Loss of IL-17-producing CD8 T cells during late chronic stage of pathogenic simian immunodeficiency virus infection

Progressive disease caused by pathogenic SIV/HIV infections is marked by systemic hyperimmune activation, immune dysregulation, and profound depletion of CD4(+) T cells in lymphoid and gastrointestinal mucosal tissues. IL-17 is important for protective immunity against extracellular bacterial infections at mucosa and for maintenance of mucosal barrier. Although IL-17-secreting CD4 (Th17) and CD8 (Tc17) T cells have been reported, very little is known about the latter subset for any infectious disease. In this study, we characterized the anatomical distribution, phenotype, and functional quality of Tc17 and Th17 cells in healthy (SIV-) and SIV+ rhesus macaques. In healthy macaques, Tc17 and Th17 cells were present in all lymphoid and gastrointestinal tissues studied with predominance in small intestine. About 50% of these cells coexpressed TNF-α and IL-2. Notably, ～50% of Tc17 cells also expressed the co-inhibitory molecule CTLA-4, and only a minority (<20%) expressed granzyme B suggesting that these cells possess more of a regulatory than cytotoxic phenotype. After SIV infection, unlike Th17 cells, Tc17 cells were not depleted during the acute phase of infection. However, the frequency of Tc17 cells in SIV-infected macaques with AIDS was lower compared with that in healthy macaques demonstrating the loss of these cells during end-stage disease. Antiretroviral therapy partially restored the frequency of Tc17 and Th17 cells in the colorectal mucosa. Depletion of Tc17 cells was not observed in colorectal mucosa of chronically infected SIV+ sooty mangabeys. In conclusion, our results suggest a role for Tc17 cells in regulating disease progression during pathogenic SIV infection.     

Appraising the roles of CBLL1 and the ubiquitin/proteasome system for flavivirus entry and replication

The ubiquitin ligase CBLL1 (also known as HAKAI) has been proposed to be a critical cellular factor exploited by West Nile virus (WNV) for productive infection. CBLL1 has emerged as a major hit in a recent RNA interference screen designed to identify cellular factors required for the early stages of the WNV life cycle. Follow-up experiments showed that HeLa cells knocked down for CBLL1 by a small interfering RNA (siRNA) failed to internalize WNV particles and resisted infection. Furthermore, depletion of a free-ubiquitin pool by the proteasome inhibitor MG132 abolished WNV endocytosis, suggesting that CBLL1 acts in concert with the ubiquitin proteasome system to mediate virus internalization. Here, we examined the effect of CBLL1 knockdown and proteasome inhibitors on infection by WNV and other flaviviruses. We identified new siRNAs that repress the CBLL1 protein and strongly inhibit the endocytosis of Listeria monocytogenes, a bacterial pathogen known to require CBLL1 to invade host cells. Strikingly, however, we detected efficient WNV, dengue virus, and yellow fever virus infection of human cells, despite potent downregulation of CBLL1 by RNA interference. In addition, we found that the proteasome inhibitors MG132 and lactacystin did not affect WNV internalization but strongly repressed flavivirus RNA translation and replication. Together, these data do not support a requirement for CBLL1 during flavivirus entry and rather suggest an essential role of the ubiquitin/proteasome pathway for flavivirus genome amplification.     

Spindle checkpoint component Mad2 contributes to biorientation of homologous chromosomes

Cell cycle checkpoints sense defects in chromosome metabolism, halt the cell cycle, and activate pathways that repair the defects. The spindle checkpoint arrests the cell cycle in response to defects in the interaction between microtubules and kinetochores (the proteinaceous complex assembled on centromeric DNA), but no repair function has been demonstrated for this checkpoint. We show that the roles of two spindle checkpoint components, Mad2 and Mad3, differ in meiosis I. In the absence of Mad2, meiosis I nondisjunction occurs at a high frequency and can be corrected by delaying the onset of anaphase. The absence of Mad3 does not induce nondisjunction, even though mad3Delta cells cannot arrest the cell cycle in response to kinetochores that lack either microtubules or tension on the linkage between chromosomes and microtubules. The two proteins have different roles in chromosome alignment. Compared to wild type and mad3Delta cells, mad2Delta mutants are slower to attach homologous chromosomes to opposite poles of the spindle. This observation suggests that Mad2 plays a role in reorienting chromosomes that are incorrectly attached to the spindle as well as delaying the cell cycle, whereas Mad3 is needed for the cell cycle delay, but not for chromosome reorientation.     

G protein-dependent CCR5 signaling is not required for efficient infection of primary T lymphocytes and macrophages by R5 human immunodeficiency virus type 1 isolates

The requirement of human immunodeficiency virus (HIV)-induced CCR5 activation for infection by R5 HIV type 1 (HIV-1) strains remains controversial. Ectopic CCR5 expression in CD4(+)-transformed cells or pharmacological inhibition of G(alpha)i proteins coupled to CCR5 left unsolved whether CCR5-dependent cell activation is necessary for the HIV life cycle. In this study, we investigated the role played by HIV-induced CCR5-dependent cell signaling during infection of primary CD4-expressing leukocytes. Using lentiviral vectors, we restored CCR5 expression in T lymphocytes and macrophages from individuals carrying the homozygous 32-bp deletion of the CCR5 gene (ccr5 Delta32/Delta32). Expression of wild-type (wt) CCR5 in ccr5 Delta32/Delta32 cells permitted infection by R5 HIV isolates. We assessed the capacity of a CCR5 derivative carrying a mutated DRY motif (CCR5-R126N) in the second intracellular loop to work as an HIV-1 coreceptor. The R126N mutation is known to disable G protein coupling and agonist-induced signal transduction through CCR5 and other G protein-coupled receptors. Despite its inability to promote either intracellular calcium mobilization or cell chemotaxis, the inactive CCR5-R126N mutant provided full coreceptor function to several R5 HIV-1 isolates in primary cells as efficiently as wt CCR5. We conclude that in a primary, CCR5-reconstituted CD4(+) cell environment, G protein signaling is dispensable for R5 HIV-1 isolates to actively infect primary CD4(+) T lymphocytes or macrophages.     

Defining a novel cis element in the 3'-untranslated region of mammalian ribonucleotide reductase component R2 mRNA: role in transforming growth factor-beta 1 induced mRNA stabilization.

Ribonucleotide reductase R2 gene expression is elevated in BALB/c 3T3 fibroblasts treated with transforming growth factor beta 1. We investigated the possibility that the 3'-UTR of ribonucleotide reductase R2 mRNA contains regulatory information for TGF-beta 1 induced message stability. Using end-labeled RNA fragments in gel shift assays and UV cross-linking analyses, we detected in the 3'-UTR a novel 9 nucleotide (nt) cis element, 5'-GAGUUUGAG-3' site, which interacted specifically with a cytosolic protease sensitive factor to form a 75 kDa complex. The cis element protein binding activity was inducible and markedly up-regulated cross-link 4 h after TGF-beta 1 treatment of mouse BALB/c 3T3 cells. Other 3'-UTRs [IRE, GM-CSF, c-myc and homopolymer (U)] were poor competitors to the cis element with regard to forming the TGF-beta 1 dependent RNA-protein complex. However, the cis element effectively competed out the formation of the R2 3'-UTR protein complex. Cytosolic extracts from a variety of mammalian cell lines (monkey Cos7, several mouse fibrosarcomas and human HeLa S3) demonstrated similar TGF-beta 1 dependent RNA-protein band shifts as cell extract from BALB/c 3T3 mouse fibroblasts. Binding was completely prevented by several different mutations within the cis element, and by substitution mutagenesis, we were able to predict the consensus sequences, 5'-GAGUUUNNN-3' and 5'-NNNUUUGAG-3' for optimal protein binding. These results support a model in which the 9 nt region functions in cis to destabilize R2 mRNA in cells; and upon activation, a TGF-beta 1 responsive protein is induced and interacts with the 9 nt cis element in a mechanism that leads to stabilization of the mRNA. This appears to be the first example of a mRNA binding site that is involved in TGF-beta 1-mediated effects.     

Tolerance ofRhizobium phaseoli to acidity,aluminium and manganese

Summary Strains ofRhizobium phaseoli were evaluated with respect to their ability to grow or survis when subjected to various stresses common to soils of the tropics. These stresses included low pH, high Al and Mn. In liquid culture 10 strains ofR. phaseoli all grew well at pH 5.0, a few strains grew at pH 4.5 and one at pH 4.0. Some strains grew at pH 4.0 after repeated transfer to medium at this pH, but this characteristic remained stable for only one strain, S-442, which combined a tolerance to low pH with an improved tolerance to Al and Mn compared to the parent strain P-442. Survival studies of S-442 and P-442 in three acidic Brazilian soils, at their natural pH (4.2–4.6) and when limed to near neutral pH, showed little difference in numbers after a 21–30 day period. Only a one log cycle decrease in numbers of P-442 occurred in the Erechim soil that had a 185 mol/l Al concentration in the soil saturation extract. Strains ofR. phaseoli were screened for their ability to grow in liquid culture at pH 5.0 in the presence of Al up to 100 mol/l (16 strains) and Mn up to 320 g/ml (13 strains). Strains differed in relative tolerance to both Al and Mn with some strains being capable of excellent growth at the highest concentrations of Al and Mn employed. With the exception ofR. phaseoli (C-12) the ability to tolerate high levels of Mn did not show any relationship to Al tolerance. It was concluded that soil stress factors need not have a serious impact on survival ofR. phaseoli in soils because sufficient variability in tolerance to these factors occurs naturally among strains.

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Resumen Se evaluaron cepas deRhizobium phaseoli en relación con su habilidad para crecer o sobrevivir bajo las condiciones de stress que son habituales de suelos tropicales. Estas condiciones comprenden bajos pH y altas concentraciones de Al y Mn. Diez cepas deR. phaseoli crecieron bien, en cultivo líquido, a pH: 5; tan solo algunas lo hicieron a pH: 4.5 y únicamente una cepa creció a pH: 4.0. Aunque varias cepas consiguieron crecer a pH: 4.0, tras transferencias sucesivas a un medio con dicho pH, esta característica solo logró establizarse en la cepa S-442 que combinó esta tolerancia a la acidez con un incremento en la tolerancia a Al y Mn comparada con la cepa parental P-442. Se realizaron estudios sobre la supervivencia de las cepas S-442 y P-442 en tres suelose ácidos del Brasil, en estado natural (pH: 4.2–4.6) y neutralizados con cal. Se observaron pocas diferencias cuantitativas al cabo de un periodo de 21–30 dias un solo ciclo Log de P-442 mostró una disminución numérica en un suelo denominado Erechim que contenía 185 mol/l de Al medido en extracto de pasta saturada. Se estudió la capacidad de cepas deR. phaseoli para crecer en cultivo liquido a pH: 5.0 en presencia de hasta 100 mol/l de Al (16 cepas) y 320 g/ml de Mn (13cepas), algunas de estas cepas fueron capaces de crecer a las máximas concentraciones de Al y Mn utilizadas. ExceptuandoR. phaseoli (C-12) la tolerancia a niveles altos de Mn no estaba correlacionada con la tolerancia al Al. Se concluyó que estos factores edáficos de stress no deberían de tener efecto alguno en la supervivencia deR. phaseoli en suelo, ya que existe una variabilidad natural de tolerancias a dichos factores suficiente para garantizar esta supervivencia.

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Résumé Des souches deRhizobium phaseoli ont été téstées pour leur aptitude à se développer et à survivre lorsqu'elles soumises à diverses contraintes habituelles dans les sols tropicaux, c'est à dire des pH acides et à de fortes teneurs en Al et Mn. En culture liquide, 10 souches deR. phaseoli ont toutes poussé à pH 5,0, un petit nombre à pH 4,5 et une seule à pH 4,0. Certaines souches parviennent, après repiquages répétés, à se developper à pH 4,0; mais ce caractère ne s'est stabilisé que dans le cas d'une seule souche (S-442), qui présente à la fois une résistance à l'acidité et une tolérance accrue à Al et Mn par rapport à la souche originelle P-442. L'étude de la survie de S-442 et P-442 dans trois sols brésiliens acides, à leur pH naturel (4,2–4,6) et après chaulage à pH neutre, n'a montré au bout de 21 à 30 jours que de faibles différences numériques. Dans le sol d'Erechim, où la concentration en Al est de 185 mol/l, on n'a constaté dans l'extrait de sol à saturation qu'une diminution de la population d'un seul facteur logarithmique. Des souches deR. phaseoli ont été téstées pour leur aptitude à pousser en milieu liquide à pH 5,0 et en présence de concentrations allant jusqu'à 100 mol/l d'Al (16 souches) et 320 g/l de Mn (13 souches). Les souches différent en ce qui concerne leur résistance à la fois à Al et à Mn, certaines étant capables de se développer très bien aux concentrations en Al et Mn les plus élevées. A l'exception deR. phaseoli C-12, la tolérance aux fortes concentrations en Mn ne présente aucune corrélation avec la résistance à Al. En conclusion, les facteurs d'agression n'ont pas d'effet important sur la survie deR. phaseoli dans le sol, étant donné que le tolérance des souches à ces facteurs peut varier de façon suffisante.

     

Environmental Control on Fish and Macrocrustacean Spring Community-Structure,on an Intertidal Sandy Beach

The inter-annual variability of the fish and macrocrustacean spring community on an intertidal sandy beach near the Canche estuary (North of France) was studied from 2000 to 2013 based on weekly spring sampling over an 11-year period. Twenty-eight species representing 21 families were collected during the course of the study. The community was dominated by a few abundant species accounting for > 99% of the total species densities. Most individuals caught were young-of-the-year indicating the importance of this ecosystem for juvenile fishes and macrocrustaceans. Although standard qualitative community ecology metrics (species composition, richness, diversity, evenness and similarity) indicated notable stability over the study period, community structure showed a clear change since 2009. Densities of P. platessa, P. microps and A. tobianus decreased significantly since 2009, whereas over the period 2010-2013, the contribution of S. sprattus to total species density increased 4-fold. Co-inertia and generalised linear model analyses identified winter NAO index, water temperature, salinity and suspended particular matter as the major environmental factors explaining these changes. Although the recurrent and dense spring blooms of the Prymnesiophyte Phaeocystis globosa is one of the main potential threats in shallow waters of the eastern English Channel, no negative impact of its temporal change was detected on the fish and macrocrustacean spring community structure.