoxicity against virus-infected cells. Moreover, they are known to activate dendritic cells, improving antigen presentation function. After HIV infection, Vgamma9Vdelta2 T-cell number and reactivity are rapidly affected and they decrease upon disease progression. Bisphosphonate drugs such as zoledronic acid (Zol), used to treat bone diseases, have been shown to induce in vivo, in combination with interleukin-2, Vgamma9Vdelta2 T-cells' activation. The aim of this work was to verify whether the administration of Zol in combination with interleukin-2 in HIV-infected patients might improve Vgamma9Vdelta2 T-cell function, including immune adjuvancy mediated by gammadelta-dendritic cell cross-talk. Design and methods In HIV patients naive to antiretroviral therapy, we analyzed the effect of combined Zol and interleukin-2 treatment, in comparison to Zol alone, on Vgamma9Vdelta2 T-cell number, maturation and function, on dendritic cell activation and on HIV-specific CD8 T-cell response. Results Zol and interleukin-2-combined treatment induced in-vivo Vgamma9Vdelta2 T-cell expansion and maturation. Paralleling Vgamma9Vdelta2 T-cell activation, increased dendritic cell maturation and HIV-specific CD8 T-cell responses were found. Conclusion The specific modulation of Vgamma9Vdelta2 T-cell number and responsiveness after HIV infection may be at least transiently restored in vivo by Zol and interleukin-2 treatment. In this way, the immune effector mechanisms, secondary to Vgamma9Vdelta2 T-cell activation, were improved, suggesting a possible adjuvancy role of Zol and interleukin-2 treatment in restoring innate and specific competence in HIV-infected persons.