Drug resistance-associated mutations in antiretroviral treatment-naïve and -experienced patients in Kuwait.

The identification of human immunodeficiency virus (HIV) mutations leading to drug resistance enables patient-specific adaptation of the treatment regimen and predicts the risk of transmission of drug-resistant HIV. In this study, we report for the first time the prevalence in Kuwait of non-polymorphic resistance-associated mutations (RAMs) in patients under first-line antiretroviral therapy. Viral RNA was extracted from plasma samples of 64 treatment-naïve (untreated) and 64 treatment-experienced patients. The HIV-1 load was determined by real-time RT-PCR. The protease- and reverse transcriptase-encoding regions were analyzed by subtyping, and for drug resistance. The HIV-1 load at sampling in treatment-naïve patients ranged from 1.61 x 104 to 1.91 x 106 copies/ml, whereas that in treatment-experienced patients ranged from bitors (PIs) and NNRTIs. These results necessitate efforts to be made for reducing emergence of resistance-associated mutations in treated patients, and highlight the need for continuous monitoring of drug resistance patterns in Kuwait.

Cryptococcus neoformans serotype A and Cryptococcus gattii serotype B isolates differ in their susceptibilities to fluconazole and voriconazole.

This study presents antifungal susceptibility data for environmental isolates of Cryptococcus neoformans serotype A (n=32) and Cryptococcus gattii serotype B (n=18) to fluconazole and voriconazole employing disc diffusion and Etest methods. The disc diffusion test was performed on Mueller-Hinton agar as recommended by the Clinical and Laboratory Standards Institute (CLSI). For comparison, the disc diffusion test and Etest were also performed on RPMI-1640 agar supplemented with 2% glucose. The plates were incubated at 35 degrees C and read after 48h. Comparison of geometric mean inhibition zone diameters revealed that C. gattii isolates were significantly less susceptible than C. neoformans isolates to fluconazole (P=0.001) and voriconazole (P<0.0001). Similar results were obtained on RPMI agar by disc diffusion test and Etest, showing significantly reduced susceptibility for C. gattii isolates. Notwithstanding differences in the susceptibilities of the two species to fluconazole and voriconazole, they appeared susceptible according to the CLSI breakpoints recommended for some Candida spp. To what extent these differences in the susceptibilities of C. neoformans and C. gattii impact on the therapeutic management of cryptococcosis is unclear, although some studies have reported less favourable response in cases caused by the latter species.

[Mechanisms of imiquimod indirect antiviral activity]. / Mécanismes de l'activité antivirale indirecte de l'imiquimod.

The potential role of an immune response in HPV-related anogenital disorders had already been anticipated by clinicians. Indeed the lesions efflorescence and the relapsing HPV infection in HIV positive patients as well as the lack of recurrence in patients with spontaneous cure, provided relevant clues for a likely immune mechanism. At present time, the role of the immune system in the development of HPV-related anogenital disorders is well established : HPV induce a humoral and cell mediated immune response. This response is mainly exerted towards infected cells; it is also exerted at the systemic level, through antibodies synthesis, but this pathway remains a secondary one. Due to the limits of the present therapies (either purely destructive and characterized by the rate of recurrences, or antiviral, but difficult to use), it was necessary to find a new treatment type which enhances the local immune response, results in the disappearance of lesions and allows for a decrease in the risk of recurrences. The original mechanism of action of the first cell-mediated immune response modifier: imiquimod, for local use (Aldara 5 % cream) is an answer to this need. The first positive results observed in vitro and in animals were confirmed in patients with HPV anogenital warts in a double blind placebo-controlled study: imiquimod inhibits HPV replication and results in the condyloma regression. Its action is based on the combined activation of the natural local immunity, by stimulating interferon alpha; and of the acquired immunity, by stimulating a T-cell mediated immune response. Thus imiquimod appears to be an original antiviral compound, because it does not act directly on the virus itself.

Antibody-dependent enhancement of coxsackievirus B4 infectivity of human peripheral blood mononuclear cells results in increased interferon-alpha synthesis.

IgG devoid of neutralizing activity and isolated from donor plasma by chromatography formed immune complexes with coxsackievirus B4 (CVB4) and significantly increased the infection of peripheral blood mononuclear cells with CVB4. The major host cells for CVB4 infection enhanced with IgG are monocytic CD14+ cells. The roles of CVB and adenovirus receptor and Fcgamma receptor II and III have been shown. Increased viral replication and the release of infectious particles were demonstrated when interferon (IFN)-alpha produced by infected cells was first neutralized by use of antibodies. The CVB4 IgG-induced synthesis of IFN-alpha by monocytes reflected entry and uncoating of CVB4 but not of viral replication and required the presence of CVB4 RNA inside the cells. Thus, CVB4 can infect monocytes by an antibody-dependent mechanism through interactions between the virus, antiviral antibodies, and specific receptors that result in IFN-alpha production.

Increased levels of antiviral MxA protein in peripheral blood of patients with a chronic disease of unknown etiology.

Interferon alpha (IFN-alpha) is synthesized in response to viral infections. MxA protein, induced specifically by IFN-alpha and beta, expressed in peripheral blood cells, is detected more consistently than circulating IFN-alpha in serum of patients with viral infections. Thus, activation of the IFN-alpha/MxA system can be used as additional marker of the presence of a virus in patients. Therefore MxA protein and IFN-alpha levels were measured in patients with multiple sclerosis (MS), a chronic neurological disease of unknown etiology, in order to investigate the possible role of viruses in the expression of this disease. The means of MxA values obtained by using an immunochemiluminescent assay were significantly higher in blood of patients with remitting (n = 197) or relapsing (n = 39) multiple sclerosis (MS) patients and in patients with viral infections than in blood from healthy controls (n = 25) and from patients with bacterial infections (n = 12). Intra-individual variance in MxA levels in seven clinically stable remitting patients with MS was observed in the course of a follow-up, and high MxA levels were detected in three of them in blood samples collected consecutively over several months. By using an ultra sensitive assay, a higher MxA-inducer activity was obtained with sera from MS patients (n = 39) than with those from healthy controls (n = 12). Experiments with neutralizing antibodies proved that this activity in serum from patients was due to IFN-alpha, whereas IFN-alpha could not be detected by other methods. Altogether these results demonstrate that there is an activation of the IFN-alpha/MxA system in MS patients, which is consistent with the hypothesis that a viral infection may be associated with MS.

Inhibition of coxsackievirus B4 replication in stably transfected cells expressing human MxA protein.

Coxsackieviruses B (CVB) (B1-B6), positive-strand RNA viruses, cause a variety of diseases. CVB4 may have a causal role in insulin-dependent diabetes mellitus. IFN-alpha inhibits CVB replication; however, the mechanism is not well known. The interferon-alpha-inducible human MxA protein exerts an antiviral activity against negative-strand RNA viruses and against Semliki Forest virus, a positive-strand RNA virus. To test the antiviral spectrum of MxA against CVB4, we took advantage of stably transfected Vero cells expressing MxA (Vero/MxA) in 98% of cells. Compared with control cells, in Vero/MxA cells, CVB4 yields were dramatically reduced and expression of the VP1 CVB protein analyzed by immunofluorescence was highly restricted. Furthermore, the accumulation of positive- and negative-strand CVB4 RNA was prevented as shown by in situ hybridization and RT-PCR. These results indicate that the antiviral activity of MxA extends to CVB4 and that its replication cycle is inhibited at an early step in Vero/MxA cells.

Persistent infection of human pancreatic islets by coxsackievirus B is associated with alpha interferon synthesis in beta cells.

The interactions of coxsackievirus B3 (CVB3), CVB4E2 (diabetogenic), and CVB4JBV (nondiabetogenic) strains with human pancreatic islets from eight adult brain-dead donors were investigated. Persistent replication of viruses in human islets was proved by detection of viral RNA by in situ hybridization, VP1 capsid protein by immunofluorescence (IF) staining, negative-strand viral RNA by reverse transcription-PCR in extracted RNA from islets, and release of infectious particles up to 30 days after infection without obvious cytolysis. By double IF staining, glucagon-containing alpha cells and insulin-containing beta cells were shown to be susceptible to CVB. The persistence of CVB3 and CVB4 in islet cells was associated with the chronic synthesis of alpha interferon (IFN-alpha), as evidenced by the detection of IFN-alpha mRNA and immunoreactive IFN-alpha with antiviral activity. By double IF staining, IFN-alpha was detected in insulin-producing beta cells only. Experiments with neutralizing anti-coxsackievirus and adenovirus receptor (CAR) antibodies provided evidence that CAR was expressed by alpha and beta cells and that it played a role in the infection of these cells with CVB and the consecutive IFN-alpha expression in beta cells. The viral replication and the expression of IFN-alpha in islets were not restricted to the CVB4E2 diabetogenic strain and did not depend on the genetic background of the host. The neutralization of endogenous IFN-alpha significantly enhanced the CVB replication in islet cells and resulted in rapid destruction of islets. Thus, human beta cells can harbor a persistent CVB infection, and CVB-induced IFN-alpha plays a role in the initiation and/or maintenance of chronic CVB infection in human islets.

Increased level of interferon-alpha in blood of patients with insulin-dependent diabetes mellitus: relationship with coxsackievirus B infection.

The activation of the interferon (IFN)-alpha system and its relationship with coxsackievirus B (CVB) infection has been analyzed in 56 patients with insulin-dependent diabetes mellitus (IDDM; 25 children and 31 adults). Elevated levels of IFN-alpha were found in plasma of 70% of patients (39/56), and a positive detection of IFN-alpha mRNA in blood cells by reverse transcriptase-polymerase chain reaction (RT-PCR) was observed in 75% of patients (42/56). Enterovirus (EV) RNA assayed by seminested RT-PCR was detected in the blood of 50% of IFN-alpha-positive patients but not in any IFN-alpha-negative patients. The results of genotype analysis of amplified EV RNA sequences (5 CVB2, 8 CVB3, and 8 CVB4) were concordant with the results of CVB-neutralization tests. The comparison between IFN-alpha, EV RNA, and serology suggested that the proportion of CVB infection associated with IFN-alpha positivity might be higher than is predicted from the investigation of EV RNA. Together, the results suggest that, in a majority of cases, a CVB infection is associated with clinical IDDM.

Ex vivo interferon-gamma response to human immunodefiency virus-1 derived peptides in human immunodeficiency virus-1 infected patients.

The pattern of human immunodeficiency virus (HIV)-1 antigen-activated production of interferon (IFN)-gamma by immunocompetent cells of HIV-1 infected patients has been studied using a simplified assay combining a small volume (25 microliter) of whole blood stimulation with various HIV-1 antigens, and cytokine measurement in the same wells of microtitre plates (enzyme-linked immunotrapping assay, ELITA). The levels of IFN-gamma were higher using this assay than in the supernatant from stimulated whole blood cultures, therefore ELITA was used in the rest of the study. Specific immune responses to HIV-1 proteins (gp120, p24) and synthetic peptides derived from these proteins and from gp41 were detected in patients, but not in healthy controls. Decreased levels of IFN-gamma were observed in CDC class B (n = 5) and C (n = 4), compared with CDC class A (n = 5), following HIV-1 antigen-specific challenge. The positive response of cells from different patients to overlapping peptides of p25 (amino acids 329-344 and 335-351) was suggestive of a new epitope of HIV-1 gag recognized by T cells in the overlap region. In conclusion, the difference in in vitro antigen-specific T-cell responses of HIV-1-infected patients was shown using the ELITA method. Our results raise the possibility of using this method in screening specific antigens in HIV-1 infection.

In HIV-1-infected patients, plasma levels of HIV-1 RNA are inversely correlated with IFN-alpha responsiveness of whole-blood cultures to sendai virus.

BACKGROUND: A diminished or totally blocked IFN-alpha production in cells from HIV-1-infected patients has been reported. OBJECTIVE: To investigate the relationship between the decreased in vitro production of IFN-alpha and the plasma level of HIV-1 RNA. STUDY DESIGN: Whole blood samples of 39 healthy subjects and 44 HIV-1-infected patients were incubated in the presence of Sendai virus for 24 h. IFN-alpha contained in supernatants was assayed by using an immunochemical method (DELFIA) and by using an antiviral assay. Plasma HIV-1 RNA was measured by the Amplicor HIV-1 monitor test. RESULTS: The levels of IFN-alpha obtained were significantly lower in cultures from HIV-1 infected patients than in control subjects (P<0.0001). The antiviral activity in supernatants of Sendai virus-activated whole-blood cultures, assayed by protection of MDBK cells against vesicular stomatitis virus (VSV), was significantly lower in cultures from HIV-1 infected patients than in corresponding controls (P<0.0001). IFN-alpha values determined by DELFIA and those determined by bioassay were significantly correlated. In vitro production of IFN-alpha by whole-blood cultures correlated well with the plasma levels of HIV-1 RNA (P<0.001). CONCLUSIONS: In HIV-infected patients an increased rate of HIV-1 replication is associated with reduced responsiveness to induction of IFN-alpha by indicator virus, suggesting that HIV-1 replication causes impaired production of IFN-alpha by blood cells or vice-versa.

[Alpha interferon, antiviral proteins and their value in clinical medicine]. / Interféron alpha, protéines antivirales et applications médicales.

Type I interferon system is an important part of host's innate defense mechanisms against viral infections. The type I interferons mediate in part their antiviral effect via induction of various proteins. Among them the most widely known are 2'-5' oligoadenylate synthetase (2'-5' OAS) and a protein kinase (PKR). MxA, an other antiviral protein, is specifically induced by the type I interferons. The MxA protein contains the dynamin signature, which is implicated in transport processes. The MxA protein appears to block the replication of certain viruses at poorly defined steps. There are substantial differences in the antiviral activity of MxA between virus types. Indeed, the replication of vesicular stomatitis virus and influenza virus is inhibited by MxA, but not the one of type I herpes simplex virus. Measurements of interferon alpha and MxA levels may be of high value in clinical practice. Interferon alpha can be detected by using a bioassay based on the interferon alpha ability to protect cultured cells from the cytopathic effect caused by a selected challenged virus, or by using immunological techniques. The current bioassays are the most sensitive methods but they are cumbersome and lengthy, even though simplifications have been proposed. Immunological techniques are easier, however they do not explore the biological activity of the circulating interferon. The presence of type I interferon in biological samples (serum, plasma, cerebro-spinal fluid, cultured cell supernatants) can be indirectly assessed by capability of interferon alpha to induce in vitro the synthesis of MxA in a dose dependent manner in cultured cells. Following to the lysis of the cells, the induced MxA can be quantitated and hence the type I-interferon concentration can be determinated in samples. The quantitation of MxA protein in peripheral blood lysates can be useful as a specific marker of acute viral infections. A minute amount of whole blood (15 mul) is sufficient which facilitates its use in pediatrics. The specifically type-I-interferons inducible MxA protein is also a potential useful marker in the management of interferon alpha-treatment. Moreover, the detection of interferon alpha and antiviral proteins constitute an indirect approach for investigating the hypothesis of the role of viruses in chronic diseases with suspected infectious aetiology.

MxA protein in capillary blood of children with viral infections.

Capillary blood of febrile children was lysed by using a lysis buffer containing ascorbic acid. MxA quantitation was performed by an immunochemiluminescent assay. The MxA values were significantly higher in capillary blood of infants with viral infections due to adenovirus (n = 5), rotavirus (n = 15), or respiratory syncytial virus (n = 28), than in capillary whole blood from infants with bacterial infections (n = 6) and healthy control patients (n = 20). A strong correlation was found between the MxA values in capillary whole blood and peripheral whole blood (r' = 0.86, P < 0.0001, n = 48). The MxA values found at these two sites were compared with the levels of IFN-alpha obtained by a dissociation enhanced lanthanide fluoroimmunoassay. A correlation between these two values was found. The results show that the combination of collection of blood by finger prick and specific immunochemiluminescent assay for MxA protein measurement may be of value for the diagnosis of viral infections in children.

[Anti-viral proteins: from interferon alpha to its receptor]. / Protéines antivirales: de l'interféron alpha à son récepteur.

The immune response against viral pathogens include specific and non specific mechanisms. Cytokines are peptides which can play a role in the non specific immunity. Type I interferons (IFNalpha/beta) are the most effective antiviral cytokines. Interferons alpha are represented by a large familly of structurally related genes while the IFNbeta is encoded by a single gene. Type I interferon genes are located on the chromosome 9, and are segregated in a "modern" and an "ancestral" group with distinctive effects onto the cells. Type I interferons consist of 5 alpha helices, 4 of which are closely held together. Type I interferons receptor is a member of the class II cytokine receptor familly. It is a heterodimer composed of polypeptidic chains naimed IFNAR-1 and IFNAR-2. The type I interferons inducers are viruses, inactivated viruses and certain synthetic molecules. The molecular mechanisms of the induction of IFNbeta have been extensively described whereas the factors that play a role in the regulation of IFNalpha are not so well characterized. Two regulating domains located on the IFNbeta gene promotor are involved. The activation of these regulating domains leads to adverse effects by interacting with two intracellular factors (IRF1 and IRF2). IRF1 enhances the synthesis of IFNbeta but other pathways may be involved as well. The different IFNalpha sub-types have not only synergistic but also antagonistic effects. The synthesis of the different subtypes depends on cell lines and IFNalpha inducers. The N terminal part of IFNalpha is a major determinant in the diversity of the biological effects of IFNalpha sub-types which probably involve changes of the conformation of the type I receptor resulting from interactions between the receptor and its ligand. The type I interferons confer resistance to viruses, especially by enhancing the synthesis of intracellular antiviral proteins.

Biological properties of interferon-alpha produced Ex vivo by whole blood of patients infected by human immunodeficiency virus-1.

We investigated the biological properties of interferon (IFN)-alpha produced by Sendai virus (SV)-activated whole blood cultures in 20 patients infected with human immunodeficiency virus (HIV)-1 and 24 healthy controls. Supernatants of cultures were assayed for IFN-alpha by using an immunological method (DELFIA), biological methods and an in-vitro MxA induction assay. The levels of intracellular MxA protein were detected by an immunochemiluminescence assay. The levels of IFN-alpha in patients measured by DELFIA were significantly lower than those in healthy controls (P < 0.0001), but the antiviral activity of IFN-alpha in patients infected with HIV-1 was lower than predicted from DELFIA. The IFN-alpha produced by cells of patients infected with HIV-1 was able to induce MxA protein in human amnions WISH cells but was unable to protect these cells against Vesicular Stomatitis Virus (VSV)-induced cytopathic effects. A relative increased capability to induce the production of MxA protein in vitro was observed with the IFN-alpha contained in culture supernatant of virus-activated whole blood of HIV-1-infected patients with increased levels of MxA in their peripheral blood. These data suggest that biological properties of IFN-alpha produced in the course of HIV-1 infection are different from those observed with IFN-alpha of healthy subjects.

Production of interleukin-4, interferon (IFN)-gamma and IFN-alpha in human immunodeficiency virus-1 infection: an imbalance of type 1 and type 2 cytokines may reduce the synthesis of IFN-alpha.

Interferon-alpha (IFN-alpha) is an important molecule in the antiviral response, but cells from HIV-1-infected individuals show a reduced ability to secrete IFN-alpha. We investigated an association between an imbalance of type 1/type2 cytokines and the production of IFN-alpha in HIV-1 infection. We used whole blood culture to study the cytokine production profile, interferon-gamma (IFN-gamma) and interleukin-4 (IL-4), in response to HIV-1 antigens and to study the Sendai Virus and HSV-1-induced-production of IFN-alpha in seven HIV-1-infected patients. An impaired synthesis of IFN-alpha was obtained in patients with a predominant IL-4 production (IL-4 > IFN-gamma), and we found a positive correlation between the ex vivo production of IFN-alpha and the IFN-gamma/IL-4 ratio but not with the HIV RNA copy number in plasma. We investigated the role of T-cell-derived cytokines in the in vitro production of IFN-alpha by PBMC from eight healthy donors, activated with Sendai Virus or HSV-1. Whereas type 2 cytokines (IL-4, IL-13) inhibited virus-induced IFN-alpha synthesis, on the contrary, type 1 cytokines (IL-2, IFN-gamma) enhanced it. A disarray in the T-cell-derived cytokine response may play a role in the defect of IFN-alpha production in HIV-1-infected individuals. Further investigations are needed to explore this hypothesis.