Antiviral responses of human Leydig cells to mumps virus infection or poly I:C stimulation.

BACKGROUND: The immuno-privileged status of the testis is essential to the maintenance of its functions, and innate immunity is likely to play a key role in limiting harmful viral infections, as demonstrated in the rat. In men mumps virus infects Leydig cells and has deleterious effects on testosterone production and spermatogenesis. The aim of this study was to test whether mumps virus infection of isolated human Leydig cells was associated with an inhibition of their innate antiviral defences. METHODS: Leydig cell production of mRNA and protein for interferons (IFNs) and of three antiviral proteins-2'5' oligoadenylate synthetase (2'5'OAS), double-stranded RNA-activated protein kinase (PKR) and MxA-was investigated, in the absence or presence of mumps virus or viral stimuli including poly I:C, a mimetic of RNA viruses replication product. RESULTS: Stimulated or not, human Leydig cells appeared unable to produce routinely detectable IFNs alpha, beta and gamma. Although the level of PKR remained unchanged after stimulation, the expression of 2'5'OAS and MxA was enhanced following either mumps virus or poly I:C exposure (P < 0.05 versus control). CONCLUSIONS: Overall, our results demonstrate that mumps virus replication in human Leydig cells is not associated with a specific inhibition of IFNs or 2'5'OAS, MxA and PKR production and that these cells display relatively weak endogenous antiviral abilities, as opposed to their rat counterparts.

Clinically validated mutation scores for HIV-1 resistance to fosamprenavir/ritonavir.

BACKGROUND: We developed clinically relevant genotypic scores for resistance to fosamprenavir/ritonavir in HIV-1 protease inhibitor (PI)-experienced patients. METHODS: PI-experienced patients with virological failure receiving fosamprenavir/ritonavir as the sole PI for at least 3 months and with detectable fosamprenavir plasma levels were included. The impact of baseline protease mutations on virological response (VR, i.e. decrease in plasma HIV-1 RNA between baseline and month 3) was analysed using the Mann-Whitney test. Mutations with prevalence >10% and P value <0.10 were retained. The Jonckheere-Terpstra test was used to select the combination of mutations most strongly associated with VR. The association between score and VR was assessed by multivariate backward regression. RESULTS: In the 73 patients included, the median baseline HIV-1 RNA was 4.6 log(10) copies/mL (range: 2.7-6.9) and the mean decrease at month 3 was -1.07 +/- 1.40 log(10) copies/mL. Ninety per cent of the patients were infected by HIV-1 subtype B variants. Two fosamprenavir/ritonavir mutation scores were constructed: score A (L10F/I/V + L33F + M36I + I54L/M/V/A/T/S + I62V + V82A/F/C/G + I84V + L90M) was based only on mutations associated with a worse VR, whereas score B (L10FIV + L33F + M36I + I54L/M/V/A/T/S + A71V - V77I - N88S + L90M) also took into account favourable mutations. Both scores were independent predictors of VR, however, co-administration of tenofovir was associated with a worse VR and the presence of the N88S protease mutation and co-administration of enfuvirtide with a better VR. CONCLUSIONS: These clinically validated mutation scores should be of interest for the clinical management of PI-experienced patients. The fosamprenavir/ritonavir score A was introduced in the 2006 ANRS algorithm along with isolated mutations I50V and V32I + I47V.

Experimental inoculation of the adult rat testis with Sendai virus: effect on testicular morphology and leukocyte population.

BACKGROUND: Surprisingly little is known about the interactions between viruses and the male uro-genital tract. These are important, as viral testicular orchitis, induced by mumps or human immunodeficiency virus (HIV) infection for example, can lead to sterility. Moreover, semen is an essential vector in the propagation of sexually transmissible viral diseases. Here, we studied the effects of testicular infection with Sendai virus, a virus related to mumps virus, on the cellular distribution of viral particles and on testicular morphology, with particular attention to the testicular leukocyte population. METHODS: At 5, 9, 11 or 24 h post-injection of Sendai virus through the scrotum, the testes were fixed for morphological and immunohistological studies. Localization of virus particles and numeration of leukocytes were performed using specific antibodies and morphological criteria. RESULTS: As early as 5 h post-injection, a rapid and massive infiltration of leukocytes was observed in the interstitial tissue. The peritubular cell layer and the most external part of the basal portion of the seminiferous tubules were altered. The virus was diffusely located within the interstitial tissue 9 h following the injection whereas, after 24 h, viral proteins were restricted to the cytoplasm of infiltrated leukocytes. The number of leukocytes increased with time post-injection. Thus, 24 h post-injection, CD3+ T-cell number was 3-fold higher, ED1+ monocyte number was 4-fold higher and polynuclear cell number was 600-fold higher than in the control testes (P<0.001 all observations). In contrast, the population of resident macrophages was unaffected by Sendai virus. CONCLUSIONS: Testicular viral infection causes inflammation including rapid recruitment of leukocytes. The experiments presented here provide a model for further studies on the etiopathology of viral orchitis, in particular that caused by mumps virus.

Ritonavir-saquinavir dual protease inhibitor compared to ritonavir alone in human immunodeficiency virus-infected patients.

The objective of this study was to evaluate the antiretroviral efficacy and safety of ritonavir (600 mg twice a day [b.i.d.])-saquinavir (400 mg b.i.d.) compared to ritonavir (600 mg b.i.d.) in patients pretreated and receiving continued treatment with two nucleoside analogs. The study was placebo controlled, randomized, and double blind. Inclusion criteria included protease inhibitor naive status and a viral load of >10,000 copies/ml. The main end point was viral load at week 24. Forty-seven patients were included (25 given ritonavir and 22 given ritonavir-saquinavir) and monitored until week 48. At inclusion, 23% had had at least one AIDS-defining event. Previous treatment durations (mean and standard deviation) were 42 +/- 25 and 37 +/- 23 months, viral loads were 4.75 +/- 0.62 and 4.76 +/- 0.50 log(10) copies/ml, and CD4 cell counts were 236 +/- 126 and 234 +/- 125/mm(3) in the ritonavir and ritonavir-saquinavir groups, respectively. At week 24, viral loads were 2.81 +/- 1.48 and 2.08 +/- 1.14 log(10) copies/ml (P = 0.04) and CD4 cell counts were 330 +/- 151 and 364 +/- 185/mm(3) (P = 0.49) in the ritonavir and ritonavir-saquinavir groups, respectively. Similar results were observed at week 48. Moreover, at week 48, 40 and 68% (P = 0.05) and 28 and 59% (P = 0.03) of patients achieved viral suppression at below 200 and 50 copies/ml in the ritonavir and ritonavir-saquinavir groups, respectively. At week 24, six patients in the ritonavir group but only one in the ritonavir-saquinavir group had key mutations conferring resistance to protease inhibitors. Clinical and biological tolerances were similar in both groups. In nucleoside analog-pretreated patients, ritonavir-saquinavir has higher antiretroviral efficacy than and is as well tolerated as ritonavir alone.

Prevalence of resistance mutations in antiretroviral-naive chronically HIV-infected patients in 1998: a French nationwide study.

OBJECTIVE: To estimate the prevalence of resistance-conferring mutations to antiretroviral drugs in previously untreated patients with chronic HIV-1 infection as a basis for French recommendations on viral genotyping before antiretroviral treatment initiation. DESIGN: Resistance mutations were sought in samples from 404 patients seen in 23 specialized centres throughout metropolitan France in 1998. METHODS: The protease and reverse transcriptase (RT) genes of plasma virions were sequenced. Primary and secondary protease and RT gene mutations were identified from the International AIDS Society resistance testing - USA panel. RESULTS: The prevalence of patients with primary and secondary mutations were 3.7% (95% CI 1.7-5.7) and 50.3% (95% CI 45.0-55.6), respectively. The prevalence of patients with mutations associated with resistance to nucleoside RT inhibitors (NRTI) and non-nucleoside RT inhibitors was 3.3% (95% CI 1.5-5.1) and 0.8% (95% CI 0.0-1.7), respectively. The prevalence of patients with NRTI primary mutations differed according to whether seropositivity had been diagnosed more or less than one year previously (0.2 versus 2.2% P = 0.023). Primary mutations associated with protease inhibitor resistance occurred at a prevalence of 1.9% (95% CI 0.5-3.4) with no difference according to the duration of known seropositivity. CONCLUSION: In France, in 1998, the prevalence of patients with primary mutations associated with resistance to antiretroviral drugs was low. Genotyping before the initiation of therapy was not recommended in chronically HIV-1-infected naive patients. A national sentinel survey of resistance in this clinical setting is performed regularly to update the recommendations for resistance testing.

Genotypic and phenotypic resistance patterns of human immunodeficiency virus type 1 variants with insertions or deletions in the reverse transcriptase (RT): multicenter study of patients treated with RT inhibitors.

Genomic rearrangements in the 5' part of the human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) have been involved in multidrug resistance to nucleoside RT inhibitors (NRTI). We carried out a retrospective, multicenter study to investigate the prevalence, variability, and phenotypic consequences of such rearrangements. Data concerning the HIV-1 RT genotype and the biological and clinical characteristics of NRTI-treated patients were collected from 10 virology laboratories. Sensitivities of the different HIV-1 variants to RT inhibitors were analyzed in a single-cycle recombinant virus assay. Fifty-two of 2,152 (2.4%) RT sequences had a rearrangement in the 5' part of the RT, with an extensive molecular variation. The number of codons inserted between positions 68 and 69 ranged from 1 (3 samples) or 2 (41 samples) to 5 and 11 in one case each. In four cases, codon 67 was deleted. High levels of phenotypic resistance to zidovudine (AZT), lamivudine (3TC), stavudine (d4T), abacavir (ABC), and didanosine (ddI) were found in 95, 92, 72, 62, and 15% of the 40 samples analyzed, respectively. Resistance to AZT, d4T, and ABC could be found in the absence of the T215Y/F mutations. Resistance to 3TC could develop in the absence of specific mutations. Low-level resistance to ddI was noticed in 40% of the patients. The deletions of codon 67 seemed to have little effect on NRTI sensitivity. Most of the rearrangements were shown to contribute to cross-resistance to NRTI. The results regarding susceptibility to ddI raise the question of the interpretation of the phenotypic data concerning this drug.

JC virus genotypes in France: molecular epidemiology and potential significance for progressive multifocal leukoencephalopathy.

JC virus (JCV) induces progressive multifocal leukoencephalopathy (PML), especially in human immunodeficiency virus (HIV)-infected patients. Although JCV genotypes have primarily been associated with geographic patterns, a distinctive neuropathogenicity was recently attributed to genotype 2. A multicenter study was conducted to describe the distribution of JCV genotypes in France and to investigate correlations between genotypes and PML. Genotypes were determined by sequencing 494 bp in the VP1 capsid gene. Peripheral JCV was studied in 65 urine samples from 43 HIV-infected patients and from 22 control subjects. Genotypes 1, 4, 2, and 3 were detected in 52.3%, 30.8%, 12.3%, and 4.6% of the samples, respectively. In 56 brain or cerebrospinal fluid samples, PML-associated JCV of genotypes 1, 2, 4, and 3 was found in 66%, 19.7%, 8.9%, and 5.4%, respectively. Infection with JCV genotypes 1 or 2 was correlated with PML (odds ratio, 3.29). On the other hand, infection with JCV genotype 4 could represent a lower risk for PML.

HIV RNA and HIV DNA in peripheral blood mononuclear cells are consistent markers for estimating viral load in patients undergoing long-term potent treatment.

The aim of this study was to evaluate residual viral replication by assessing the HIV load of circulating infected cells in patients given an effective antiprotease-containing treatment for 1 year. PBMC HIV RNA and HIV DNA was quantified by techniques standardized and evaluated by interlaboratory quality control testing. Viral markers identified in a multicenter study were validated in a cross-sectional study of 121 patients beginning treatment. A longitudinal study of 3 viral markers was carried out in 18 patients, each of whom had fewer than 200 copies of HIV RNA per milliliter of plasma after 12 months of treatment. The cross-sectional study showed that viral replication in PBMCs was correlated with the number of circulating infected cells (Spearman rank correlation; p = 0.0001, r = 0.35) and the concentration of virus particles in the plasma (Spearman; p = 0.0001, r = 0.54). The longitudinal study showed that the decrease in HIV RNA levels was smaller in PBMCs than in the plasma. The largest decrease in HIV DNA levels after 12 months of treatment was recorded in patients with low levels of intracellular replication (Spearman; p = 0.005, r = 0.69). PBMC HIV RNA and HIV DNA levels were very informative markers, complementary to plasma HIV RNA levels. They should be used in future trials evaluating the long-term efficacy of new associations of highly active antiretroviral treatments.

Safety and efficacy of ritonavir and saquinavir in combination with zidovudine and lamivudine.

BACKGROUND: Ritonavir is a potent inhibitor of cytochrome P4503A4 that strongly increases saquinavir bioavailability. In this study we assessed the safety and antiretroviral efficacy of the combination of these two compounds in patients pretreated and receiving continued treatment with zidovudine and lamivudine who were protease inhibitor naive and who had a CD4 cell counts below 200/mm3. METHODS: In this 48-week pilot study, all patients received 600 mg ritonavir and 400 mg saquinavir twice daily. Administration of zidovudine and lamivudine was continued without a change in previous doses. Viral load, CD4 cell count, and the emergence of resistance to the two protease inhibitors were evaluated repeatedly up to week 48. RESULTS: Sixteen patients were included in the study. Previous nucleoside analog treatment duration was 48+/-22 months (mean +/- SD). Two patients quit taking both protease inhibitors within 2 weeks. The ritonavir dose had to be reduced in 10 other patients because of side effects. Between inclusion and week 48, plasma viremia varied from 4.87+/-0.43 to 3.00+/-1.29 log10 copies/mL and CD4 cell counts ranged from 98+/-61 to 250+/-139/mm3. Ten patients (63%) had viral loads below 200 copies/mL and 7 (44%) had viral loads below 50 copies/mL. A single key mutation that conferred ritonavir resistance I84V and V82A/V developed in two patients. A mutation at codon 54 developed in another patient. These mutations were associated with repeated cessations of antiretroviral treatment. No lipodystrophy was observed. CONCLUSION: Ritonavir and saquinavir in combination are quite well tolerated and induce a high and sustained antiretroviral efficacy. A four-drug combination that includes these two protease inhibitors should be considered as a first line of treatment in patients with low CD4 cell counts.

[Beta interferon in multiple sclerosis: pharmacological differences]. / Les interférons bêta dans la sclérose en plaques: les différences pharmacologiques.

Interferons beta (INF) would be a good recombinant therapeutic choice for the management of relapsing remitting multiple sclerosis (RRMS). Three forms are approved in various countries including one IFN beta-1b (Betaféron) and two IFN beta-1a (Avonex and Ribif). These agents are apparently similar although they are not identical. Differences may be of clinical relevance. Important differences are described here in terms of pharmacokinetics, the spectrum of side effects and molecular chemistry. The clinical consequences on the benefit/risk ratio are discussed. This review recalls the difficulty in developing such compounds and in reaching marketing approval. An improvement in the acceptability and efficiency of IFN beta could by obtained when analyzing basic research data.

Zidovudine resensitization and dual HIV-1 resistance to zidovudine and lamivudine in the delta lamivudine roll-over study.

OBJECTIVE: To study zidovudine resensitization and dual resistance to zidovudine/lamivudine in HIV-1 isolates from nucleoside reverse transcriptase (RT) inhibitor-experienced patients during selective pressure exerted by zidovudine/lamivudine combination therapy. DESIGN AND METHODS: HIV-1 isolates from 29 patients receiving zidovudine/lamivudine combination therapy in the Delta roll-over study were analysed at entry and during a 1 year follow-up period for phenotypic susceptibility to zidovudine and lamivudine in the ANRS PBMC assay. The RT gene from codon 20 to 230 and at codon 333 was analysed by nucleotide sequencing of the corresponding isolates. RESULTS: HIV-1 isolates from 23 of the 29 patients were phenotypically resistant to zidovudine at baseline; 61% of these patients showed significant zidovudine resensitization during follow-up. The zidovudine IC50 value correlated positively with log10 plasma HIV-1 RNA (P = 0.02) and negatively with the CD4 cell count (P = 0.004). Zidovudine resensitization (related to acquisition of the M184V mutation) was transient, with evolution towards dual resistance to zidovudine and lamivudine in 20 of the 29 patients. The phenotype of certain dually resistant isolates coincided with the emergence of multiple mutations in the 5' part of the RT gene. CONCLUSIONS: M184V-mediated zidovudine resensitization of HIV-1 is transient in most patients who are given zidovudine/lamivudine combination therapy when zidovudine resistance has already emerged. The subsequent evolution towards dual phenotypic resistance to zidovudine/lamivudine corresponds to complex genotypic profiles.

Prevalence of JC virus viraemia in HIV-infected patients with or without neurological disorders: a prospective study.

Progressive Multifocal Leukoencephalopathy (PML) is a severe demyelinating disease, which is rapidly fatal and is due to JC virus (JCV) infection, which especially occurs in HIV-infected patients. To investigate JCV pathophysiology and to evaluate the predictive value of JCV detection in blood, we looked for JCV DNA in leukocytes and plasma of 96 patients without any neurological symptoms and 109 patients with neurological diseases, among whom 19 were suffering from PML. JCV genome was detected in about 18% of all patients, i.e. 15.6% of patients with central nervous system disorders except PML, 13.5% of patients without neurological symptoms and significantly more often in PML patients (47.6%). Both leukocytes and plasma were tested; in plasma, JCV DNA was found in 36.1% of positive patients and in cells in 80.5%. Surprisingly in seven instances only the plasma contained JCV genome. One-year follow-up of these patients showed that the absence of JCV DNA in blood was associated with a very low probability of developing PML (negative predictive value=0.99).

Longitudinal study of HIV-specific cytotoxic lymphocytes in HIV type 1-infected patients: relative balance between host immune response and the spread of HIV type 1 infection.

To evaluate the contribution of a specific cytotoxic response in the control of HIV infection in relation to clinical status, we performed serial analysis of anti-Env and anti-Gag cytotoxic activity in 13 infected individuals over a 6- to 10-year period, using cryopreserved peripheral blood mononuclear cells (PBMCs). Autologous EBV-transformed B cell lines infected in vitro with recombinant vaccinia viruses expressing HIV-1 env and gag genes were used as targets. Without any stimulation of the effector cells, we were able to show an anti-HIV cytotoxic activity in the PBMCs of 12 of 13 HIV-1-infected patients, consistent with chronic immune activation in HIV infection. Different patterns of HIV-specific cytotoxic activity were observed, and the extent of this cytotoxic response varied between the clinically defined groups of individuals. No direct relationship was observed with the number of CD4 and CD8 lymphocytes during the observation period. However, patients who remained asymptomatic had a more vigorous cytotoxic response than patients with clinical deterioration during the observation period, and a significant difference was observed for HIV Gag-specific CTL activity. From these data, we suggest that the HIV-specific cytotoxic response has a protective role in the course of HIV infection.

New types of primers (stair primers) for PCR amplification of the variable V3 region of the human immunodeficiency virus.

The aim of the study was to develop a reliable PCR method for the detection of viral genomes with frequent mutations like HIV and hepatitis C virus. A system of 'stair' primers is suggested which allows amplification of a genomic sequence despite the presence of mutations in the region of the primers. In this system, classical primers are replaced with primers composed of a mixture of equimolar oligonucleotides in which the 5' end remains constant (single sized fragment) and the 3' end is displaced base by base. By PCR, 'stair' primers (HIV set) were compared to single-sequence primers of 20 and 25 nucleotides chosen in the same hypervariable region of the HIV gp120 (on both sides of V3 region), as well as to classical primers chosen in the conserved pol (polV2) and gag (SK38-39) regions of the genome. Of 17 HIV isolates obtained by co-culture of lymphocytes from HIV-seropositive patients, 17/17 (100%) were amplified using stair primers, 14/17 (82%) with 25-nucleotide primers, and 12/17 (70%) with 20-nucleotide primers. Amplification occurred in 17/17 instances with polV2 primers and in 16/17 instances with SK38-39. In addition, 55 other isolates were tested comparatively using stair, polV2 and SK38-39 primers. All isolates were amplified using stair and SK38-39 primers and 54/55 isolates with polV2 primers. When applied to 22 extracts of patients' lymphocytes DNA, stair primers amplified all 22 extracts to the same degree as polV2 and SK38-39, whereas the 20 and 25 nucleotide primers chosen in the variable region were not as reliable. This new primer system allows reliable detection of variable genomic regions of the HIV genome and amplification of such regions directly in patient leukocytes. In addition, the contribution of this system to microbiology and human genetics in general may be important.

Interferon-alpha and -gamma expression in the rat testis.

Interferon-alpha (IFN alpha), -beta, and -gamma are well known for their antiviral, antiproliferative, and immunoregulatory activities. Although several studies suggest an involvement of IFNs in the spermatogenic process, nothing is known about the possible production of these molecules within the testis. Moreover, the antiviral capabilities of testicular cells have not yet been explored despite their importance in the context of sexually transmissible diseases. Using reverse transcription-polymerase chain reaction, a cytopathic inhibition micromethod assay, and an enzyme-linked immunosorbent assay, the present study demonstrates for the first time that IFN alpha and -gamma are produced by testicular cells. IFN alpha protein and corresponding messenger RNA are expressed by peritubular, Sertoli, and germ cells. In vitro, IFN alpha production by Sertoli cells, peritubular cells, and early spermatids was inducible by the Sendai virus, whereas pachytene spermatocyte IFN alpha production was not triggered by this virus. Of all the testicular cell types tested, Sertoli cells by far produced the highest concentrations of IFN alpha/beta, followed by peritubular cells. Both IFN gamma messenger RNA and IFN gamma protein were found in early spermatids, but, in contrast, were not produced by peritubular cells, Sertoli cells, or pachytene spermatocytes. In conclusion, our study establishes the cellular distribution of IFNs within the seminiferous tubules and provides the basis for research into the possible involvement of IFNs in regulation of the spermatogenic process. To the best of our knowledge, our results afford the first insight on how the testicular antiviral defense system is organized.

The prognostic value of plasma viremia in HIV-infected patients under AZT treatment: a two-year follow-up study.

To determine the prognostic value of plasma viremia in long-term zidovudine (AZT)-treated HIV-infected patients, HIV-1 plasma viremia (PV) was quantified in 28 HIV-infected patients before and during AZT long-term treatment; the follow-up also included p24 antigenemia and CD4 cell counts. The variations of these markers during the follow-up period, the correlation with the clinical outcome (progressors versus nonprogressors), and the discrepancies between PV and surrogate markers were then analyzed. A significant and stable decrease in PV titer was observed in only nonprogressors (Friedman test, p < 0.005). At the end of follow-up, 11 (73%) of the 15 non-progressors were PV responders (patients who remained or became PV- long-term), whereas all the 13 progressors were PV nonresponders (patients who remained or became PV+). These results indicated a strong correlation between PV and clinical outcome (Fischer's exact test, p < 0.0001). The persistence, increase, or reappearance of viral replication appeared to be an important predictor of poor clinical outcome in HIV-infected patients under AZT treatment. This finding could provide a rational basis to help the clinician's decision in the clinical treatment of HIV-infected patients.

Genotypic evolution of HIV-1 isolates from patients after a switch of therapy from zidovudine to didanosine.

The existence of zidovudine (ZDV)-resistant and didanosine (ddI)-resistant human immunodeficiency-1 (HIV-1) variants mutated in the reverse transcriptase (RT) gene has been previously demonstrated. In this study, we tried to follow up the genotypic changes in the RT after the switch of therapy from ZDV to ddI. We studied HIV-1 isolates from 11 patients undergoing ddI therapy. Genotypic data were obtained with differential polymerase chain reaction (PCR) and with direct sequencing after PCR. The prevalence of ZDV resistance-related mutations showed a very slow decrease, particularly when patients had been treated with ZDV for a long time. The appearance of a mutation at codon 74 seemed to be independent of the presence or absence of ZDV resistance-related mutations. The broad genotypic heterogeneity of the isolates and the complexity of the evolution in one patient's isolates plead for large sequencing studies of the RT genome in new therapeutic approaches.