Sub-epitopic dissection of HCV E1315-328HRMAWDMMMNWSPT sequence by similarity analysis.

Our labs are focused on identifying amino acid sequences having the ability to react specifically with the functional binding site of a complementary antibody. Our epitopic definition is based on the analysis of the similarity level of antigenic amino acid sequences to the host proteome. Here, the similarity profile to the human proteome of an HCV E1 immunodominant epitope, i.e. the HCV E1(315-328)HRMAWDMMMNWSPT sequence, led to i) characterizing the immunoreactive HCV E1 315-328 region as a sequence endowed with a low level of similarity to human proteins; ii) defining 2 contiguous immunodominant linear determinants respectively located at the NH(2) and COOH terminus of the conserved viral antigenic sequence. This study supports the hypothesis that low sequence similarity to the host's proteome modulates the pool of epitopic amino acid sequences in a viral antigen, and appears of potential value in defining immunogenic viral peptide sequences to be used in immunotherapeutic approaches for HCV treatment.

Effect of HCV infection on THP-1 monocytoid cells.

Hepatitis C virus (HCV) is the main cause of hepatocellular carcinoma in industrialized countries. HCV-HIV-1 co-infection occurs frequently among users of illicit intravenous drugs, thereby increasing the severity of HIV disease and the evolution of chronic active hepatitis towards cirrhosis and hepatocellular carcinoma. The present work shows that THP-1 monocytoid cells are susceptible to HCV infection, of strain 1b, and that this strain can induce cellular modifications in this cell line. Infection of HCV was demonstrated by positivity for the E2 antigen within THP-1 cells and by indirect immunofluorescence; moreover, HCV-RNA was detected in supernatants of THP-1 cells from day 7 post-inoculation. Cell shape and membrane surface antigens varied upon viral infection, which is also capable of inducing oxygen radicals. In particular we underline the relevant intracellular accumulation of ferritin that paralleled an increase of cell surface expression of the transferrin receptor. Evaluation of cellular events upon HCV infection in THP-1 cells may represent a useful tool with which to identify alteration in monocytes metabolism and to study therapeutic approaches for such alterations.

HCV infective virions can be carried by human platelets.

It has been previously demonstrated that platelets (PLTs) can bind and transport HIV-1 infectious virions. Hepatitis C virus (HCV)-HIV-1 co-infection occurs frequently among users of illicit intravenous drugs, thereby increasing the severity of HIV disease and the evolution towards chronic active hepatitis and hepatocellular carcinoma of HCV-related hepatitis. In the present study we investigated whether or not PLTs can carry HCV, and studied the binding mechanisms. Purified PLTs, obtained from healthy donors, HCV negative and HIV negative, were adsorbed with HCV-containing serum and then employed to infect a THP-1 monocytoid cell line. Replication of HCV was observed as shown by positivity for the E2 antigen within THP-1 cells, by indirect immunofluorescence; moreover, HCV-RNA was detected in supernatants of THP-1 cells at day 7 post-incubation with HCV-adsorbed PLTs. The binding of HCV to PLTs seems to involve fibronectin (FN), as already shown in the case of HIV-1. Indeed, treatment with RGD (Gly-Arg-Gly-Asp-Ser), the key oligopeptide of FN binding, inhibits the ability of HCV to be carried by PLTs in infective forms; the same phenomenon occurs with Mabs to FN. Moreover the infection of THP-1 cells seems to increase FN surface expression, as demonstrated by immunofluorescence tests.

A review of cardiovascular complications accompanying AIDS.

Manifestations of cardiovascular system involvement are not uncommon complications of HIV infection, especially in AIDS patients. However, the frequency of these manifestations is influenced by different variables including: survival prolongation in HIV-infected patients, because of advances in antiretroviral treatment; improvement of immunodepression and reduction in the occurrence of opportunistic infections; adverse effects of some drugs. At present, on the whole cardiovascular complications that are HIV correlated in the western world, including Italy, occur less frequently than in the past. However complications associated with alterations in lipometabolism prevail because they can be promoted by some protease inhibitors in predisposed subjects. The most frequently reported questions and a careful analysis of recent data in the medical literature regarding the most common HIV-correlated cardiovascular complications are discussed in this review.

Cognitive and affective disorders associated to HIV infection in the HAART era: findings from the NeuroICONA study. Cognitive impairment and depression in HIV/AIDS. The NeuroICONA study.

OBJECTIVE: To assess the natural story of HIV-associated affective and cognitive disorders and the relationship with clinical, pharmacological, immunological and behavioural factors. METHOD: A total of 395 HIV-positive patients, naive to Highly Active Antirectroviral therapy (HAART), with no severe psychiatric disorders have been enrolled in the Neuro-ICONA Study. All participants were administered a comprehensive data collection instrument including an addiction behaviour survey, a medical problem list, a psychiatric assessment, a validated neuropsychological test battery. RESULTS: The global prevalence of cognitive impairment and of prominent depressive symptomatology were 17.9 and 15.5%, respectively. A significant difference in the prevalence of prominent depressive symptomatology was observed between patients in HAART and those not taking HAART(14.1 vs. 23.8%; P = 0.05). CONCLUSION: Depressive and cognitive disorders affect a substantial proportion of HIV-seropositive subjects. The prevalence of prominent depressive symptomatology appears to significantly vary in relationship to the therapeutic protocol.

Seroprevalence study of tick borne encephalitis in Turin province.

BACKGROUND: Tick borne encephalitis (TBE) virus is diffused in some European countries and it is transmitted by tick bites. In Italy Isodex ricinus represents the main vector of the infection, that rarely produces the neurologic manifestations, characterising the secondary phase of the same. METHODS: In Italy TBE has been little studied and this only in the Middle and Northern regions of the country. Seroepidemiological researches were done prevalently on subjects at high risk of tick bite, such as hunters or forest guards and especially in Trentino and Tuscany. No precise information about TBE virus diffusion was disposable in the Piedmont region and particularly in the Susa valley where, before our investigation failed the data about it. RESULTS: We found that usual hunters and wild boar breeders seem to be particularly exposed to the risk of TBE virus infection, but none neurologic involvement was detected in the anamnesis of the significantly seropositive subjects and also of the borderline ones, that we have studied, despite the limited number of these subjects. CONCLUSIONS: Nevertheless we hope for a following extension of our case report, also in consideration that rare cases of encephalitis of unknown etiology, are signalled in Piedmont.

Cervico-vaginal dysplasia--papillomavirus-induced and HIV-1 infection: role of correlated markers for prognostic evaluation.

Sexually-transmitted diseases (STD) can facilitate the progression of HIV-1 infection. Among them, as we have previously demonstrated, cervico-vaginal dysplasia-papillomavirus (HPV)-induced, together with HSV-2 co-infection, seems to be correlated with a more evident immunodepression in HIV-positive women, compared with other sexually transmitted diseases. Here we have analysed some of the main correlated markers of HIV-1 infection progression: CD4 + T lymphocyte concentration, CD4 +/CD8 + T cells ratio, HIV-1 RNA loads and haemoglobin (Hb) concentration in 30 HIV-1 positive women co-infected with HPV, and suffering from cervico-vaginal dysplasia, in different stages. In particular, we noticed a positive correlation, evaluated by Spearman's test, between the degree of progression of dysplastic stages (CIN1 --> 3) until invasive carcinoma (IC) and HIV-1 RNA loads (C(s) = +0.78; p < 0.001), and in contrast, a negative correlation between the same stages of progression and respectively CD4 + T cell concentration (C(s) = -0.54; p = 0.01), ratio (C(s) = - 0.63; p = 0.002) and Hb concentration (C(s) = -0.85; p < 0.001). In conclusion, it is important to underline that low levels of Hb generally paralleled the degree of immunodepression. In fact CD4 + T cell levels and ratio positively correlated with Hb concentrations respectively, with C(s) = + 0.83; p < 0.001 and C(s) = + 0.90; p < 0.001. Finally, the most efficacious antiretroviral combined therapy (HAART = Highly Active Antiretroviral Therapy) can improve the above described laboratory parameters in HIV-1/HPV co-infected women and seems to prevent the progression of CIN1 to the following stages of the dysplastic disease.

Serum citrate levels, haptoglobin haplotypes and transferrin receptor (CD71) in patients with HIV-1 infection.

BACKGROUND: The progression of HIV-1 infection towards its more advanced stages is accompanied by changes in iron metabolism and increased body iron stores. PATIENTS AND METHODS: Given the ability of HIV to alter iron metabolism, we studied the principal (transferrin system) and alternative (citrate system) iron pathways in a group of 65 HIV-infected patients (symptomatic stage B1-B3) and in a group of 36 healthy seronegative individuals. We determined serum citrate levels, haptoglobin (Hp) haplotypes, expression of transferrin receptor (CD71) on cell lines infected with HIV-1 as well as iron markers including blood iron, transferrin and ferritin. RESULTS: Our data showed decreased serum citrate levels in the HIV-infected patients compared to controls (92.9 +/- 22.4 microM/l vs 126.2 +/- 29.2 microM/L; p < 0.01). In particular, the serum citrate levels negatively correlated with HIV-1 RNA copy number (mean: 2.53 +/- 1.88 x 10(5)/ml, r(s) = 0.70, p < 0.01) and positively correlated with CD4+ T-lymphocyte count (mean: 241 +/- 168/ml, r(s) = 0.64, p > 0.05). Accordingly, blood iron, transferrin and red cell concentrations were lower in HIV-infected patients compared to the controls, whereas serum ferritin levels were higher in HIV-infected patients. Moreover, the Hp haplotype distribution showed significant differences only in the group of HIV-infected patients (p = 0.02; chi2 test). CONCLUSION: Our results show that iron metabolism is altered in patients with HIV-1 infection. The alternative pathway (citrate system) is particularly affected, since when citrate levels are low, both aconitase activity and HIV-1 replication need iron.

Anti-HIV effects of chloroquine: mechanisms of inhibition and spectrum of activity.

OBJECTIVE: To investigate the mechanisms and spectrum of the anti-HIV activity of chloroquine. DESIGN AND METHODS: MT-4 cells or peripheral blood mononuclear cells were infected with X4, R5 or R5/X4 HIV-1 strains from clades A-E and HIV-2. The cells were then treated with clinically relevant and achievable chloroquine concentrations (i.e. 0-12.5 microM), so as to determine the EC50. The effects of chloroquine on reverse transcription and integration were tested using a replication-defective reporter HIV-1 construct (pRRL.sin.hPGK.GFP). The effects of the drug on the viral envelope were assessed by syncytium assays and immunoprecipitation, using antibodies to different epitopes of gp120. RESULTS: In de-novo infected MT-4 cells, chloroquine selectively inhibited HIV-1 IIIB replication but not pRRL.sin.hPGK.GFP. In chronically HIV-1-infected H9 IIIB cells, chloroquine decreased the infectivity of the newly produced virus and the ability of these cells to form syncytia in co-culture with MT-2 cells. These effects were associated with structural changes in the gp120 glycoprotein, such as a reduction of reactivity with antibodies directed against the glycosylated 2G12 epitope. Although affecting a variable target such as gp120, chloroquine was capable of inhibiting X4, R5 and R5/X4 primary HIV-1 isolates from subtypes A, B, C, D, E and HIV-2. CONCLUSION: At clinically achievable concentrations chloroquine inhibits HIV-1 post-integrationally by affecting newly produced viral envelope glycoproteins, and the drug has broad-spectrum anti-HIV-1 and HIV-2 activity.

The anti-HIV-1 activity of chloroquine.

BACKGROUND: there is a dramatic need for drugs with anti-HIV-1 activity that are affordable for resource-poor countries. Chloroquine (CQ) is one such drug. OBJECTIVES: to review the data indicating that CQ has anti-HIV-1 activity. RESULTS: chloroquine (CQ) and its derivative hydroxychloroquine (HCQ) are endowed with a broad anti-HIV-1 activity inhibiting X4, R5, and X4/R5 stains in lymphocytic and monocytic cells. Interestingly, CQ is capable of inhibiting HIV-1 replication at concentrations within the range reported in plasma of individuals chronically treated with doses of the drug which have well-known and limited toxicity. These effects have been confirmed in vivo in two clinical trials. The principal mechanism of HIV-1 inhibition by CQ seems to be an effect on gp120 on a post-transcriptional level. Because CQ and HCQ appear to have a novel site of action (i.e. post-transcriptional inhibition of gp120), these drugs may be particularly useful in combination with other anti-retroviral agents (e.g. ZDV, ddI and HU). CONCLUSION: combining these drugs with other anti-HIV-1 agents, especially HU and ddI, may be an interesting option for the treatment for HIV-1 infected individuals in the developing world.

Human herpesvirus 8 and associated diseases in a group of 67 human immunodeficiency virus-seropositive individuals.

Human herpesvirus 8 (HHV-8) is involved in the pathogenesis of Kaposi's sarcoma, of B-cells lymphomas, and of Castelman's disease. However, the role of this virus is not yet well known. To investigate the relationship between HHV-8 infection and diseases correlated with human immunodeficiency virus (HIV), we studied a cohort of 67 HIV-seropositive subjects, some of them coinfected with HHV-8. An indirect immunofluorescence test was employed to detect the antibodies against this virus. Positive cases were 31 (46.3%); among the 67 patients, 14 were weakly positive, or + (20.9%); 11 were significantly positive, or ++ (16.4%); and 6 were strongly positive, or (8.9%). These last six patients were the most affected by opportunistic infections, and all were affected by neoplastic pathologies. Moreover, the HHV-8 positive subjects showed hematologic and martial alterations more severe than those in the negative subjects. HHV-8 seroprevalence in HIV-seropositive patients of our cohort was higher (46.3%) than in normal population (0-10%). The presence of disseminated Kaposi's sarcoma and other neoplasms associated with high HIV-RNA levels in HHV-8-positive patients, and particularly in those with strong positivity, corroborates the hypothesis that the virus is correlated with the progression of HIV infection and with its related diseases, especially those that are neoplastic. Last, the severe alterations of iron metabolism found in the patients coinfected with HHV-8 and the negative effect of this virus on the lymphocytic populations can contribute to the unfavorable evolution of HIV infection and also might facilitate tumor development.

Effects of the exogenous Nef protein on HIV-1 target cells.

Nef is a multifunctional gene of HIV which can increase virus replication either directly or by modulating the target cell's metabolism. Nevertheless the role of the exogenous Nef protein is not yet well understood. To investigate it, we studied the effects of the recombinant Nef protein on the expression of some antigens of lymphoid T-cells permissive to HIV-1 replication, and on their proliferation and on apoptosis induction. For this purpose, we utilised MT-4 and H9 T-cell lines. We evaluated FN (fibronectin), CD4 and CD71 expression in uninfected and acutely or chronically infected cells, both untreated and treated with Nef. Our studies showed a significant up-regulation of FN especially in uninfected cells, with a dose of 2.5 microg ml(-1); in contrast, a significant down-modulation of CD4 and CD71 both in uninfected and in acutely or chronically infected cells, was detected. The proliferation of H9 uninfected cells was significantly reduced 24 h after treatment with Nef protein in a dose-dependent manner (ranging from 0.02 to 2.5 microg ml(-1)); likewise a significant inhibition of proliferation of acutely and chronically infected cells was evident with 2.5 microg ml(-1). Moreover, we demonstrated a dose-dependent activity of Nef on inducing apoptosis in H9 uninfected cells and no effects of this protein on modulation of INF alpha and gamma production in peripheral blood mononucleated cells of health donors. Nef appeared to be able to increase the effect of apoptotic stimuli. In conclusion, our data suggest that in our experimental system, the exogenous Nef protein can inhibit cellular synthesis facilitating the metabolic pathway involved in virus replication. In addition it modulates the susceptibility to the HIV-1 infection and finally, that apoptosis induction or enhancement can facilitate the release of neoformed virions.

Modulation of surface transferrin receptors in lymphoid cells de novo infected with human immunodeficiency virus type-1.

To investigate whether transferrin receptor (CD71) expression is affected by acute HIV-1 infection, three different lymphoid cell lines (MT-4, SUPT-1, H9) were infected with HIV-1 and tested for surface CD71 expression after different incubation periods depending on cell survival after infection. We found that expression of surface CD71 was lower in cells infected with HIV-1 than in uninfected controls: the timing and extent of this down-modulation depended apparently on the different susceptibility of the cell lines to HIV-1 infection and cytopathogenicity. Citrate, a molecule capable of chelating iron, dose-dependently prevented down-modulation of surface CD71 in HIV-1 infected cells as well as viral cytopathic effects. We conclude that (i) expression of surface transferrin receptors is down-modulated by acute HIV-1 infection in T lymphoid cells, that (ii) this cell phenotypic modulation is associated with the cytopathic effects of the virus, and that (iii) these phenomena are modulated by iron chelation. These results support the view that iron metabolism may be an important area for interaction between HIV-1 and human cells.