Both the Fas ligand and inducible nitric oxide synthase are needed for control of parasite replication within lesions in mice infected with Leishmania major whereas the contribution of tumor necrosis factor is minimal.

Following infection with the protozoan parasite Leishmania major, C57BL/6 mice develop a small lesion that heals spontaneously. Resistance to infection is associated with the development of CD4(+) Th1 cells producing gamma interferon (IFN-gamma) and tumor necrosis factor (TNF), which synergize in activating macrophages to their microbicidal state. We show here that C57BL/6 mice lacking both TNF and Fas ligand (FasL) (gld TNF(-/-) mice) infected with L. major neither resolved their lesions nor controlled Leishmania replication despite the development of a strong Th1 response. Comparable inducible nitric oxide synthase (iNOS) activities were detected in lesions of TNF(-/-), gld TNF(-/-), and gld mice, but only gld and gld TNF(-/-) mice failed to control parasite replication. Parasite numbers were high in gld mice and even more elevated in gld TNF(-/-) mice, suggesting that, in addition to iNOS, the Fas/FasL pathway is required for successful control of parasite replication and that TNF contributes only a small part to this process. Furthermore, FasL was shown to synergize with IFN-gamma for the induction of leishmanicidal activity within macrophages infected with L. major in vitro. Interestingly, TNF(-/-) mice maintained large lesion size throughout infection, despite being able to largely control parasite numbers. Thus, IFN-gamma, FasL, and iNOS appear to be essential for the complete control of parasite replication, while the contribution of TNF is more important in controlling inflammation at the site of parasite inoculation.

Inflammation And Autoimmune Responses Are Independent Of Peripheral Mhc Class Ii Expression Driven By Ciita Piv In Collagen Induced Arthritis

Background In rheumatoid arthritis (RA), non-professional antigen presenting cells (APCs) such as fi broblast-like synoviocytes (FLS) can express MHC class II (MHCII) molecules and function as non-professional APCs in vitro.Objective To examine the regulation of MHCII expression in FLS and to investigate the role of FLS as non-professional APCs in collagen-induced arthritis (CIA). Methods Expression of MHCII, CIITA and Ciita isoforms pI, pIII and pIV was examined by RT-qPCR, immunohistochemistry and fl ow cytometry in human synovial tissues, arthritic mouse joints and human as well as mouse FLS. CIA was induced in mice knockout for the isoform IV of Ciita (pIV-/-), in pIV-/- mice transgenic for CIITA in the thymus (pIV-/- K14 CIITA) and in control littermates in the DBA/1 background by immunising with bovine collagen type II (CII) in complete Freund's adjuvant.Results HLA-DRA, total CIITA and CIITA pIII mRNA levels were signifi cantly increased in the synovial tissues from RA compared to osteoarthritis patients. Human FLS expressed surface MHCII via CIITA pIII and pIV, while MHCII expression in murine FLS was entirely mediated by pIV. pIV-/- mice lacked both inducible MHCII expression on non-professional APCs including FLS, and in the thymic cortex. The thymic defect in pIV-/- mice impaired CD4+ positive selection, thus protecting pIV-/- mice from CIA by preventing CD4+ T cells immune responses against CII and blocking the release of IFN-γ and IL-17 in ex vivo stimulated lymph node cells. The production of T dependent, arthritogenic anti-CII antibodies was also impaired in pIV-/- mice. A normal thymic expression of MHCII and CD4+ T cell repertoire was obtained in pIV-/- K14 CIITA Tg mice. Immune responses against CII were restored in pIV-/- K14 CIITA Tg mice, as well as the arthritis incidence and clinical severity despite the lack of MHCII expression by mouse FLS. At histology, infl ammation andneutrophils infi ltration scores were not reduced in pIV-/- K14 CIITA Tg mice, while the bone erosion score was signifi cantly lower than in controls.Conclusion Over expression of MHCII is tightly correlated with CIITA pIII in the arthritic human synovium. MHCII is induced via CIITA pIII and pIV in human FLS. In the mouse, MHCII expression in the thymic cortex and in FLS is strictly dependent upon Ciita pIV. The lack of Ciita pIV in the periphery of pIV-/- K14 CIITA Tg mice lowered the bone erosion score but did not signifi cantly protect from infl ammation and autoimmune responses in CIA.

Comparative gene finding in chicken indicates that we are closing in on the set of multi-exonic widely expressed human genes.

The recent availability of the chicken genome sequence poses the question of whether there are human protein-coding genes conserved in chicken that are currently not included in the human gene catalog. Here, we show, using comparative gene finding followed by experimental verification of exon pairs by RT-PCR, that the addition to the multi-exonic subset of this catalog could be as little as 0.2%, suggesting that we may be closing in on the human gene set. Our protocol, however, has two shortcomings: (i) the bioinformatic screening of the predicted genes, applied to filter out false positives, cannot handle intronless genes; and (ii) the experimental verification could fail to identify expression at a specific developmental time. This highlights the importance of developing methods that could provide a reliable estimate of the number of these two types of genes.

Ethnic differences in proximal and distal tubular sodium reabsorption are heritable in black and white populations.

BACKGROUND: Segmental handling of sodium along the proximal and distal nephron might be heritable and different between black and white participants. METHODS: We randomly recruited 95 nuclear families of black South African ancestry and 103 nuclear families of white Belgian ancestry. We measured the (FENa) and estimated the fractional renal sodium reabsorption in the proximal (RNaprox) and distal (RNadist) tubules from the clearances of endogenous lithium and creatinine. In multivariable analyses, we studied the relation of RNaprox and RNadist with FENa and estimated the heritability (h) of RNaprox and RNadist. RESULTS: Independent of urinary sodium excretion, South Africans (n = 240) had higher RNaprox (unadjusted median, 93.9% vs. 81.0%; P < 0.001) than Belgians (n = 737), but lower RNadist (91.2% vs. 95.1%; P < 0.001). The slope of RNaprox on FENa was steeper in Belgians than in South Africans (-5.40 +/- 0.58 vs. -0.78 +/- 0.58 units; P < 0.001), whereas the opposite was true for the slope of RNadist on FENa (-3.84 +/- 0.19 vs. -13.71 +/- 1.30 units; P < 0.001). h of RNaprox and RNadist was high and significant (P < 0.001) in both countries. h was higher in South Africans than in Belgians for RNaprox (0.82 vs. 0.56; P < 0.001), but was similar for RNadist (0.68 vs. 0.50; P = 0.17). Of the filtered sodium load, black participants reabsorb more than white participants in the proximal nephron and less postproximally. CONCLUSION: Segmental sodium reabsorption along the nephron is highly heritable, but the capacity for regulation in the proximal and postproximal tubules differs between whites and blacks.

Treatment-Naive Individuals Are the Major Source of Transmitted HIV-1 Drug Resistance in Men Who Have Sex With Men in the Swiss HIV Cohort Study.

Background. Human immunodeficiency virus type 1 (HIV-1) transmitted drug resistance (TDR) can compromise antiretroviral therapy (ART) and thus represents an important public health concern. Typically, sources of TDR remain unknown, but they can be characterized with molecular epidemiologic approaches. We used the highly representative Swiss HIV Cohort Study (SHCS) and linked drug resistance database (SHCS-DRDB) to analyze sources of TDR. Methods. ART-naive men who have sex with men with infection date estimates between 1996 and 2009 were chosen for surveillance of TDR in HIV-1 subtype B (N = 1674), as the SHCS-DRDB contains pre-ART genotypic resistance tests for >69% of this surveillance population. A phylogeny was inferred using pol sequences from surveillance patients and all subtype B sequences from the SHCS-DRDB (6934 additional patients). Potential sources of TDR were identified based on phylogenetic clustering, shared resistance mutations, genetic distance, and estimated infection dates. Results. One hundred forty of 1674 (8.4%) surveillance patients carried virus with TDR; 86 of 140 (61.4%) were assigned to clusters. Potential sources of TDR were found for 50 of 86 (58.1%) of these patients. ART-naive patients constitute 56 of 66 (84.8%) potential sources and were significantly overrepresented among sources (odds ratio, 6.43 [95% confidence interval, 3.22-12.82]; P < .001). Particularly large transmission clusters were observed for the L90M mutation, and the spread of L90M continued even after the near cessation of antiretroviral use selecting for that mutation. Three clusters showed evidence of reversion of K103N or T215Y/F. Conclusions. Many individuals harboring viral TDR belonged to transmission clusters with other Swiss patients, indicating substantial domestic transmission of TDR in Switzerland. Most TDR in clusters could be linked to sources, indicating good surveillance of TDR in the SHCS-DRDB. Most TDR sources were ART naive. This, and the presence of long TDR transmission chains, suggests that resistance mutations are frequently transmitted among untreated individuals, highlighting the importance of early diagnosis and treatment.

The impact of cyclin D1 mRNA isoforms, morphology and p53 in mantle cell lymphoma: p53 alterations and blastoid morphology are strong predictors of a high proliferation index.

BACKGROUND: Mantle cell lymphoma is a clinically heterogeneous disease characterized by overexpression of cyclin D1 protein. Blastoid morphology, high proliferation, and secondary genetic aberrations are markers of aggressive behavior. Expression profiling of mantle cell lymphoma revealed that predominance of the 3'UTR-deficient, short cyclin D1 mRNA isoform was associated with high cyclin D1 levels, a high "proliferation signature" and poor prognosis. DESIGN AND METHODS: Sixty-two cases of mantle cell lymphoma were analyzed for cyclin D1 mRNA isoforms and total cyclin D1 levels by real-time reverse transcriptase polymerase chain reaction, and TP53 alterations were assessed by immunohistochemistry and molecular analysis. Results were correlated with proliferation index and clinical outcome. RESULTS: Predominance of the short cyclin D1 mRNA was found in 14 (23%) samples, including four with complete loss of the standard transcript. TP53 alterations were found in 15 (24%) cases. Predominance of 3'UTR-deficient mRNA was significantly associated with high cyclin D1 mRNA levels (P=0.009) and more commonly found in blastoid mantle cell lymphoma (5/11, P=0.060) and cases with a proliferation index of >20% (P=0.026). Both blastoid morphology (11/11, P<0.001) and TP53 alterations (15/15, P<0.001) were significantly correlated with a high proliferation index. A proliferation index of 10% was determined to be a significant threshold for survival in multivariate analysis (P=0.01). CONCLUSIONS: TP53 alterations are strongly associated with a high proliferation index and aggressive behavior in mantle cell lymphoma. Predominance of the 3'UTR-deficient transcript correlates with higher cyclin D1 levels and may be a secondary contributing factor to high proliferation, but failed to reach prognostic significance in this study.

A high proportion of users of low-threshold facilities with needle exchange programmes in Switzerland are currently on methadone treatment: implications for new approaches in harm reduction and care.

BACKGROUND: Increasingly, patients receiving methadone treatment are found in low threshold facilities (LTF), which provide needle exchange programmes in Switzerland. This paper identifies the characteristics of LTF attendees receiving methadone treatment (MT) compared with other LTF attendees (non-MT). METHODS: A national cross-sectional survey was conducted in 2006 over five consecutive days in all LTF (n=25). Attendees were given an anonymous questionnaire, collecting information on socio-demographic indicators, drug consumption, injection, methadone treatment, and self-reported HIV and HCV status. Univariate analysis and logistic regression were performed to compare MT to non-MT. The response rate was 66% (n=1128). RESULTS: MT comprised 57.6% of the sample. In multivariate analysis, factors associated with being on MT were older age (OR: 1.38), being female (OR: 1.60), having one's own accommodation (OR: 1.56), receiving public assistance (OR: 2.29), lifetime injecting (OR: 2.26), HIV-positive status (OR: 2.00), and having consumed cocaine during the past month (OR: 1.37); MT were less likely to have consumed heroin in the past month (OR: 0.76, not significant) and visited LTF less often on a daily basis (OR: 0.59). The number of injections during the past week was not associated with MT. CONCLUSIONS: More LTF attendees were in the MT group, bringing to light an underappreciated LTF clientele with specific needs. The MT group consumption profile may reflect therapeutic failure or deficits in treatment quality and it is necessary to acknowledge this and to strengthen the awareness of LTF personnel about potential needs of MT attendees to meet their therapeutic goals.

Leishmania major induces distinct neutrophil phenotypes in mice that are resistant or susceptible to infection.

Polymorphonuclear neutrophils (PMN) are key components of the inflammatory response contributing to the development of pathogen-specific immune responses. Following infection with Leishmania major, neutrophils are recruited within hours to the site of parasite inoculation. C57BL/6 mice are resistant to infection, and BALB/c mice are susceptible to infection, developing unhealing, inflammatory lesions. In this report, we investigated the expression of cell surface integrins, TLRs, and the secretion of immunomodulatory cytokines by PMN of both strains of mice, in response to infection with L. major. The parasite was shown to induce CD49d expression in BALB/c-inflammatory PMN, and expression of CD49d remained at basal levels in C57BL/6 PMN. Equally high levels of CD11b were expressed on PMN from both strains. In response to L. major infection, the levels of TLR2, TLR7, and TLR9 mRNA were significantly higher in C57BL/6 than in BALB/c PMN. C57BL/6 PMN secreted biologically active IL-12p70 and IL-10. In contrast, L. major-infected BALB/c PMN transcribed and secreted high levels of IL-12p40 but did not secrete biologically active IL-12p70. Furthermore, IL-12p40 was shown not to associate with IL-23 p19 but formed IL-12p40 homodimers with inhibitory activity. No IL-10 was secreted by BALB/c PMN. Thus, following infection with L. major, in C57BL/6 mice, PMN could constitute one of the earliest sources of IL-12, and in BALB/c mice, secretion of IL-12p40 could contribute to impaired, early IL-12 signaling. These distinct PMN phenotypes may thus influence the development of L. major-specific immune response.

Grabbing your ear: rapid auditory-somatosensory multisensory interactions in low-level sensory cortices are not constrained by stimulus alignment.

Multisensory interactions are observed in species from single-cell organisms to humans. Important early work was primarily carried out in the cat superior colliculus and a set of critical parameters for their occurrence were defined. Primary among these were temporal synchrony and spatial alignment of bisensory inputs. Here, we assessed whether spatial alignment was also a critical parameter for the temporally earliest multisensory interactions that are observed in lower-level sensory cortices of the human. While multisensory interactions in humans have been shown behaviorally for spatially disparate stimuli (e.g. the ventriloquist effect), it is not clear if such effects are due to early sensory level integration or later perceptual level processing. In the present study, we used psychophysical and electrophysiological indices to show that auditory-somatosensory interactions in humans occur via the same early sensory mechanism both when stimuli are in and out of spatial register. Subjects more rapidly detected multisensory than unisensory events. At just 50 ms post-stimulus, neural responses to the multisensory 'whole' were greater than the summed responses from the constituent unisensory 'parts'. For all spatial configurations, this effect followed from a modulation of the strength of brain responses, rather than the activation of regions specifically responsive to multisensory pairs. Using the local auto-regressive average source estimation, we localized the initial auditory-somatosensory interactions to auditory association areas contralateral to the side of somatosensory stimulation. Thus, multisensory interactions can occur across wide peripersonal spatial separations remarkably early in sensory processing and in cortical regions traditionally considered unisensory.

Circulating matrix γ-carboxyglutamate protein (MGP) species are refractory to vitamin K treatment in a new case of Keutel syndrome.

Summary.  Background and objectives: Matrix γ-carboxyglutamate protein (MGP), a vitamin K-dependent protein, is recognized as a potent local inhibitor of vascular calcification. Studying patients with Keutel syndrome (KS), a rare autosomal recessive disorder resulting from MGP mutations, provides an opportunity to investigate the functions of MGP. The purpose of this study was (i) to investigate the phenotype and the underlying MGP mutation of a newly identified KS patient, and (ii) to investigate MGP species and the effect of vitamin K supplements in KS patients. Methods: The phenotype of a newly identified KS patient was characterized with specific attention to signs of vascular calcification. Genetic analysis of the MGP gene was performed. Circulating MGP species were quantified and the effect of vitamin K supplements on MGP carboxylation was studied. Finally, we performed immunohistochemical staining of tissues of the first KS patient originally described focusing on MGP species. Results: We describe a novel homozygous MGP mutation (c.61+1G>A) in a newly identified KS patient. No signs of arterial calcification were found, in contrast to findings in MGP knockout mice. This patient is the first in whom circulating MGP species have been characterized, showing a high level of phosphorylated MGP and a low level of carboxylated MGP. Contrary to expectations, vitamin K supplements did not improve the circulating carboxylated MGP levels. Phosphorylated MGP was also found to be present in the first KS patient originally described. Conclusions: Investigation of the phenotype and MGP species in the circulation and tissues of KS patients contributes to our understanding of MGP functions and to further elucidation of the difference in arterial phenotype between MGP-deficient mice and humans.

Hepatitis C virus-mediated mitochondrial dysfunctions are prevented and rescued by cyclophilin inhibition

Alisporivir (Debio-025) is an analogue of cyclosporine A andrepresents the prototype of a new class of non-immunosuppressivecyclophilin inhibitors. In vitro and in vivo studies have shownthat alisporivir inhibits hepatitis C virus (HCV) replication andongoing clinical trials are exploring its therapeutic potential inpatients with chronic hepatitis C. Recent data suggest that theantiviral effect is mediated by inhibition of cyclophilin A whichis an essential host factor in the HCV life cycle. However, alisporiviralso inhibits mitochondrial permeability transition by bindingto cyclophilin D. As HCV is known to affect mitochondrialfunction, we explored the effect of alisporivir on HCV proteinmediatedmitochondrial dysfunction. By the use of inducible celllines, which allow to investigate the effects of HCV polyproteinexpression independent from viral RNA replication and whichrecapitulate the major alterations of mitochondrial bioenergeticsobserved in infectious cell systems, we show that alisporivir preventsHCV protein-mediated cytochrome c redistribution,decrease of cell respiration, collapse of mitochondrial membranepotential, overproduction of reactive oxygen species and mitochondrialcalcium overload. Strikingly, some of the HCV-mediatedmitochondrial dysfunctions could even be rescued byalisporivir. These observations provide new insights into thepathogenesis of HCV-related liver disease and reveal an additionalmechanism of action of alisporivir that is likely beneficialin the treatment of chronic hepatitis C.

Male body size and breeding tubercles are both linked to intra-sexual dominance and reproductive success in the minnow

Male dominance hierarchies are usually linked to relative body size and to weapon size, that is, to determinants of fighting ability. Secondary sexual characters that are not directly used as weapons could still be linked to dominance if they reveal determination or overall health and vigour and hence, indirectly, fighting ability. We studied the mating behaviour of the minnow, Phoxinus phoxinus, a cyprinid fish in which males develop breeding tubercles during the spawning season. The function of these breeding tubercles is still not clear. Using microsatellite markers, we determined male reproductive success under controlled conditions. The minnows were territorial and quickly established a dominance hierarchy at the beginning of the spawning season. Dominance was strongly and positively linked to fertilization success. Although body size and number of breeding tubercles were not significantly correlated in our sample, both large males and males with many breeding tubercles were more dominant and achieved higher fertilization success than small males or males with few tubercles. We found multimale fertilization in most clutches, suggesting that sperm competition is important in this species. Females showed behaviour that may be linked to spawning decision, that is, male dominance might not be the only determinant of male reproductive success in minnows

One hundred base pairs of 5' flanking sequence of a vaccinia virus late gene are sufficient to temporally regulate late transcription.

A vaccinia virus late gene coding for a major structural polypeptide of 11 kDa was sequenced. Although the 5' flanking gene region is very A+T rich, it shows little homology either to the corresponding region of vaccinia early genes or to consensus sequences characteristic of most eukaryotic genes. Three DNA fragments (100, 200, and 500 base pairs, respectively), derived from the flanking region and including the late gene mRNA start site, were inserted into the coding sequence of the vaccinia virus thymidine kinase (TK) early gene by homologous in vivo recombination. Recombinants were selected on the basis of their TK- phenotype. Cells were infected with the recombinant viruses and RNA was isolated at 1-hr intervals. Transcripts initiating either from the TK early promoter, or from the late gene promoter at its authentic position, or from the translocated late gene promoters within the early gene were detected by nuclease S1 mapping. Early after infection, only transcripts from the TK early promoter were detected. Later in infection, however, transcripts were also initiated from the translocated late promoters. This RNA appeared at the same time and in similar quantities as the RNA from the late promoter at its authentic position. No quantitative differences in promoter efficiency between the 100-, 200-, and 500-base-pair insertions were observed. We conclude that all necessary signals for correct regulation of late-gene expression reside within only 100 base pairs of 5' flanking sequence.

Human alveolar macrophages infected by virulent bacteria expressing SipB are a major source of active interleukin-18.

Recent publications have demonstrated that the protease caspase-1 is responsible for the processing of pro-interleukin 18 (IL-18) into the active form. Studies on cell lines and murine macrophages have shown that the bacterial invasion factor SipB activates caspase-1, triggering cell death. Thus, we investigated the role of SipB in the activation and release of IL-18 in human alveolar macrophages (AM), which are the first line of defense against inhaled pathogens. Under steady-state conditions, AM are a more important source of IL-18 than are dendritic cells (DC) and monocytes. Cytokine production by AM and DC was compared after both types of cells had been infected with a virulent strain of Salmonella enterica serovar Typhimurium and an isogenic sipB mutant, which were used as an infection model. Infection with virulent Salmonella led to marked cell death with features of apoptosis while both intracellular activation and release of IL-18 were demonstrated. In contrast, the sipB mutant did not induce such cell death or the release of active IL-18. The specific caspase-1 inhibitor Ac-YVAD-CMK blocked the early IL-18 release in AM infected with the virulent strain. However, the type of Salmonella infection did not differentially regulate IL-18 gene expression. We concluded that the bacterial virulence factor SipB plays an essential posttranslational role in the intracellular activation of IL-18 and the release of the cytokine in human AM.

T helper 1 (Th1) and Th2 characteristics start to develop during T cell priming and are associated with an immediate ability to induce immunoglobulin class switching.

The respective production of specific immunoglobulin (Ig)G2a or IgG1 within 5 d of primary immunization with Swiss type mouse mammary tumor virus [MMTV(SW)] or haptenated protein provides a model for the development of T helper 1 (Th1) and Th2 responses. The antibody-producing cells arise from cognate T cell B cell interaction, revealed by the respective induction of Cgamma2a and Cgamma1 switch transcript production, on the third day after immunization. T cell proliferation and upregulation of mRNA for interferon gamma in response to MMTV(SW) and interleukin 4 in response to haptenated protein also starts during this day. It follows that there is minimal delay in these responses between T cell priming and the onset of cognate interaction between T and B cells leading to class switching and exponential growth. The Th1 or Th2 profile is at least partially established at the time of the first cognate T cell interaction with B cells in the T zone. The addition of killed Bordetella pertussis to the hapten-protein induces nonhapten-specific IgG2a and IgG1 plasma cells, whereas the anti-hapten response continues to be IgG1 dominated. This indicates that a Th2 response to hapten-protein can proceed in a node where there is substantial Th1 activity.