Single nucleus and spatial transcriptomic profiling of healthy human hamstring tendon.

The molecular and cellular basis of health in human tendons remains poorly understood. Among human tendons, hamstring tendon has markedly low pathology and can provide a prototypic healthy tendon reference. The aim of this study was to determine the transcriptomes and location of all cell types in healthy hamstring tendon. Using single nucleus RNA sequencing, we profiled the transcriptomes of 10 533 nuclei from four healthy donors and identified 12 distinct cell types. We confirmed the presence of two fibroblast cell types, endothelial cells, mural cells, and immune cells, and identified cell types previously unreported in tendons, including different skeletal muscle cell types, satellite cells, adipocytes, and undefined nervous system cells. The location of these cell types within tendon was defined using spatial transcriptomics and imaging, and potential transcriptional networks and cell-cell interactions were analyzed. We demonstrate that fibroblasts have the highest number of potential cell-cell interactions in our dataset, are present throughout the tendon, and play an important role in the production and organization of extracellular matrix, thus confirming their role as key regulators of hamstring tendon homeostasis. Overall, our findings underscore the complexity of the cellular networks that underpin healthy human tendon function and the central role of fibroblasts as key regulators of hamstring tendon tissue homeostasis.

Comprehensive epigenomic profiling reveals the extent of disease-specific chromatin states and informs target discovery in ankylosing spondylitis.

Ankylosing spondylitis (AS) is a common, highly heritable inflammatory arthritis characterized by enthesitis of the spine and sacroiliac joints. Genome-wide association studies (GWASs) have revealed more than 100 genetic associations whose functional effects remain largely unresolved. Here, we present a comprehensive transcriptomic and epigenomic map of disease-relevant blood immune cell subsets from AS patients and healthy controls. We find that, while CD14+ monocytes and CD4+ and CD8+ T cells show disease-specific differences at the RNA level, epigenomic differences are only apparent upon multi-omics integration. The latter reveals enrichment at disease-associated loci in monocytes. We link putative functional SNPs to genes using high-resolution Capture-C at 10 loci, including PTGER4 and ETS1, and show how disease-specific functional genomic data can be integrated with GWASs to enhance therapeutic target discovery. This study combines epigenetic and transcriptional analysis with GWASs to identify disease-relevant cell types and gene regulation of likely pathogenic relevance and prioritize drug targets.

Chromosome conformation capture approaches to investigate 3D genome architecture in Ankylosing Spondylitis.

Ankylosing Spondylitis (AS) is a chronic inflammatory arthritis of the spine exhibiting a strong genetic background. The mechanistic and functional understanding of the AS-associated genomic loci, identified with Genome Wide Association Studies (GWAS), remains challenging. Chromosome conformation capture (3C) and derivatives are recent techniques which are of great help in elucidating the spatial genome organization and of enormous support in uncover a mechanistic explanation for disease-associated genetic variants. The perturbation of three-dimensional (3D) genome hierarchy may lead to a plethora of human diseases, including rheumatological disorders. Here we illustrate the latest approaches and related findings on the field of genome organization, highlighting how the instability of 3D genome conformation may be among the causes of rheumatological disease phenotypes. We suggest a new perspective on the inclusive potential of a 3C approach to inform GWAS results in rheumatic diseases. 3D genome organization may ultimately lead to a more precise and comprehensive functional interpretation of AS association, which is the starting point for emerging and more specific therapies.

Atendimentos em saúde na perspectiva de transexuais / Transsexual's perspective on health care / Atención en salud desde la perspectiva de transexuales

Este artigo teve como objetivo compreender como transexuais significam os atendimentos em saúde, em um hospital público, na microrregião do Triângulo Mineiro. Nove transexuais foram entrevistados, e os conteúdos foram organizados em quatro categorias para análise temática (concepções sobre transexualidade; informações; percepções sobre atenção à saúde; concepções sobre direitos e preconceitos). Os principais resultados apontaram para: a compreensão da transexualidade como experiência identitária discordante com o corpo biológico; a busca por modificações corporais; as insuficiências e ausências de informações e organização para acesso, adesão e permanência naquele serviço de saúde; respeito ao nome social, apesar de pontuais discriminações por parte de alguns profissionais de saúde. A não consolidação das políticas de atenção básica de saúde (especialmente da integralidade) pode afastar e dificultar essa população de um direito que lhe é garantido, porém nem sempre observado.

The aim of this paper is to understand how transsexuals signify health care in a public hospital in the Triângulo Mineiro (Brazil) microregion. Nine transexuals were interviewed about contents which were organized into four categories of thematic analysis (conception on transsexuality, information, perception of health care, conception on rights and prejudices). The main results highlighted: transsexuality as an identity experience dissenting from the biological body; the search for body modification; shortcomings and lack of information regarding access to health services and continuity of attendance; occasional discriminatory attitudes by a few health professionals. We consider that the non-consolidation of the basic health care policies can hinder this population from their rights.

Este artículo tuvo como objetivo comprender cómo los transexuales significan la atención en salud en un hospital público en la micro región del Triángulo Minero (Brasil). Se reclutaron nueve transexuales para entrevistas cuyos contenidos se organizaron en cuatro categorías de análisis temático (concepciones sobre transexualidad; información; percepciones sobre la atención en salud; concepciones sobre derechos y prejuicios). Los principales resultados desvelaron una comprensión de la transexualidad como una experiencia identitaria discordante con el cuerpo biológico; la búsqueda por cambios corporales; insuficiencias y faltas de información para acceso y permanencia en los servicios de salud; respeto al nombre social, a pesar de algunas conductas discriminatorias por parte de los profesionales de salud. Si no se consolidan las políticas de atención básica en la salud (especialmente la integralidad) se puede alejar y dificultar que esta población goce de un derecho que es garantizado, pero no siempre observado.

Perspectives on the Genetic Associations of Ankylosing Spondylitis.

Ankylosing spondylitis (AS) is a common form of inflammatory spinal arthritis with a complex polygenic aetiology. Genome-wide association studies have identified more than 100 loci, including some involved in antigen presentation (HLA-B27, ERAP1, and ERAP2), some in Th17 responses (IL6R, IL23R, TYK2, and STAT3), and others in macrophages and T-cells (IL7R, CSF2, RUNX3, and GPR65). Such observations have already helped identify potential new therapies targeting IL-17 and GM-CSF. Most AS genetic associations are not in protein-coding sequences but lie in intergenic regions where their direct relationship to particular genes is difficult to assess. They most likely reflect functional polymorphisms concerned with cell type-specific regulation of gene expression. Clarifying the nature of these associations should help to understand the pathogenic pathways involved in AS better and suggest potential cellular and molecular targets for drug therapy. However, even identifying the precise mechanisms behind the extremely strong HLA-B27 association with AS has so far proved elusive. Polygenic risk scores (using all the known genetic associations with AS) can be effective for the diagnosis of AS, particularly where there is a relatively high pre-test probability of AS. Genetic prediction of disease outcomes and response to biologics is not currently practicable.

Functional Genomic Analysis of a RUNX3 Polymorphism Associated With Ankylosing Spondylitis.

OBJECTIVE: To investigate the functional consequences of the single-nucleotide polymorphism rs4648889 in a putative enhancer upstream of the RUNX3 promoter associated with susceptibility to ankylosing spondylitis (AS). METHODS: Using nuclear extracts from Jurkat cells and primary human CD8+ T cells, the effects of rs4648889 on allele-specific transcription factor (TF) binding were investigated by DNA pull-down assay and quantitative mass spectrometry (qMS), with validation by electrophoretic mobility shift assay (EMSA), Western blotting of the pulled-down eluates, and chromatin immunoprecipitation (ChIP)-quantitative polymerase chain reaction (qPCR) analysis. Further functional effects were tested by small interfering RNA knockdown of the gene for interferon regulatory factor 5 (IRF5), followed by reverse transcription-qPCR (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA) to measure the levels of IFNγ messenger RNA (mRNA) and protein, respectively. RESULTS: In nuclear extracts from CD8+ T cells, results of qMS showed that relative TF binding to the AS-risk A allele of rs4648889 was increased 3.7-fold (P < 0.03) for Ikaros family zinc-finger protein 3 (IKZF3; Aiolos) and components of the NuRD complex, including chromodomain helicase DNA binding protein 4 (CHD4) (3.6-fold increase; P < 0.05) and retinoblastoma binding protein 4 (RBBP4) (4.1-fold increase; P < 0.03). In contrast, IRF5 bound significantly more to the AS-protective G allele compared to the AS-risk A allele (fold change 8.2; P = 0.003). Validation with Western blotting, EMSA, and ChIP-qPCR confirmed the differential allelic binding of IKZF3, CHD4, RBBP4, and IRF5. Silencing of IRF5 in CD8+ T cells increased the levels of IFNγ mRNA as measured by RT-qPCR (P = 0.03) and IFNγ protein as measured by ELISA (P = 0.02). CONCLUSION: These findings suggest that the association of rs4648889 with AS reflects allele-specific binding of this enhancer-like region to certain TFs, including IRF5, IKZF3, and members of the NuRD complex. IRF5 may have crucial influences on the functions of CD8+ lymphocytes, a finding that could reveal new therapeutic targets for the management of AS.

Disruption of c-MYC Binding and Chromosomal Looping Involving Genetic Variants Associated With Ankylosing Spondylitis Upstream of the RUNX3 Promoter.

Background: Ankylosing Spondylitis (AS) is a common form of inflammatory spinal arthritis with a complex aetiology and high heritability, involving more than 100 genetic associations. These include several AS-associated single nucleotide polymorphisms (SNPs) upstream of RUNX3, which encodes the multifunctional RUNT-related transcription factor (TF) 3. The lead associated SNP rs6600247 (p = 2.6 × 10-15) lies ∼13kb upstream of the RUNX3 promoter adjacent to a c-MYC TF binding-site. The effect of rs6600247 genotype on DNA binding and chromosome looping were investigated by electrophoretic mobility gel shift assays (EMSA), Western blotting-EMSA (WEMSA) and Chromosome Conformation Capture (3C). Results: Interrogation of ENCODE published data showed open chromatin in the region overlapping rs6600247 in primary human CD14+ monocytes, in contrast to the Jurkat T cell line or primary human T-cells. The rs6600247 AS-risk allele is predicted to specifically disrupt a c-MYC binding-site. Using a 50bp DNA probe spanning rs6600247 we consistently observed reduced binding to the AS-risk "C" allele of both purified c-MYC protein and nuclear extracts (NE) from monocyte-like U937 cells. WEMSA on U937 NE and purified c-MYC protein confirmed these differences (n = 3; p < 0.05). 3C experiments demonstrated negligible interaction between the region encompassing rs6600247 and the RUNX3 promoter. A stronger interaction frequency was demonstrated between the RUNX3 promoter and the previously characterised AS-associated SNP rs4648889. Conclusion: The lead SNP rs6600247, located in an enhancer-like region upstream of the RUNX3 promoter, modulates c-MYC binding. However, the region encompassing rs6600247 has rather limited physical interaction with the promoter of RUNX3. In contrast a clear chromatin looping event between the region encompassing rs4648889 and the RUNX3 promoter was observed. These data provide further evidence for complexity in the regulatory elements upstream of the RUNX3 promoter and the involvement of RUNX3 transcriptional regulation in AS.

Corpo feminino e violência de gênero: uma análise do documentário "chega de fiu fiu" / Cuerpo femenino y violencia de género: un análisis del documental "chega de fiu fiu" / Female body and gender violence: an analysis of the documentary "chega de fiu fiu"

Resumo Historicamente nota-se que a domesticação e a normatização do corpo feminino podem ser reconhecidas como uma estratégia consideravelmente durável e flexível de controle social, cumprindo também com uma função ideológica que pode vir a se desdobrar em inúmeras formas de violência. Nesse contexto, o presente artigo teve como objetivo compreender como o corpo feminino e a violência de gênero são abordados no documentário brasileiro de 2018 Chega de Fiu Fiu. A partir de uma análise de conteúdo temática os principais resultados destacam a objetificação e a submissão das mulheres por parte dos homens que, naturalizados, reforçam e reiteram os assédios morais e violências sexuais que vitimam as mulheres na nossa sociedade.

Resumen Históricamente, se ha señalado que la domesticación y normalización del cuerpo femenino pueden ser reconocidas como una estrategia de control social considerablemente duradera y flexible, cumpliendo además una función ideológica que puede desplegarse en innumerables formas de violencia. En este contexto, este artículo tuvo como objetivo comprender cómo se abordan el cuerpo femenino y la violencia de género en el documental brasileño Chega de Fiu Fiu 2018. A partir de un análisis de contenido temático, los principales resultados destacan la cosificación y sometimiento de las mujeres por parte de los hombres que, naturalizados, refuerzan y reiteran los hostigamientos morales y la violencia sexual que victimizan a las mujeres en nuestra sociedad.

Abstract Historically, it has been noted that the domestication and standardization of the female body can be recognized as a considerably long-lasting and flexible strategy of social control, also fulfilling an ideological function that can unfold into several forms of violence. In this context, this article aimed to comprehend how the female body and gender violence are addressed in the 2018 Brazilian documentary Chega de Fiu Fiu. From a thematic content analysis, the main results highlight the objectification and submission of women by men, which, when naturalized, reinforce and reiterate the moral harassment and sexual violence that victimize women in our society.

The severity of ankylosing spondylitis and responses to anti-tumour necrosis factor biologics are not influenced by the tumour necrosis factor receptor polymorphism incriminated in multiple sclerosis.

Genetic polymorphism (rs1800693) of TNFRSF1A (type 1 tumour necrosis factor receptor) encodes a potentially anti-inflammatory soluble truncated form of the p55 receptor, which is associated with predisposition to multiple sclerosis but protection against ankylosing spondylitis (AS). We analysed 2917 UK Caucasian cases by linear and logistic regression for associations of rs1800693 with disease severity assessed by the Bath Ankylosing Spondylitis measures of disease activity and function (BASDAI, BAS-G and BASFI) and/or responses to anti-TNF therapy. In contrast to predictions, rs1800693 GG homozygotes actually had significantly worse BASDAI (mean 4.2, 95% CI: 4-4.5) than AA homozygotes (mean 3.8, 95% CI: 3.7-4) in both the unadjusted (difference = 0.4, p = 0.006) and adjusted analyses (difference = 0.2-0.5, p = 0.002-0.04 depending on the adjustment model). We found no evidence that rs1900693 predicted functional status (BASFI) or global disease scores (BAS-G), and it exerted no influence on either the intention to treat with or efficacy of anti-TNF treatment.

Quantifying the genetic risk for the development of axial spondyloarthropathy: could this become a diagnostic tool?

PURPOSE OF REVIEW: To assess the utility of recent genetic findings in ankylosing spondylitis (AS) and axial spondyloarthropathy (SpA) in relation to diagnostic testing, prognosis and responses to biologic treatment and the development of new therapies. RECENT FINDINGS: AS and other forms of SpA are polygenic with more than 100 genes contributing to disease susceptibility. The role of genes in determining the outcome of the disease and response to treatment is less clear. Here, we review some of the progress that has been made over the past decade in understanding the genetic contribution to these diseases and how this may be used to inform the development of new treatments. In those with a high pretest probability of SpA human leukocyte antigen (HLA)-B27 testing can increase the posttest predictive value to almost 100% in some cases. There are currently no reliable genetic predictors of disease severity or response to treatment. SUMMARY: The utility of HLA-B27 as a diagnostic tool when coupled with careful clinical assessment is well established but other genetic markers probably have relatively little to add. In contrast, novel drug targets are likely to be identified from genetic association studies.

Evidence for a second ankylosing spondylitis-associated RUNX3 regulatory polymorphism.

OBJECTIVES: To explore the functions of RUNX3 single nucleotide polymorphisms (SNPs) associated with ankylosing spondylitis (AS). METHODS: Individual SNP associations were evaluated in 4230 UK cases. Their effects on transcription factor (TF) binding, transcription regulation, chromatin modifications, gene expression and gene interactions were tested by database interrogation, luciferase reporter assays, electrophoretic mobility gel shifts, chromatin immunoprecipitation and real-time PCR. RESULTS: We confirmed the independent association of AS with rs4265380, which was robust (P=4.7×10-6) to conditioning on another nearby AS-associated RUNX3 SNP (rs4648889). A RUNX3 haplotype incorporating both SNPs was strongly associated with AS (OR 6.2; 95% CI 3.1 to 13.2, P=1.4×10-8). In a large UK cohort, rs4265380 is associated with leucocyte counts (including monocytes). RUNX3 expression is lower in AS peripheral blood mononuclear cells than healthy controls (P<0.002), independent of rs4265380 genotype. Enhancer function for this RUNX3 region was suggested by increased luciferase activity (approximately tenfold; P=0.005) for reporter constructs containing rs4265380. In monocytes, there was differential allelic binding of nuclear protein extracts to a 50 bp DNA probe containing rs4265380 that was strongly augmented by lipopolysaccharide activation. TF binding also included the histone modifier p300. There was enrichment for histone modifications associated with active enhancer elements (H3K27Ac and H3K79Me2) that may be allele dependent. Hi-C database interrogation showed chromosome interactions of RUNX3 bait with the nearby RP4-799D16.1 lincRNA. CONCLUSIONS: The association of AS with this RUNX3 regulatory region involves at least two SNPs apparently operating in different cell types. Monocytes may be potential therapeutic targets in AS.

RUNX3 and T-Bet in Immunopathogenesis of Ankylosing Spondylitis-Novel Targets for Therapy?

Susceptibility to ankylosing spondylitis (AS) is polygenic with more than 100 genes identified to date. These include HLA-B27 and the aminopeptidases (ERAP1, ERAP2, and LNPEPS), which are involved in antigen processing and presentation to T-cells, and several genes (IL23R, IL6R, STAT3, JAK2, IL1R1/2, IL12B, and IL7R) involved in IL23 driven pathways of inflammation. AS is also strongly associated with polymorphisms in two transcription factors, RUNX3 and T-bet (encoded by TBX21), which are important in T-cell development and function. The influence of these genes on the pathogenesis of AS and their potential for identifying drug targets is discussed here.

An ankylosing spondylitis-associated genetic variant in the IL23R-IL12RB2 intergenic region modulates enhancer activity and is associated with increased Th1-cell differentiation.

OBJECTIVES: To explore the functional basis for the association between ankylosing spondylitis (AS) and single-nucleotide polymorphisms (SNPs) in the IL23R-IL12RB2 intergenic region. METHODS: We performed conditional analysis on genetic association data and used epigenetic data on chromatin remodelling and transcription factor (TF) binding to identify the primary AS-associated IL23R-IL12RB2 intergenic SNP. Functional effects were tested in luciferase reporter assays in HEK293T cells and allele-specific TF binding was investigated by electrophoretic mobility gel shift assays. IL23R and IL12RB2 mRNA levels in CD4+ T cells were compared between cases homozygous for the AS-risk 'A' allele and the protective 'G' allele. The proportions of interleukin (IL)-17A+ and interferon (IFN)-γ+ CD4+ T-cells were measured by fluorescence-activated cell sorting and compared between these AS-risk and protective genotypes. RESULTS: Conditional analysis identified rs11209032 as the probable causal SNP within a 1.14 kb putative enhancer between IL23R and IL12RB2. Reduced luciferase activity was seen for the risk allele (p<0.001) and reduced H3K4me1 methylation observed in CD4+ T-cells from 'A/A' homozygotes (p=0.02). The binding of nuclear extract to the risk allele was decreased ∼3.5-fold compared with the protective allele (p<0.001). The proportion of IFN-γ+ CD4+ T-cells was increased in 'A/A' homozygotes (p=0.004), but neither IL23R nor IL12RB2 mRNA was affected. CONCLUSIONS: The rs11209032 SNP downstream of IL23R forms part of an enhancer, allelic variation of which may influence Th1-cell numbers. Homozygosity for the risk 'A' allele is associated with more IFN-γ-secreting (Th1) cells. Further work is necessary to explain the mechanisms for these important observations.

The genetic association of RUNX3 with ankylosing spondylitis can be explained by allele-specific effects on IRF4 recruitment that alter gene expression.

OBJECTIVES: To identify the functional basis for the genetic association of single nucleotide polymorphisms (SNP), upstream of the RUNX3 promoter, with ankylosing spondylitis (AS). METHODS: We performed conditional analysis of genetic association data and used ENCODE data on chromatin remodelling and transcription factor (TF) binding sites to identify the primary AS-associated regulatory SNP in the RUNX3 region. The functional effects of this SNP were tested in luciferase reporter assays. Its effects on TF binding were investigated by electrophoretic mobility gel shift assays and chromatin immunoprecipitation. RUNX3 mRNA levels were compared in primary CD8+ T cells of AS risk and protective genotypes by real-time PCR. RESULTS: The association of the RUNX3 SNP rs4648889 with AS (p<7.6×10(-14)) was robust to conditioning on all other SNPs in this region. We identified a 2 kb putative regulatory element, upstream of RUNX3, containing rs4648889. In reporter gene constructs, the protective rs4648889 'G' allele increased luciferase activity ninefold but significantly less activity (4.3-fold) was seen with the AS risk 'A' allele (p≤0.01). The binding of Jurkat or CD8+ T-cell nuclear extracts to the risk allele was decreased and IRF4 recruitment was reduced. The AS-risk allele also affected H3K4Me1 histone methylation and associated with an allele-specific reduction in RUNX3 mRNA (p<0.05). CONCLUSION: We identified a regulatory region upstream of RUNX3 that is modulated by rs4648889. The risk allele decreases TF binding (including IRF4) and reduces reporter activity and RUNX3 expression. These findings may have important implications for understanding the role of T cells and other immune cells in AS.

Human Th1 and Th17 cells exhibit epigenetic stability at signature cytokine and transcription factor loci.

The linear model of Th cell lineage commitment is being revised due to reports that mature Th cells can trans-differentiate into alternate lineages. This ability of Th cells to reprogram is thought to be regulated by epigenetic mechanisms that control expression of transcription factors characteristic of opposing lineages. It is unclear, however, to what extent this new model of Th cell plasticity holds true in human Th cell subsets that develop under physiological conditions in vivo. We isolated in vivo-differentiated human Th1 and Th17 cells, as well as intermediate Th1/17 cells, and identified distinct epigenetic signatures at cytokine (IFNG and IL17A) and transcription factor (TBX21, RORC, and RORA) loci. We also examined the phenotypic and epigenetic stability of human Th17 cells exposed to Th1-polarizing conditions and found that although they could upregulate TBX21 and IFN-γ, this occurred without loss of IL-17 or RORC expression, and resulted in cells with a Th1/17 phenotype. Similarly, Th1 cells could upregulate IL-17 upon enforced expression of RORC2, but did not lose expression of IFN-γ or TBX21. Despite alterations in expression of these signature genes, epigenetic modifications were remarkably stable aside from the acquisition of active histone methylation marks at cytokine gene promoters. The limited capacity of human Th17 and Th1 cells to undergo complete lineage conversion suggests that the bipotent Th1/17 cells may arise from Th1 and/or Th17 cells. These data also question the broad applicability of the new model of Th cell lineage plasticity to in vivo-polarized human Th cell subsets.

Placenta-specific expression of the interleukin-2 (IL-2) receptor ß subunit from an endogenous retroviral promoter.

The long terminal repeat (LTR) sequences of endogenous retroviruses and retroelements contain promoter elements and are known to form chimeric transcripts with nearby cellular genes. Here we show that an LTR of the THE1D retroelement family has been domesticated as an alternative promoter of human IL2RB, the gene encoding the ß subunit of the IL-2 receptor. The LTR promoter confers expression specifically in the placental trophoblast as opposed to its native transcription in the hematopoietic system. Rather than sequence-specific determinants, DNA methylation was found to regulate transcription initiation and splicing efficiency in a tissue-specific manner. Furthermore, we detected the cytoplasmic signaling domain of the IL-2Rß protein in the placenta, suggesting that IL-2Rß undergoes preferential proteolytic cleavage in this tissue. These findings implicate novel functions for this cytokine receptor subunit in the villous trophoblast and reveal an intriguing example of ancient LTR exaptation to drive tissue-specific gene expression.

SHIP-deficient dendritic cells, unlike wild type dendritic cells, suppress T cell proliferation via a nitric oxide-independent mechanism.

BACKGROUND: Dendritic cells (DCs) not only play a crucial role in activating immune cells but also suppressing them. We recently investigated SHIP's role in murine DCs in terms of immune cell activation and found that TLR agonist-stimulated SHIP-/- GM-CSF-derived DCs (GM-DCs) were far less capable than wild type (WT, SHIP+/+) GM-DCs at activating T cell proliferation. This was most likely because SHIP-/- GM-DCs could not up-regulate MHCII and/or co-stimulatory receptors following TLR stimulation. However, the role of SHIP in DC-induced T cell suppression was not investigated. METHODOLOGY/PRINCIPAL FINDINGS: In this study we examined SHIP's role in DC-induced T cell suppression by co-culturing WT and SHIP-/- murine DCs, derived under different conditions or isolated from spleens, with αCD3+ αCD28 activated WT T cells and determined the relative suppressive abilities of the different DC subsets. We found that, in contrast to SHIP+/+ and -/- splenic or Flt3L-derived DCs, which do not suppress T cell proliferation in vitro, both SHIP+/+ and -/- GM-DCs were capable of potently suppressing T cell proliferation. However, WT GM-DC suppression appeared to be mediated, at least in part, by nitric oxide (NO) production while SHIP-/- GM-DCs expressed high levels of arginase 1 and did not produce NO. Following exhaustive studies to ascertain the mechanism of SHIP-/- DC-mediated suppression, we could conclude that cell-cell contact was required and the mechanism may be related to their relative immaturity, compared to SHIP+/+ GM-DCs. CONCLUSIONS: These findings suggest that although both SHIP+/+ and -/- GM-DCs suppress T cell proliferation, the mechanism(s) employed are different. WT GM-DCs suppress, at least in part, via IFNγ-induced NO production while SHIP-/- GM-DCs do not produce NO and suppression can only be alleviated when contact is prevented.

Endogenous retroviral LTRs as promoters for human genes: a critical assessment.

Gene regulatory changes are thought to be major factors driving species evolution, with creation of new regulatory regions likely being instrumental in contributing to diversity among vertebrates. There is growing appreciation for the role of transposable elements (TEs) in gene regulation and, indeed, laboratory investigations have confirmed many specific examples of mammalian genes regulated by promoters donated by endogenous retroviruses (ERVs) or other TEs. Bioinformatics studies have revealed hundreds of additional instances where this is likely to be the case. Since the long terminal repeats (LTRs) of retroviruses naturally contain abundant transcriptional regulatory signals, roles for ERV LTRs in regulating mammalian genes are eminently plausible. Moreover, it seems reasonable that exaptation of an LTR regulatory module provides opportunities for evolution of new gene regulatory patterns. In this Review we summarize known examples of LTRs that function as human gene alternative promoters, as well as the evidence that LTR exaptation has resulted in a pattern of novel gene expression significantly different from the pattern before LTR insertion or from that of gene orthologs lacking the LTR. Available data suggest that, while new expression patterns can arise as a result of LTR usage, this situation is relatively rare and is largely restricted to the placenta. In many cases, the LTR appears to be a minor, alternative promoter with an expression pattern similar to that of the native promoter(s) and hence likely exerts a subtle overall effect on gene expression. We discuss these findings and offer evolutionary models to explain these trends.

An aptamer that neutralizes R5 strains of HIV-1 binds to core residues of gp120 in the CCR5 binding site.

We have previously isolated nucleic acid ligands (aptamers) that bind the surface envelope glycoprotein, gp120, of HIV-1, and neutralize infection of diverse sub-types of virus. Our earlier studies have identified the overall structure of one of these aptamers, B40, and have indicated that it binds to gp120 in a manner that competes with that of the HIV-1 coreceptor, CCR5, and select "CD4i" antibodies with epitopes overlapping this region. Here, we sought to map the B40 binding site on gp120 more precisely by analysing its interaction with a panel of alanine substitution mutants of gp120. Furthermore, we tested our hypothesis concerning the structure of the 40 nucleotide functional core of the aptamer by the solid-phase synthesis of truncated and chemically modified derivatives. The results confirm our structural predictions and demonstrate that aptamer B40 neutralizes a diverse range of HIV-1 isolates as a result of binding to relatively conserved residues on gp120 at the heart of the CCR5-binding site. These structural insights may provide the basis for the development of potential anti-viral agents with high specificity and robustness.