[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.

[Evaluation of a quantitative HBV-DNA PCR assay in lamivudine treated hepatitis B-infected patients]. / Intérêt de la quantification par PCR de l'ADN du VHB chez les patients traités par lamivudine.

Lamivudine (3TC) is a nucleoside analogue which inhibits replication of HIV and HBV and which is used in the treatment of chronic hepatitis B-infected patients with safety and efficacy. The activity of lamivudine was evaluated by the measurement of DNA-HBV concentration in plasma using a very sensitive assay (1,000 copies/mL) (Amplicor VHB Monitor. Roche). Ten patients chronically infected with hepatitis B (group A) and 24 patients with HIV-1 co-infection (group B) were enrolled. In 9 patients of group A, HBVDNA load was undetectable a median of 3.5 months after the beginning of treatment and remained negative for 2 years with hepatitis Be antigen disappearing and normal alanine aminotransferase concentration. In the last immunodeficient patient, the virus which had been resistant to three interferon treatments, was also resistant to lamivudine. In five patients of group B, HBV DNA load remained undetectable after 18 months with HBe antigen disappearing and baseline concentration of alanine aminotransferase. In the remaining 19 patients after a transient decrease of HBV DNA concentration for one year, HBV DNA load increased again without disappearing of HBe antigen and without decrease of alanine aminotransferase concentration showing lamivudine resistant hepatitis B virus. Mutations in the YMDD motif of the DNA polymerase gene were identified in 11 patients (3 with M550V/I mutation; 7 with M550V/I and L256M mutations; 1 with M550V/I, L526M and V519L mutations). In 6 of these patients, was found a M184V mutation in the VIH polymerase. No correlation could be observed between the mutations detected in the two viruses. Using a sensitive HBV-DNA assay, efficacy of lamivudine for a long time in HBV infected patients was proved. However, the prevalence of lamivudine resistance is related to duration of treatment and it may be necessary to use a multitherapy.

[Congenital human cytomegalovirus infection: value of human cytomegalovirus DNA quantification in amniotic fluid]. / Infections congénitales à cytomégalovirus humain: valeur de la quantification de l'ADN du CMVH dans le liquide amniotique.

A quantitative PCR assay (RS Elosa CMV, Lambdatech) was used to quantitate HCMV DNA in maternal amniotic fluid of 12 fetuses with congenital infection (group 1) and of 10 fetuses without congenital infection (group 2). HCMV detection was performed for both groups using culture and qualitative PCR. Histologic examinations of fetal tissues and placenta were carried out for 9 patients from group 1. The amniotic fluid viral loads were negative in all patients of group 2. In group 1, all viral loads were high (from 1.105 to > 107 cop/mL) and no difference was observed between symptomatic and asymptomatic foetuses. Further evaluation on larger samples is needed to define more precisely the pronostic value of HCMV DNA quantification in amniotic fluid.

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.

Detection of enteroviruses ribonucleic acid sequences in endomyocardial tissue from adult patients with chronic dilated cardiomyopathy by a rapid RT-PCR and hybridization assay.

A rapid reverse transcription polymerase chain reaction (RT-PCR) and microwell capture hybridisation assay with general specificity for enteroviruses was developed and compared with an improved nested RT-PCR for the detection of enteroviral RNA sequences in endomyocardial tissue from patients with chronic dilated cardiomyopathy. This method could detect as few as 20 genomic RNA copies per 100 mg of heart tissue homogenate and results could be obtained within 8 hours. Of the 55 biopsy specimens aseptically collected from the explanted hearts of 55 patients, 21 (38.2%) were positive by RT-PCR microplate assay, whereas only 19 (34.5%) were positive by nested RT-PCR assay and none were positive by classical cell culture assays. No enterovirus was detectable by RT-PCR or classical cell culture assays in any of the 55 heart biopsy specimens taken from organ donors without any known heart disease. Moreover, the nucleotide sequences of EV nested RT-PCR products showed greatest similarity to group B Coxsackieviruses [CVB3 (n = 12) or CVB5 (n = 3)], but also to group A Coxsackieviruses (CVA21 (n = 1) or CVA9 ( n= 3)]. The described RT-PCR and microwell capture hybridisation assay can be applied to the virological diagnosis of human enteroviral cardiac infections. Moreover our findings suggest that group B and group A Coxsackieviruses can persist in heart tissue from patients with end-stage chronic cardiomyopathy, supporting the hypothesis that these viruses could be implicated in the etiology of idiopathic dilated cardiomyopathy.

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.

Simultaneous detection of 6 human herpesviruses in cerebrospinal fluid and aqueous fluid by a single PCR using stair primers.

A Herpes Consensus allows the simultaneous detection of 6 human herpesviruses: herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2), human cytomegalovirus (HCMV), varicella-zoster virus (VZV), Epstein-Barr virus (EBV), and human herpes virus 6 (HHV-6). This technique was used first to examine retrospectively 100 DNA extracts from 95 CSF and 5 aqueous fluids, prepared by treatment by saturated NaCl followed by ethanol precipitation (n = 63) or by simple boiling (n = 37) and stored at -80 degrees C, and secondly to test prospectively 38 CSF samples for which two DNA extracts were prepared with commercially available DNA extraction kits. In all cases, the results were compared with those of an "in-house" PCR. Concordant results between both PCR and the Herpes Consensus techniques were obtained in 61 of 63 DNA extracts prepared by treatment by saturated NaCl (97%) and in only 31 of 37 boiled samples (84%). Both commercially available methods of DNA extraction examined appear to be suitable for Herpes Consensus PCR, although they cannot remove completely PCR inhibitors that must be sought in case of negative results. This preliminary study shows that the Herpes Consensus method should be of value for rapid diagnosis of herpesvirus infections on condition that it is performed on purified DNA extracts.

Enteroviruses can persist with or without active viral replication in cardiac tissue of patients with end-stage ischemic or dilated cardiomyopathy.

To investigate enterovirus replication versus persistence in end-stage cardiac diseases, endomyocardial biopsies from explanted hearts of 70 patients with idiopathic dilated cardiomyopathy (IDCM), 64 patients with chronic coronary disease (CCD), and 45 donors of healthy hearts (controls) were examined by reverse transcriptase-polymerase chain reaction for genomic and antigenomic enterovirus RNA and by VP1 antigen immunohistochemistry. Enterovirus genome was detected in 25 of 70 patients with IDCM and in 21 of 64 patients with CCDs (35.7 vs. 32.8%, respectively; P=.12). Of the 46 patients positive for genomic RNA, only 3 exhibited antigenomic RNA and VP1 antigen that demonstrated active viral replication, whereas 43 had latent infection characterized by the absence of antigenomic RNA associated with or not with VP1 antigen expression. No viral component was detected in control subjects. The findings demonstrate that a small percentage of patients with end-stage chronic cardiac diseases had active enterovirus replication in their myocardium.

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.

Differential detection of rhinoviruses and enteroviruses RNA sequences associated with classical immunofluorescence assay detection of respiratory virus antigens in nasopharyngeal swabs from infants with bronchiolitis.

To define the role of enteroviruses and human rhinoviruses as etiological agents in childhood bronchiolitis, clinical aspirates from 84 infants admitted to hospital with symptoms of obstructive bronchiolitis were tested by picornavirus RT-PCR assay, adenovirus PCR assay and classical immunofluorescence antigen detection of common respiratory viral agents. Respiratory syncytial viruses (A&B) were detectable in 45 of 84 (53.6%) nasopharyngeal aspirates from infants with bronchiolitis, whereas coronaviruses, influenza viruses, and parainfluenza viruses were not detectable in the same samples. Adenoviruses were detectable by PCR in 11 of 84 (13.1%) nasopharyngeal swabs. By using a picornavirus RT-PCR assay followed by a differential molecular hybridisation, rhinovirus and enterovirus RNA sequences were detected in 16 of 84 (19%) and in 10 of 84 (11.9%) of the nasopharyngeal swabs tested. Positive human rhinovirus or enterovirus RT-PCR assay, however, was the only evidence of respiratory infection in 8 of 84 (9.5%) and in 7 of 84 (8.33%) of the studied patients. Respiratory syncytial viruses, human rhinoviruses, adenoviruses, and enteroviruses occur in dual infections detected in 18 of 84 (21.4%) respiratory samples tested. The median duration of stay in hospital was not significantly different between the patients demonstrating a single viral infection and those with a dual viral infection (6.22 +/- 2.07 vs. 5. 04 +/- 0.95 days; P > 0.05). In summary, combination of molecular and classical detection assays of common viruses can be used to demonstrate enterovirus and human rhinovirus respiratory infection in childhood bronchiolitis, and provides an improved approach to obtain new insights into concomitant viral respiratory tract infection in infants.

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.

[Detection of human papillomavirus DNA by molecular hybridization in tube: interest in cervical neoplasia]. / Détection de l'ADN des papillomavirus humains par hybridation moléculaire en tube: intérêt dans les dysplasies cervicales.

The presence of human papillomavirus (HPV) DNA in 79 cervical specimens obtained from 70 patients was studied by using a molecular hybridization technique performed in tube. The results were compared to those of the cytological and histological studies. The molecular hybridization technique in tube (Hybrid Capture I) detects two groups of HPV types. One group is highly associated with the development of cancer (types 16, 18, 31, 33, 35, 45, 51, 52, 56) whereas the second group (types 6, 11, 42, 43, 44) is not. Among 42 patients with cervical lesions before any treatment, high risk DNA of HPV was found in 50% of those with low grade cytology and 90% with high grade cytology. In total, 32 out of the 42 patients (76%) who presented histological lesions, were actually infected by HPV. Samples were obtained before and after treatment from 9 patients. Seven out of 9 presented high grade cervical intraepithelial neoplasia (CIN) and 2 other patients had low grade CIN. HPV DNA was not detected in any of the patients after treatment. Detection of HPV DNA by molecular hybridization in tube is simple, sensitive, standardized, inexpensive and is well adapted to screening programs. It can be used in complement of the cytological diagnosis, in the surveillance of equivocal cytological abnormalities, and in the follow-up of treated patients.

Human polyomavirus JC latency and reactivation status in blood of HIV-1-positive immunocompromised patients with and without progressive multifocal leukoencephalopathy.

BACKGROUND: Human polyomavirus JC (JCV) induces human progressive multifocal leukoencephalopathy (PML) in patients with AIDS. Peripheral blood mononuclear cells (PBMC) of HIV-1-positive immunocompetent and immunocompromised patients can harbour JCV genome, but their precise role in JCV latency or reactivation status before the onset of PML remains hypothetical. OBJECTIVES: To assess JCV latency or reactivation status in PBMC of HIV-1-positive immunocompromised patients without PML. DESIGN: A group of 82 HIV-1-positive immunocompromised patients who did not have PML were compared with 10 patients with AIDS and PML and with 69 HIV-1-positive immunocompetent patients without PML. METHODS: DNA and total RNA were extracted from PBMC. The presence of JCV DNA was demonstrated by a semi-nested polymerase chain reaction (PCR). By using primer pairs specific for an early gene,T, and a late gene, VP1, the expression of both early and late gene mRNA in PBMC could be identified using reverse transcriptase (RT) PCR. RESULTS: JCV DNA was detected by PCR in 17.4% of 69 HIV-1-positive immunocompetent patients, in 23.2% of 82 HIV-1-positive immunocompromised patients, and in 60% of 10 patients with AIDS and PML. No correlation could be drawn between the detection of JCV DNA in the PBMC and the clinical or biological status of the HIV-1-positive patients. By using RT-PCR procedures, no expression of JCV early and late mRNA in PBMC was found in any patients. CONCLUSIONS: JCV DNA is detectable in the PBMC of 20.5% of 151 HIV-1-infected patients independently of the CDC (Centers for Disease Control and Prevention) stages of the infection. Moreover, our results suggest that active replication of JCV in PBMC appears to be absent or at least a very rare event in HIV-1-positive immunocompromised patients with and without PML.

Specific immune response to human immunodeficiency virus (HIV)-1 in patients assessed through the production of interferon-gamma and interleukin-4 in HIV-1 p24-activated whole blood cultures: relationship with the viral load in plasma.

We studied the in vitro HIV-1 antigen-stimulated production of IFN-gamma and IL-4 in HIV-1-infected patients and its relationship with viral replication as assessed through the plasma level of HIV-1 RNA. The levels of IFN-gamma and IL-4 were higher in supernatants of stimulated whole blood cultures than in stimulated peripheral blood mononuclear cell cultures, therefore whole blood cultures were used in the rest of the study. Specific IFN-gamma and IL-4 responses to HIV-1 p24 antigen were observed in HIV-1-infected patients but not in healthy controls (n = 23). A lower proportion of individuals with a positive IFN-gamma response to HIV-1 p24 was observed in patients at a declining clinical stage: 62% in asymptomatic patients (CDC group A, n = 16) versus 19% in symptomatic patients (CDC groups B and C, n = 21; P = 0.007, chi2 testing), whereas the proportion of individuals with a positive IL-4 response to HIV-1 p24 was almost similar in both groups of patients (25% versus 23.8%). Increased IL-4 production by HIV-1 p24-activated immunocompetent cells of patients and a predominant IL-4 response to HIV-1 p24 (with IL-4/IFN-gamma > 1) were positively correlated with an increased viral load. In contrast, there was no correlation between the mitogen-stimulated production of IL-4 and IFN-gamma and the viral load in plasma. The CD8 T cells from whole blood of patients, but not from controls played a significant role in the HIV-1 p24-activated production of IFN-gamma and IL-4. In conclusion, HIV-1-antigen-stimulated whole blood appears to be a valuable tool to study the production of IL-4 in HIV-1-infected patients. The cytokine profile pattern in response to epitopes of HIV-1 gag p24 may play an important role in the host immune response to HIV-1.

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.