Ferdinando Dianzani

Neutralizing Antibodies to Interferon-α: Relative Frequency in Patients Treated with Different Interferon Preparations

Abstract The frequencies of antibody development so far reported in patients treated with different interferons (IFNs) are not readily comparable because of differences in treatment regimens and assay methods. Thus the frequency of neutralizing antibody development was analyzed in a large sample of sera derived from a relatively homogeneous group of patients treated with different IFN-α preparations. The frequency of developing neutralizing antibody to IFN varied according to the IFN given. Particularly, the seroconversion frequency was significantly higher in patients treated with recombinant IFN-α2a (20.2%) than in patients treated with either recombinant IFN-α2b (69%) or IFN-αN1 (1.2%), a lymphoblastoid IFN-α. Furthermore, sera obtained from patients treated with either recombinant IFN neutralized both types of recombinant IFNs but failed to neutralize IFN-αN1.

Spontaneous release of interferon gamma by intestinal lamina propria lymphocytes in Crohn's disease. Kinetics of in vitro response to interferon gamma inducers.

The spontaneous induced release of interferon gamma (IFN gamma) by cultured intestinal lamina propria lymphocytes was investigated in patients with Crohns disease. In contrast to normal lymphocytes, intestinal lymphocytes from these patients spontaneously released IFN gamma and seemed to contain IFN gamma in their cytoplasm. Autologous peripheral lymphocytes did not release IFN gamma. When stimulated with interferon inducers lamina propria lymphocytes from Crohns disease tissue showed an increase in IFN gamma release 24 hours after induction with no appreciable further increase over the next two days of culture, while in control cells, either peripheral or intestinal, IFN gamma release progressively increased, peaking 72 hours after induction. These findings indicate that in Crohns disease the intestinal lymphocytes are stimulated in vivo to produce IFN gamma and that the spontaneous IFN gamma production is compartmentalised to the gut lymphocytes. These data support the concept that locally released IFN gamma has a crucial role in cell interactions in the lamina propria and contribute to the locally occurring immune phenomena in Crohns disease, including the increased epithelial expression of major histocompatibility complex class II antigens.

Secondary Mutations in the Protease Region of Human Immunodeficiency Virus and Virologic Failure in Drug-Naive Patients Treated with Protease Inhibitor-Based Therapy

The role of mutations in protease (PR) and reverse-transcriptase (RT) of human immunodeficiency virus (HIV) in predicting virologic failure was assessed in 248 antiretroviral-naive HIV-positive patients who began a PR inhibitor-containing antiretroviral regimen. Genotypic testing was performed on plasma samples stored before the start of therapy. Twenty-seven patients (10.9%) had mutations in the RT, 5 (2%) carried primary mutations in the PR, and 131 (52.8%) showed only secondary PR mutations. Virologic failure at week 24 occurred in 62 (25.0%) of 248 patients. There was a statistically significant correlation between virologic failure and the number of PR mutations (P= .04, chi(2) test). Mutations at codons 10 and 36 of PR (present in 39.3% and 40.0% of patients in whom treatment failed, respectively) were identified by stepwise logistic regression as the strongest predictors of virologic failure (odds ratio, 2.20; 95% confidence interval, 1.30-3.75; P= .004). If confirmed in independent studies, this result may justify the increased use of HIV genotyping in drug-naive patients requiring antiretroviral therapy.

Unidirectional budding of HIV-1 at the site of cell-to-cell contact is associated with co-polarization of intercellular adhesion molecules and HIV-1 viral matrix protein

Objectives: To explore the possibility that HIV‐1 budding and cellular adhesion molecules co‐polarize at cell‐to‐cell contact sites. To investigate the incorporation of host‐cell‐derived adhesion molecules into HIV‐1. Methods: The cellular sites involved in HIV‐1 budding were examined by transmission electron microscopy. Single and double immunocytochemistry staining was used to evaluate the cellular distribution of the viral matrix protein and adhesion molecules. Quantitative flow cytometry was used to measure the cellular expression of adhesion molecules. An immunocapture technique was used to measure the presence of cell‐derived proteins on HIV‐1. The captured virus was measured by a p24 antigen assay. The infectivity of virus captured by monoclonal antibodies was tested by measuring the virus antigen yield in supernatants after the addition of sensitive cells. Results: Released and budding HIV‐1 was mainly localized at the cell‐to‐cell contact regions. This feature was consistent with a polarized staining for the virus matrix protein p18 at cell‐to‐cell contact regions. Intercellular adhesion molecules (ICAM)‐1 in HIV‐1‐infected cells were polarized on both isolated cells and syncytia, co‐localizing with HIV‐1 matrix protein. HIV‐1 incorporated all the adhesion molecules expressed by the host cells, although without quantitative correlation with their cellular expression. Conclusions: HIV‐1 is released at cell‐to‐cell membrane contact sites. Both ICAM‐1 and virus matrix protein co‐polarized on isolated cells and syncytia at the sites involved in the recruitment of uninfected cells. The impressive concentration of ICAM at cell sites where most virions are released may account for the acquisition of these membrane proteins by the HIV‐1 progeny, and may be important for the cell‐mediated spread. AIDS 1995, 9:329‐335

Increased replication of T-cell-tropic HIV strains and CXC-chemokine receptor-4 induction in T cells treated with macrophage inflammatory protein (MIP)-1α, MIP-1β and RANTES β-chemokines

Objective and design: To study, in T-lymphoid cells, the effects of macrophage inflammatory protein (MIP)-1α, MIP-1β and RANTES β-chemokines on the replication of T-cell-tropic HIV-1 strains, since it has been reported that β-chemokines interfere with the replication of macrophage-tropic HIV-1 strains, but not T-cell-tropic strains. Methods: Freshly phytohaemagglutinin (PHA)-activated peripheral blood lymphocytes (PBL) and cultured PHA-activated T cells from healthy volunteers, as well as the C8166 T-cell line, were treated overnight with β-chemokines before infection with T-cell-tropic HIV-1 isolates, or human T-lymphotropic virus type IIIB. HIV replication was followed by detecting the production of infectious particles, p24 antigen, and viral sequences. CXC-chemokine receptor (CXCR)-4 expression was followed by detection and quantification of specific transcripts. Results: Pretreatment of T cells with MIP-1α, MIP-1β and RANTES affected T-cell-tropic strains, increased the replication of HIV-1P1 and HIV-1RPdT strains dose-dependently, as well as virus absorption and provirus DNA accumulation. These findings were associated with increased accumulation of CXCR-4 transcripts, and mediated by the protein tyrosine kinase signalling. Moreover, β-chemokines stimulated PBL proliferation. Conclusions: β-Chemokines increase the adsorption and replication of at least some T-cell-tropic HIV-1 strains, and this is related to stimulated expression of the CXCR-4 coreceptor.

Breakthrough during recombinant interferon alfa therapy in patients with chronic hepatitis C virus infection : prevalence, etiology, and management

Recombinant interferon alfa (r-IFN alpha 2) has been shown to normalize the aminotransferase levels in approximately 50% of patients with chronic hepatitis C virus (HCV). Few patients experience a relapse during the treatment, in spite of a complete initial response (breakthrough). We studied 191 HCV Ab-positive patients with histologically proven chronic hepatitis. All of them were treated with r-IFN alpha 2 (3 MU three times a week). A complete response was seen in 54.4%. However, 12 of 104 responders experienced a breakthrough. At the time of breakthrough, neutralizing IFN antibodies were positive in 6 of 12 patients. Binding IFN antibodies were positive in all of these 12 patients. Continued treatment with r-IFN alpha 2, even at higher doses, did not restore the previous response in any patient. All of them were then switched to natural lymphoblastoid IFN, and this rapidly restored a complete response in all of the patients.

Antibodies to interferon (IFN) in hepatitis C patients relapsing while continuing recombinant IFN-α2 therapy

A number of trials have demonstrated that IFN‐α is effective in chronic hepatitis C virus infection. It is known, however, that a number of chronic hepatitis C patients experience, after an initial response to IFN, disease reactivation or relapse (also called ‘breakthrough’) while IFN therapy is still ongoing. Since in a number of clinical conditions a significant correlation between development of antibodies to IFN and failure of therapy has been established, we addressed the possibility that the development of antibodies to IFN may take part in the relapse occurring in hepatitis C patients during recombinant IFN‐α (rIFN‐α) therapy. The prevalence of neutralizing (NA) and binding antibodies (BA) to rIFN‐α2 has been evaluated in 45 patients with chronic hepatitis C treated with rIFN‐α2a who first normalized aminotransferase (ALT) levels, and subsequently showed disease reactivation while on treatment. The presence of NA and BA was tested before therapy, during the response to IFN treatment, and at the time when ALT started to rise again to abnormal levels. The results showed that no patients had detectable antibodies to IFN before therapy and during the period of response to the therapy, while most of them (88.9%) developed NA and/or BA to IFN‐α2 concomitantly with disease reactivation. In particular, in 29 of the 45 patients (64.4%) ALT normalized on treatment and rose to abnormal levels when NA appeared in their serum, while in 11 of the 16 (68.8%) remaining patients the relapse was associated with BA development. The frequency of seroconversion in these patients is significantly higher than that observed in the control group. These data indicate that antibodies to IFN may be responsible for breakthrough in the majority of patients showing disease reactivation while rIFN‐α therapy is still ongoing.

Correlation of Interferon-Induced Expression of MxA mRNA in Peripheral Blood Mononuclear Cells with the Response of Patients with Chronic Active Hepatitis C to IFN-alpha Therapy

MxA, a protein with selective activity against certain viruses, is an accepted specific indicator of type I interferon (IFN) activity. We have developed an internally controlled quantitative-competitive PCR to measure the amounts of MxA mRNA expressed in peripheral blood mononuclear cells (PBMC). This assay is more sensitive, quantitative, and easily applied to serial clinical samples than previously described methods. We have applied this assay retrospectively to 27 patients with chronic active hepatitis C given IFN-alpha2. Most such patients gain no sustained benefit but nevertheless suffer from the side effects, expense, and inconvenience of the treatment. Fourteen of the 27 had been classified on clinical grounds as responders and 13 as nonresponders at the end of a 6 month treatment period. We measured MxA mRNA in PBMC obtained before and after 8 weeks of IFN-alpha2 treatment. All the patients expressed some level of mRNA before treatment began, and after 8 weeks of treatment, the level rose in 19. This increase was significant (p < 0.001) only in patients classified as responders. This strongly suggests that hepatitis C virus (HCV) patients who express increased amounts of MxA mRNA in their PBMC during IFN-alpha treatment are most likely to obtain long-term benefit. If this finding is confirmed in future prospective studies, it will provide an extremely important predictive marker for managing IFN-alpha therapy in patients with HCV.

The interferons: a biological system with therapeutic potential in viral infections.

Successful medical use of interferon for chronic viral infections is increasingly dependent on understanding the biologic and molecular mechanisms of the interferon system. Interferon (IFN) is one of the bodys natural defenses. Production of IFN is a defensive response to foreign components of microbes, tumors and antigens. This IFN response begins with the production of the IFN proteins (alpha, beta and gamma) which then induce antiviral, antimicrobial, antitumor, and immunomodulatory actions. Thus, the initial production or administration of IFN(s) does not protect directly but instead reacts with specific receptors on cell surfaces to activate cytoplasmic transduction signals that then enter the nucleus to stimulate cellular genes encoding a number of effector proteins which lead to the defensive actions. The known molecular, humoral and cellular mechanisms by which these effector proteins exert their antiviral activities are presented. In addition, the pathogenesis of chronic infections is overviewed in the context of the interferon defenses.

Biological and clinical significance of neutralizing and binding antibodies to interferon-alpha (IFN-α) during therapy for chronic hepatitis C

It is known that IFN therapy can induce the development of anti‐IFN antibodies. In order to evaluate the biological and clinical significance of both neutralizing (NA) and non‐neutralizing (binding) antibodies, 123 patients with chronic hepatitis C treated with recombinant IFN‐α were examined. Among them, 15 were positive for NA and 24 for binding antibodies. The kinetics of NA appearance show that, in general, they develop early during the first 3 months of treatment. Moreover, NA seem to be clinically relevant, since they may be responsible for non‐responsiveness to treatment in 53% of patients who develop them. The evaluation of the clinical significance of binding antibodies is more difficult. They appear significantly earlier in non‐responders than in responders, but no differences were observed in the overall percentage of seroconversion between responders and non‐responders. Thus, it is not possible at the moment to establish their possible role in inducing non‐responsiveness.