Cellular activation and intracellular HCV load in peripheral blood monocytes isolated from HCV monoinfected and HIV-HCV coinfected patients.

BACKGROUND: During HCV infection, the activation status of peripheral blood monocytes and its impact on HCV replication are poorly understood. We hypothesized that a modified activation of peripheral blood monocytes in HIV-HCV coinfected compared to HCV monoinfected patients may contribute to different monocytes reservoirs of HCV replication. METHODS: We performed a case-control analysis involving HCV-infected patients with and without HIV coinfection. In peripheral blood mononuclear cells (PBMCs), peripheral blood lymphocytes (PBLs) and peripheral blood monocytes isolated from HCV monoinfected and HIV-HCV coinfected patients, intracellular HCV load and a marker of cellular activation, nuclear factor-kappaB (NF-κB) activation, were quantified using intracellular detection of HCV-core protein and electrophoretic mobility shift assay, respectively. RESULTS: Intracellular HCV loads were higher in monocytes isolated from HIV-HCV coinfected patients than in those of monoinfected patients. Among PBMCs isolated from HIV-HCV coinfected patients, intracellular HCV loads were higher in monocytes compared to PBLs. Cellular activation as measured by NF-κB activation was higher in monocytes isolated from HIV-HCV coinfected patients than in those of monoinfected patients. CONCLUSIONS: Our results reveal the peripheral blood monocytes as an important extrahepatic reservoir for HCV in HIV-HCV coinfected patients and indicate a potential association between the activation state of monocytes and the size of the HCV reservoir in HIV-HCV coinfected patients.

Human papillomavirus 16 oncoprotein E7 stimulates UBF1-mediated rDNA gene transcription, inhibiting a p53-independent activity of p14ARF.

High-risk human papillomavirus oncoproteins E6 and E7 play a major role in HPV-related cancers. One of the main functions of E7 is the degradation of pRb, while E6 promotes the degradation of p53, inactivating the p14ARF-p53 pathway. pRb and p14ARF can repress ribosomal DNA (rDNA) transcription in part by targeting the Upstream Binding Factor 1 (UBF1), a key factor in the activation of RNA polymerase I machinery. We showed, through ectopic expression and siRNA silencing of p14ARF and/or E7, that E7 stimulates UBF1-mediated rDNA gene transcription, partly because of increased levels of phosphorylated UBF1, preventing the inhibitory function of p14ARF. Unexpectedly, activation of rDNA gene transcription was higher in cells co-expressing p14ARF and E7, compared to cells expressing E7 alone. We did not find a difference in P-UBF1 levels that could explain this data. However, p14ARF expression induced E7 to accumulate into the nucleolus, where rDNA transcription takes place, providing an opportunity for E7 to interact with nucleolar proteins involved in this process. GST-pull down and co-immunoprecipitation assays showed interactions between p14ARF, UBF1 and E7, although p14ARF and E7 are not able to directly interact. Co-expression of a pRb-binding-deficient mutant (E7C24G) and p14ARF resulted in EC24G nucleolar accumulation, but not in a significant higher activation of rDNA transcription, suggesting that the inactivation of pRb is involved in this phenomenon. Thus, p14ARF fails to prevent E7-mediated UBF1 phosphorylation, but could facilitate nucleolar pRb inactivation by targeting E7 to the nucleolus. While others have reported that p19ARF, the mouse homologue of p14ARF, inhibits some functions of E7, we showed that E7 inhibits a p53-independent function of p14ARF. These results point to a mutually functional interaction between p14ARF and E7 that might partly explain why the sustained p14ARF expression observed in most cervical pre-malignant lesions and malignancies may be ineffective.

HIV-1 Nef interacts with HCV Core, recruits TRAF2, TRAF5 and TRAF6, and stimulates HIV-1 replication in macrophages.

Tumor necrosis factor receptor-associated factor (TRAF) signaling plays a central role in many biological activities, such as the regulation of immune and inflammatory responses and control of apoptosis, which are key events in the pathogenesis of the human immunodeficiency virus (HIV)-1 and the hepatitis C virus (HCV) infections. Here we show that TRAF2, TRAF5 and TRAF6 interact with the HIV-1 Nef protein, an immunomodulatory viral protein expressed and released by cells infected by the virus. We also found that TRAF2 and TRAF5 interact with the HCV Core protein. Interestingly, we observed that HIV-1 Nef interacts with HCV Core. The activation of TRAF (2, 5, 6) - mediated by HIV-1 Nef and HCV Core - enhanced the activation of the nuclear factor-kappa B (NF-κB) and increased HIV-1 replication in monocyte- derived macrophages (MDMs). The knockdown of TRAF2, TRAF5 and TRAF6 resulted in decreased NF-κB activation and reduced HIV-1 replication in MDMs. Our results reveal a mechanism by which the activation of the TRAF pathway by HIV-1 Nef and HCV Core favors the replication of HIV-1 in macrophages and could be a critical factor for optimal replication of HIV-1 in macrophages of HIV-HCV-coinfected patients.

The HIV-1 Nef protein interacts with two components of the 40S small ribosomal subunit, the RPS10 protein and the 18S rRNA.

BACKGROUND: Human immunodeficiency virus type 1 (HIV-1) Nef-encoded protein plays key functions at almost all stages of the viral life cycle, but its role in translation is largely unknown. METHODS: To determine the effect of Nef on translation we used an in vitro translation assay. The detection of Nef/RPS10 complexes and the presence of 18S rRNA and tRNAs in the complexes were performed by coimmunoprecipitation and RT-PCR assay. RESULTS: We observed that the HIV-1 Nef protein specifically impaired translation in vitro. We observed the interaction of Nef with RPS10 by coimmunoprecipitation assay. In addition 18S rRNA and tRNAs were present in the Nef/RPS10 complexes. CONCLUSIONS: Our results are consistent with a model in which the Nef protein by binding to two components of the 40S small ribosomal subunit, RPS10 and 18S rRNA, and to a lesser extent to tRNAs, could lead to decreased protein synthesis.

Immortalized sebocytes can spontaneously differentiate into a sebaceous-like phenotype when cultured as a 3D epithelium.

Sebocytes originate from the same lineage as keratinocytes, and both cell types may have similarities in terms of growth and differentiation. We were interested in studying the behaviour of human sebocytes when cultured in conditions validated for epidermal reconstruction. For this purpose, we established a HPV16-E6/7-immortalized human sebocyte cell line (SEBO662) growing in keratinocyte defined media. Postconfluent SEBO662 cells in monolayers express the early sebocyte marker, cytokeratin 7 (K7), do not express Epithelia Membrane Antigen (EMA) and do not exhibit strong lipogenic activity. However, when placed at the air-liquid interface, SEBO662 multilayers spontaneously differentiate into a sebaceous-like structure as shown by the strong polarized expression of the late sebaceous marker EMA, the overexpression of some lipogenic markers and lipid production on the upper side of the epithelium. This work highlights the value of simple 3D models for exhibiting spontaneous differentiation and polarization.

Infantile hypertrophic pyloric stenosis: are viruses involved?

Infantile hypertrophic pyloric stenosis (IHPS) is characterized by abnormal thickening of the internal circular muscle layer. IHPS is known to be due to a combination of genetic and environmental factors, but its precise causes and pathophysiology are poorly understood. The objective of the study is to determine the prevalence of the principal viruses targeting the respiratory and digestive tracts in children with IHPS. Nasopharyngeal fluids, stools, vomit, and surgical pyloric muscle fragments and swabs were tested by cell culture, viral antigen assay and PCR. IHPS was diagnosed in 23 boys and 8 girls with a mean (± SD) age of 42 ± 15 days (range 20-88 days). There was no seasonal pattern of diagnosis. Twenty-two children (71%) lost weight (mean 246 ± 164 g, range 30-600 g) after the onset of vomiting, and five (16.1%) were dehydrated. Seven (22.6%) infants had been exposed to an infectious contact within 15 days before admission, and one on the day of admission (3.2%). Ear, nose and throat samples and pyloric muscle specimens were negative for all the viruses tested. An adenovirus type 3 was recovered from one stool sample, and RT-PCR was positive for an enterovirus on one vomit sample. This study suggests that the principal viruses targeting the respiratory and digestive tracts are not responsible for IHPS.

NF-kappaB-dependent control of HIV-1 transcription by the second coding exon of Tat in T cells.

HIV-1 two-exon transactivator protein (Tat) is a 101-aa protein. We investigated the possible contribution of the extreme C terminus of HIV-1 Tat to maximize nuclear transcription factor NF-kappaB activation, long terminal repeat (LTR) transactivation, and viral replication in T cells. C-terminal deletion and substitution mutants made with the infectious clone HIV-89.6 were assayed for their ability to transactivate NF-kappaB-secreted alkaline phosphatase and HIV-1 LTR-luciferase reporter constructs for low concentrations of Tat. A mutant infectious clone of HIV-89.6 engineered by introducing a stop codon at aa 72 in the Tat open-reading frame (HIVDeltatatexon2) replicated at a significantly lower rate than the wild-type HIV-89.6 in phytohemagglutinin-A/IL-2-stimulated primary peripheral blood lymphocytes. Altogether, our results suggest a critical role for the glutamic acids at positions 92, 94, and 96 or lysines at positions 88, 89, and 90, present in the second encoding Tat exon in activating NF-kappaB, transactivating the HIV-1 LTR and enhancing HIV-1 replication in T cells.

Increased nuclear factor-kappaB activation in peripheral blood monocytes of patients with rheumatoid arthritis is mediated primarily by tumor necrosis factor-alpha.

OBJECTIVE: Rheumatoid arthritis (RA) is a disease characterized by prolonged production of tumor necrosis factor-alpha (TNF-alpha), which is regulated by the Rel/nuclear factor-kappaB (NF-kappaB) transcription factors. We assessed NF-kappaB activation in peripheral blood mononuclear cells (PBMC), peripheral blood lymphocytes (PBL), and monocytes from patients with RA, patients with ankylosing spondylitis (AS), and healthy subjects. METHODS: NF-kappaB activation was determined by electrophoretic mobility shift assays and by Western blotting in PBMC, monocytes, and PBL isolated from peripheral blood of patients with RA, patients with AS, and healthy subjects and determined after ex vivo pretreatment of PBMC, PBL, and monocytes of patients with RA and healthy subjects with infliximab and with etanercept. RESULTS: Enhanced NF-kappaB activation was observed in monocytes, PBL, and PBMC isolated from patients with RA, but not in PBMC, PBL, and monocytes of patients with AS and healthy subjects. The NF-kappaB complex was composed of p50 and p65 subunits and its activation required inhibitor of NF-kappaBalpha degradation. We observed a positive correlation between the NF-kappaB activation in monocytes, PBL, and PBMC, and TNF-alpha levels in peripheral blood of patients with RA. Ex vivo treatment with infliximab and etanercept decreased NF-kappaB activation in monocytes of patients with RA, but not in PBL and PBMC, and not in healthy subjects. CONCLUSION: Our results indicate a role for NF-kappaB activation and TNF-alpha in the activation of monocytes of patients with RA, and suggest an important role of circulating monocytes in RA pathogenesis.

HIV and inflammation.

A main feature of HIV infection is the expression of several proinflammatory cytokines. Proinflammatory cytokines expressed as soluble factors or membrane-bound molecules regulate both HIV replication and T cell apoptosis. Proinflammatory cytokines have key roles in the HIV lifecycle, especially at the level of transcription, favouring the ability of HIV to establish latent reservoirs. In addition, proinflammatory cytokines are involved in both CD4+ T cell and CD8+ T cell apoptosis, resulting in immune suppression. Moreover, several HIV proteins such as Nef, Tat, and Vpr hijack proinflammatory cytokine signaling, further underlining the potential importance of inflammation in HIV pathogenesis. In vivo chronic inflammatory conditions have been correlated to increased levels of viremia and accelerated disease progression. This article raises the possibility that inflammation plays a crucial role in both immune suppression and the formation of viral reservoirs during HIV infection. Understanding the role of inflammation in HIV infection could lead to new therapeutic strategies that could ultimately enhance immune restoration and limit the formation of viral reservoirs in HIV-infected patients.