Peg-IFNα/ribavirin/protease inhibitor combination in hepatitis C virus associated mixed cryoglobulinemia vasculitis: results at week 24.

BACKGROUND: The standard-of-care treatment of patients with hepatitis C virus (HCV)-mixed cryoglobulinemia (MC) vasculitis includes pegylated interferon α (PegIFN)-α plus ribavirin and/or rituximab. About 30-40% of patients are non-responders or relapsers to such combination. OBJECTIVE: To analyse the safety and efficacy of Peg-IFNα/ribavirin/protease inhibitor combination in HCV-MC vasculitis. PATIENTS AND METHODS: Open-label, prospective, cohort study including 23 patients with HCV-MC vasculitis. Peg-IFNα/ribavirin was associated to telaprevir (375 mg three times daily, for 12 weeks, (n=15)) or boceprevir (800 mg three times daily, for 44 weeks, (n=8)) for 48 weeks. RESULTS: The median age was 59 (52.5-66) years, with 48.8% women. Thirteen patients (56.5%) were complete clinical responders, and 10 (43.5%) were partial responders at week 24. The virological response (ie, HCV RNA negativation) was of 69.6% at week 24 (p=0.005). The cryoglobulin level decreased from 0.44 to 0.06 g/l (p=0.0006) and the C4 level increased from 0.09 to 0.15 g/l (p=0.045). Grades 3 and 4 adverse events (mainly anaemia, neutropenia and thrombocytopenia) were observed in 10 cases (43.5%). Twenty patients (87%) received erythropoietin, 9 (39.1%) had red cell transfusion, and 2 (8.7%) had granulocyte stimulating agents. Antiviral therapy discontinuation was required in 8 (34.7%) patients for virological non-response (n=5), virological relapse (n=2) and depression (n=1). CONCLUSIONS: Peg-IFNα/ribavirin/protease inhibitor combination seems highly effective in HCV-MC. Such therapeutic regimen should be administered cautiously considering the high rate of side effects.

Preexposure prophylaxis (PrEP) of HIV infection in France: a nationwide cross-sectional study (PREVIC study).

Although preliminary studies showed that preexposure prophylaxis (PrEP) lowers the HIV transmission in individuals with HIV, confirmative trials are ongoing and PrEP is not routinely recommended. The aim of this study was to assess whether individuals with HIV share antiretroviral (ARV) drugs for PrEP and to describe awareness and discussion on PrEP in this population. A cross-sectional survey was conducted in France in 23 representative departments of infectious diseases and internal medicine. Physicians administered an anonymous standardized questionnaire to all individuals with HIV receiving ARVs and followed between 24 and 31 October 2011. The questionnaire included items regarding PrEP (awareness; discussion with their close circle, physician or patients' association; experience), personal sociodemographic characteristics, risk behaviors and HIV status of the participants. Five hundred and ninety three participants were recruited: male 74.2% (men who have sex with men 52.4%, heterosexuals 21.6%), member of patient's association 9.8%. Half of them (50.6%) lived with a stable partner and 35.2% with an HIV-negative partner. Almost half (41.8%) were aware and 29.5% had had discussion about PrEP. In logistic regression, awareness and discussion on PrEP were more frequent: (1) among males, in patients' association members (p< 0.001 for both) and in nonheterosexuals (p=0.023 and 0.057, respectively); (2) among women, in those not living with a stable partner (p=0.035 and p=0.03, respectively) or living with an HIV-negative partner (p=0.049 and p=0.083, respectively). One percent of the participants declared having shared ARVs with someone and 8.3% reported PrEP in their close circle. Men reporting PrEP in their close circle shared ARVs more frequently than those who did not (10.3% vs. 0.2%, p < 0.001). Today, individuals with HIV do not seem to widely share personal ARVs for PrEP with seronegative people. A significant number of individuals with HIV are aware of and commonly discuss PrEP.

[Implication of mitochondrial apoptosis in neural degeneration of cochlea in a murine model for presbycusis]. / Implication de l'apoptose mitochondriale dans la dégénérescence neurale de la cochlée dans un modèle murin de presbyacousie.

HYPOTHESIS: Pathologies of senescence, in particular those of neurosensory organs represent an important health problem. The improvement of the life expectation entails the fast increase of the frequency of the presbyacusis in the population. The biological and molecular causes of this degenerative pathology of the inner ear are linked to the disappearance of the sensory cells (inner and outer hair cells) and are associated to nervous damages of the spiral ganglion in the cochlea. We were interested in mechanisms causing the cochlear degeneration in a model of mouse CD 1 presenting prematurely auditive losses. MATERIALS AND METHODS: We tried to correlate the evolution of the hearing and the appearance of apoptotic phenomena by marking with specific antibody, activated anti-caspase-3, in the cochlea during time. We studied the role and the involvement of proteins controlling the apoptosis as the P53 protein and from an energy point of view at the level of the mitochondria such as proteins of the Bcl-2 family and the cytochrome c in the various structures of the cochlea. RESULTS: After implantation of electrodes for auditory nerve acoustic thresholds measurements, the audition of mice CD 1 presented a characteristic profile of hearing losses which begins in the high frequencies from the age of 1 month and which quickly evolves towards the low frequencies. The observation (between the 1st and 3rd month of age) of spiral ganglion cells revealed an unchanged number of cellular bodies of type 1 neurons, on the other hand a characteristic morphology of apoptosis of glial cells with the formation of apoptotic body was noted. Indeed, glial cells expressed activated caspase-3. Furthermore, this phenomenon seems to be under the control of the pro-apoptotic protein Bax by its overexpression and a increased release of the cytochrome c. This phenomenon was followed at 3 and 6 months by the disappearance of the outer hair cells by 9 and 48% respectively. CONCLUSION: The apparition of the deafness in the murin model CD 1 allowed us to demonstrate that the degeneration of cochlear structure begins at the level of glial cells of the spiral ganglion from 3 months, followed thereafter by the deterioration of the nervous conduction between the spiral ganglion and the sensory cells. As a consequence, because of the impoverishment in nervous signals, the outer hair cells would begin to disappear during the 6th month. In conclusion, the understanding of the sequence and the cause of these mechanisms responsible for the neural degeneration and the loss of hearing could eventually, allow us to optimize the various treatments of the presbyacusis.

[Diagnostic value of needle biopsy and frozen section histological examination in the surgery of primary parotid tumors]. / Valeur diagnostique de la cytoponction et de l'examen histologique extemporané dans les tumeurs parotidiennes primitives opérées.

The necessity of fine-needle aspiration biopsy (FNAB) in the diagnosis and treatment of parotid gland lesions is still controversial. We examined the accuracy of cytology and histology in a review of 128 parotid gland tumors who underwent surgery with FNAB, n = 102 and/or frozen section examination (FS), n = 94. The diagnostic sensibility and specificity for malignant or benign lesions was respectively 81.5% and 97.5% for FNAB and 75% and 100% for FS as compared with definite histology (110 tumors were benign and 18 malignant). Insufficient material for FNAB evaluation was found in 12 patients mainly with small tumors (p = 0.043) or with tumors located in the deep process of the parotid gland (p = 0.029). Surgery was inappropriate (superficial lobe resection for malignant tumor) because of 4 false negative FS diagnoses. FNAB offers valuable information in the diagnosis of nonsurgical lesions and permits to avoid FS if FNAB identify a benign lesion. FS remains mandatory if FNAB evaluation is not possible or suggests a neoplastic tumor.

Two dimerization domains in the trans-activation response RNA-binding protein (TRBP) individually reverse the protein kinase R inhibition of HIV-1 long terminal repeat expression.

Trans-activation response (TAR) RNA-binding protein (TRBP) is a cellular protein that binds to the human immunodeficiency virus-1 (HIV-1) TAR element RNA. It has two double-stranded RNA binding domains (dsRBDs), but only one is functional for TAR binding. TRBP interacts with the interferon-induced protein kinase R (PKR) and inhibits its activity. We used the yeast two-hybrid assay to map the interaction sites between the two proteins. We show that TRBP and PKR-N (178 first amino acids of PKR) interact with PKR wild type and inhibit the PKR-induced yeast growth defect in this assay. We characterized two independent PKR-binding sites in TRBP. These sites are located in each dsRBD in TRBP, indicating that PKR-TRBP interaction does not require the RNA binding activity present only in dsRBD2. TRBP and its fragments that interact with PKR reverse the PKR-induced suppression of HIV-1 long terminal repeat expression. In addition, TRBP activates the HIV-1 long terminal repeat expression to a larger extent than the addition of each domain. These data suggest that TRBP activates gene expression in PKR-dependent and PKR-independent manners.

[A voluminous exteriorized odontogenic keratocyst]. / A propos d'un volumineux kératokyste odontogénique extériorisé.

The authors report the case of a 63 years old man who presented a voluminous exteriorized keratocyst of the jaw. We have first made a punction of the cyst in order to facilitate the operation. Anatomopathological examination of the liquid found epithelial cells. We collected the keratocyst by a non interrupting mandibulotomy via cervical incision.

A novel genital human papillomavirus (HPV), HPV type 74, found in immunosuppressed patients.

The genome of a novel human papillomavirus (HPV) type, HPV74, was cloned from an iatrogenically immunosuppressed woman with persisting low-grade vaginal intraepithelial neoplasia. HPV74 was found to be phylogenetically related to the low-risk HPV types 6, 11, 44, and 55. HPV74 or a variant of this type was found in specimens from three additional immunosuppressed women but not in about 3,000 anogenital specimens from immunocompetent patients.

Two novel genital human papillomavirus (HPV) types, HPV68 and HPV70, related to the potentially oncogenic HPV39.

The genomes of two novel human papillomavirus (HPV) types, HPV68 and HPV70, were cloned from a low-grade cervical intraepithelial neoplasia and a vulvar papilloma, respectively, and partially sequenced. Both types are related to HPV39, a potentially oncogenic virus. HPV68 and HPV70 were also detected in genital intraepithelial neoplasia from three patients and one patient, respectively. Comparison with sequence data in the literature indicates that the subgenomic ME180-HPV DNA fragment, cloned from a carcinoma cell line, corresponds to an HPV68 subtype and that several HPV DNA fragments amplified by PCR from genital neoplasia represent worldwide distributed variants of HPV68 and HPV70.

[Evaluation of a new rapid ELISA technique for detecting human cytomegalovirus antibodies (CMV). Applications in emergency blood transfusion and in organ transplantation]. / Evaluation d'une nouvelle technique rapide par Elisa de dépistage des anticorps anticytomégalovirus humain (CMV). Applications pour les transfusions sanguines en urgence et dans un contexte de transplantation d'organes.

Immunocubes can detect very low HCMV IgG levels with at least the same sensitivity as the reference technique: ELISA. Samples containing bacteria showed no particular interference on the test, and results for non-infected material were in agreement with those of ELISA. The rapid test did not give any false positive reactions even with samples from patients with very high antiherpes immunoglobulins levels. Samples from transplant patients at the beginning of seroconversion or diluted to IgG titers corresponding to ELISA cut-off values were always detected as positive by immunocubes. In view of the rapidity of the test, its high specificity, its ability to detect levels corresponding to extremely low IgG signals in ELISA, and the possibility of conserving objective proof of the test, we conclude that this rapid immunocube technique is of great interest for current serologic screening in an emergency context, especially for transfusion and organ transplantation.

[Serological confirmation of HIV infections in hospital practice: analysis of indeterminate western blot results]. / Sérologie de confirmation de l'infection par le VIH en pratique hospitalière: analyse des western blots indéterminés.

Among 770 Western blots for HIV-1 confirmation on sera from subjects at high risk for HIV infection, 4.3% (33 cases) were indeterminate. Isolated, stable, reproducible anti-gp 160 reactivity, highly suggestive of a nonspecific reaction, was found in 16% of cases. There were three other probably nonspecific patterns with anti-gp 160 and either anti-gp 41 or anti-gp 120 reactivities and thin, atypical bands. Two patterns with anti-p24 and either anti-gp 160 or anti-gp 120 reactivities were consistent with HIV-1 seroconversion. Reactivity directed solely against gag products was seen in 18% of cases. The repeat test, performed in 16 cases, showed an identical pattern in 3 cases, a modified pattern in 3 cases, negative results in 9 cases, and seroconversion in 1 case. No case of HIV-2 infection was detected. Indeterminate Western blot results reflect nonspecific reactivity in most instances but should nevertheless lead to the exclusion of technical artefacts, seroconversion, and HIV-2 infection.

[Estimated biological markers of progression in human immunodeficiency virus infection]. / Marqueurs biologiques prévisionnels d'évolution au cours de l'infection par le virus de l'immunodéficience humaine.

The biological markers for determining as early as possible the progression in the infection by the human immunodeficiency virus (HIV) are very important for the health care of patients, and to adapt their anti-retroviral treatment. Among those, four independent biological markers for predicting a pejorative evolution in the following 36 months are used in medical practice: two specific for HIV, p24 antigenemia and serum titre of antibodies to the p24 core antigen, and two non-HIV specific surrogate markers, the beta 2-microglobulinemia and the absolute number of CD4 T cell in blood. P24 antigenemia corresponds to an active retroviral in vivo replication. The cut off for detection is about 10 pg/ml. It is difficult to detect in black people, and in the asymptomatic or pauci-symptomatic stages of the disease. The apparition or the increase of the serum p24 antigen levels suggest the occurrence of opportunistic infections. P24 antigenemia decreases or disappears during the treatment by zidovudine. The diminution or the disappearance of serum antibodies directed to the p24 core protein are secondary to the deficiency of the humoral immunity, and to an increase of the viral replication, which occur at the late stage of the disease. The diminution or the disappearance of serum antibodies to p24 precede the occurrence of AIDS by several months. The increase of the serum beta 2-microglobulin level is associated with the severity of the disease. In the San Francisco prospective cohort, the progression to AIDS in 36 months was 69% when beta 2-microglobulinemia was more than 5 mg/l, 33% when it was between 3.1 to 5 mg/l, and 12% when it was less than 3 mg/l. The beta 2-microglobulin intra-thecal synthesis level could serve as a marker for the specific HIV encephalitis. The CD4 lymphocyte count constitutes an independent provisional marker for progression to AIDS, probably the most important, but mainly of statistical value. A lymphocyte count of 200 CD4/mm3 is considered as the threshold of full blown AIDS. Beside these classic biological markers, numerous other parameters have been evaluated, without knowing their practical interest. Although the predictive markers for AIDS have a real statistical significance, their interpretation could be difficult or hazardous when applied to a sole individual. In a relatively short delay, the actual biological markers will probably be completed or changed, in the routine medical practice, by the use of direct virological markers evaluating the viral load (plasmatic or cellular viremia).

Transformed NIH 3T3 cells expressing human melanoma N-ras oncogene metastasize to lymph node in nude mice.

The effect of the N-ras oncogene on the propensity of transformed cells to disseminate from the tumor and to metastasize, using NIH 3T3 cells transformed either with human melanoma DNA containing the N-ras oncogene or with the cloned N-ras from human neuroblastoma, was investigated. The results show that NIH 3T3 expressing these genes readily formed tumors after subcutaneous injection in nude mice. Spontaneous lymph node metastasis was observed after a first cycle of transfection in one animal inoculated with cells containing human melanoma N-ras oncogene, and in 95 per cent of the animals after the second and third rounds of transfection, indicating that the metastatic capacity was transferred. In all cases human N-ras oncogene was found in both the metastases and the associated tumors. No control NIH 3T3 cells formed tumors or metastases in nude mice, and NIH 3T3 cells transfected with cloned N-ras activated oncogene formed tumors in 100 per cent of injected mice, but no spontaneous metastases. Thus human activated N-ras gene may not be sufficient to confer metastatic behavior in nude mice and the metastatic ability of human melanoma DNA transfected cells may be due to, among other possibilities, expression of other gene sequences from melanoma DNA co-transfected with the N-ras oncogene, or to specific activated murine sequences switched on during the initial process of transfection.

Study of oncogenic potentialities of human melanoma: identification of N-ras oncogene after DNA transfer and tumour induction.

The oncogenic potentialities of human melanoma cells derived from two different patients were studied using DNA-mediated gene transfer into NIH 3T3 cells followed by tumor induction into athymic nude nice. 64% of the mice injected subcutaneously with selected cells which had been co-transfected with human melanoma DNA and the selective marker NeoR developed tumors within 3-4 weeks, while up to 100% of those injected with cells transfected three days before with melanoma DNA developed tumors within 4-6 weeks. Southern blots analysis of the tumors indicated that almost all of them contained human sequences. Hybridization with different oncogene probes showed the presence of an human Eco RI N-ras-hybridizing fragment in the primary and secondary derived tumors, indicating that a transforming N-ras oncogene in human melanoma had been transferred to recipient cells and that transformed cells induced tumors in nude mice.

Isolation from the spleen of myeloproliferative sarcoma virus-infected mice of a myelomonocytic tumor cell line secreting hematopoietic colony stimulating factors.

Clonogenic tumor cells were detected in the spleen of DBA/2 mice infected with the myeloproliferative sarcoma virus (MPSV). These cells could be isolated because of their ability to proliferate on the greater omentum of sublethally irradiated isogenic recipient mice. We have established a permanent suspension myelomonocytic cell line in vitro (TE8), from a tumor obtained after subcutaneous transplantation of the omental tumor masses. The TE8 cells are clonogenic in semi-solid medium containing agarose. The colonies thus obtained gave rise to myelomonocytic cell lines growing in suspension in liquid medium. One of those cloned cell lines, C1(1011) was studied in details. It is exclusively composed of initial donor cells, it is tumorigenic in vivo, clonogenic in vitro and produces MPSV. It also secretes a colony stimulating activity (CSA), and an activity termed mixed colony promoting activity (MPA), which enables pluripotent hematopoietic stem cells to proliferate and differentiate. It differentiates through the granulomacrophage cell line independently of exogenous factors other than those which may be present in the serum.

Study of the 78A1 isolate of Moloney murine sarcoma virus. I. Molecular cloning and characterization.

The 78A1 isolate of Moloney murine sarcoma virus (78A1 Mo-MuSV) was cloned from a genomic library obtained from virus producer rat cells, in the lambda vector L47. Among the recombinants hybridizing with a probe specific for the v-mos sequences, we recovered a recombinant which contained leukaemia virus (MuLV) sequences and was able to transform both mouse and rat cells in transfection experiments. The cloned provirus could be rescued by both Mo-MuLV ecotropic and amphotropic viruses in mouse cells, but only with the amphotropic helper virus in rat cells. Comparative restriction mapping indicates that the 78A1 provirus is 200 bp longer than the HT1 provirus. The difference lies in the gag-pol junction region of Mo-MuSV. Other minor differences were found in the gag region, whereas the restriction patterns of the 3' parts of the proviruses were identical.

[Characterization of a subgenomic viral RNA in the polysomes of cells infected with the Moloney murine sarcoma virus]. / Caractérisation d'un ARN sous-génomique viral dans les polysomes de cellules infectées par le Virus du Sarcome Murin de Moloney.

We have detected a 1.6 kb RNA species in the polysomes of cells infected with the m1 strain of Moloney Murine Sarcoma Virus. This RNA contains the cellular transforming sequences (mos) as well as sequences located at both ends of the viral genome and is devoid of other internal viral sequences. This RNA appears to be spliced and might function as the messenger for the transforming protein of the Moloney Murine Sarcoma Virus.

Kinetic studies on ribosomal proteins assembly in preribosomal particles and ribosomal subunits of mammalian cells.

Proteins were isolated from 80-S preribosomal particles and ribosomal subunits of murine L5178Y cells after short and longer periods of incubation with tritiated amino acids. The labeling patterns of ribosomal proteins were compared by two-dimensional polyacrylamide gel electrophoresis. The analysis of isotopic ratios in individual protein spots showed marked differences in the relative kinetics of protein appearance within nucleolar peribosomes and cytoplasmic subunits. Among the about 60 distinct proteins characterized in 80-S preribosomes, 9 ribosomal proteins appeared to incorporate radioactive amino acids more rapidly. These proteins become labeled gradually in the cytoplasmic ribosomal subunits. It was found that one non-ribosomal protein associated with 80-S preribosomes takes up label far more quickly than other preribosomal polypeptides. It is suggested that this set of proteins could associate early with newly transcribed pre-rRNA, more rapidly than others after their synthesis on polyribosomes, and could therefore play a role in the regulation of ribosome synthesis. In isolated 60-S and 40-S ribosomal subunits, we detected five proteins from the large subunit and four proteins from the small subunit which incorporate tritiated amino acids more quickly than the remainder. These proteins were shown to be absent or very faintly labeled in 80-S preribosomal particles, and would associate with ribosomal particles at later stages of the maturation process.

Studies on the distribution of ribosomal proteins in mammalian ribosomal subunits.

Murine L5178Y cell ribosomes were dissociated into subunits either with potassium chloride in the presence of puromycin or with the chelating agent EDTA. The proteins of ribosomal subunits obtained by these different methods were compared by means of bidimensional polyacrylamide gel electrophoresis. KCl-derived 60S and 40S subunits were shown to contain 38 and 31 proteins respectively, 3 of them having identical electrophoretic mobilities. Preparations of EDTA-dissociated ribosomal subparticles contained different proportions of these proteins, and 11 major spots were shared between the EDTA-derived large and small ribosomal subunits. Furthermore, 10 proteins absent from subunits treated by high concentrations of KCl were reproducibly found in EDTA-treated ribosomal subparticles.

Characterization of proteins from nucleolar preribosomes of mouse leukemia cells by two-dimensional polyacrylamide gel electrophoresis.

Proteins of isolated 80-S and 60-S nucleolar preribosomal particles were characterized by means of two-dimensional polyacrylamide gel electrophoresis, in the lymphocytic mouse leukemia cells L5178Y. Their identification and metabolic relationships with ribosomal subunit proteins were investigated using co-electrophoresis of unlabeled polysomal proteins with labeled proteins of either nucleolar preribosomes or ribosomal subunits. The large and small ribosomal subunits contain 40 and 31 proteins, respectively. The nucleolar 80-S preribosomes were analysed after 2 and 5 h of incubation with tritiated valine and leucine and were shown to contain about 55 proteins. Most of them were identical to cytoplasmic ribosomal subunit proteins. The nucleolar 60-S preribosomes contain all the proteins which are common to 80-S preribosomes and large ribosomal subunits, and one additional protein (L10). The ribosomal proteins which were absent from nucleolar particles were found to be labeled in the cytoplasmic ribosomes after the same incubation period. Thus, in addition to the association of the bulk of ribosomal proteins with 45-S RNA within the 80-S preribosomes, results indicate that a group of ribosomal proteins and particularly from the small subunits, become associated at later stages of the maturation process of mammalian ribosomes. It was further shown that a set of 10 proteins, different from ribosomal polypeptides, were present in nucleolar preribosomal particles. Several of them were associated with polyribosomes in the cytoplasm, whereas the others were unique to the nucleolus.