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Objective: Insulin resistance and other features of the metabolic syndrome have been causally linked to adipose tissue macrophages (ATMs) in mice with diet-induced obesity. We aimed to characterize macrophage phenotype and function in human subcutaneous and omental adipose tissue in relation to insulin resistance in obesity.Research Design and Methods: Adipose tissue was obtained from lean and obese women undergoing bariatric surgery. Metabolic markers were measured in fasting serum and ATMs characterized by immunohistology, flow cytometry, and tissue culture studies. RESULTS ATMs comprised CD11c(+)CD206(+) cells in "crown" aggregates and solitary CD11c(-)CD206(+) cells at adipocyte junctions. In obese women, CD11c(+) ATM density was greater in subcutaneous than omental adipose tissue and correlated with markers of insulin resistance. CD11c(+) ATMs were distinguished by high expression of integrins and antigen presentation molecules; interleukin (IL)-1beta, -6, -8, and -10; tumor necrosis factor-alpha; and CC chemokine ligand-3, indicative of an activated, proinflammatory state. In addition, CD11c(+) ATMs were enriched for mitochondria and for RNA transcripts encoding mitochondrial, proteasomal, and lysosomal proteins, fatty acid metabolism enzymes, and T-cell chemoattractants, whereas CD11c(-) ATMs were enriched for transcripts involved in tissue maintenance and repair. Tissue culture medium conditioned by CD11c(+) ATMs, but not CD11c(-) ATMs or other stromovascular cells, impaired insulin-stimulated glucose uptake by human adipocytes.Conclusions: These findings identify proinflammatory CD11c(+) ATMs as markers of insulin resistance in human obesity. In addition, the machinery of CD11c(+) ATMs indicates they metabolize lipid and may initiate adaptive immune responses. [ABSTRACT FROM AUTHOR]

Type 1 diabetes (T1D) is caused by T cell-mediated destruction of the pancreatic insulin-producing β cells. While the role of CD4+ T cells in the pathogenesis of T1D is accepted widely, the epitopes recognized by pathogenic human CD4+ T cells remain poorly defined. None the less, responses to the N-terminal region of the insulin A-chain have been described. Human CD4+ T cells from the pancreatic lymph nodes of subjects with T1D respond to the first 15 amino acids of the insulin A-chain. We identified a human leucocyte antigen-DR4-restricted epitope comprising the first 13 amino acids of the insulin A-chain (A1-13), dependent upon generation of a vicinal disulphide bond between adjacent cysteines (A6–A7). Here we describe the analysis of a CD4+ T cell clone, isolated from a subject with T1D, which recognizes a new HLR-DR4-restricted epitope (KRGIVEQCCTSICS) that overlaps the insulin A1-13 epitope. This is a novel epitope, because the clone responds to proinsulin but not to insulin, T cell recognition requires the last two residues of the C-peptide (Lys, Arg) and recognition does not depend upon a vicinal disulphide bond between the A6 and A7 cysteines. The finding of a further CD4+ T cell epitope in the N-terminal A-chain region of human insulin underscores the importance of this region as a target of CD4+ T cell responses in human T1D. [ABSTRACT FROM AUTHOR]

Changes in the number of Weyl nodes in Weyl semimetals occur through merging processes, usually involving a pair of oppositely charged nodes. More complicated processes involving multiple Weyl nodes are also possible, but they typically require fine tuning and are thus less stable. In this Letter, we study how symmetries affect the allowed merging processes and their stability, focusing on the combination of a twofold rotation and time-reversal (C_{2}T) symmetry. We find that, counterintuitively, processes involving a merging of three nodes are more generic than processes involving only two nodes. Our Letter suggests that multi-Weyl merging may be observed in a large variety of quantum materials, and we discuss SrSi_{2} and bilayer graphene as potential candidates.

Aims: Self-collection of a blood sample for autoantibody testing has potential to facilitate screening for type 1 diabetes risk. We sought to determine the feasibility and acceptability of this approach and the performance of downstream antibody assays. Methods: People living with type 1 diabetes and their family members ( N  = 97) provided paired capillary blood spot and serum samples collected, respectively, by themselves and a health worker. They provided feedback on the ease, convenience, and painfulness of blood spot collection. Islet antibodies were measured in blood spots by antibody detection by agglutination PCR (ADAP) or multiplex enzyme-linked immunoassay (ELISA), and in serum by radioimmunoassay (RIA) or ELISA. Results: Using serum RIA and ELISA to define antibody status, 50 antibody-negative (Ab neg ) and 47 antibody-positive (Ab pos ) participants enrolled, of whom 43 and 47, respectively, returned testable blood spot samples. The majority indicated that self-collection was easier, more convenient, and less painful than formal venesection. The sensitivity and specificity for detection of Ab pos by blood spot were, respectively, 85% and 98% for ADAP and 87% and 100% for multiplex ELISA. The specificities by ADAP for each of the four antigen specificities ranged from 98% to 100% and areas under the receiver operator curve from 0.841 to 0.986. Conclusions: Self-collected blood spot sampling is preferred over venesection by research participants. ADAP and multiplex ELISA are highly specific assays for islet antibodies in blood spots with acceptable performance for use alone or in combination to facilitate screening for type 1 diabetes risk. Clinical Trial Registration number: ACTRN12620000510943.

Regulatory T cells (Tregs) have potential for the treatment of autoimmune diseases and graft rejection. Antigen specificity and functional stability are considered critical for their therapeutic efficacy. In this study, expansion of human Tregs in the presence of porcine PBMCs (xenoantigen-expanded Tregs, Xn-Treg) allowed the selection of a distinct Treg subset, coexpressing the activation/memory surface markers HLA-DR and CD27 with enhanced proportion of FOXP3+Helios+ Tregs. Compared with their unsorted and HLA-DR+CD27+ double-positive (DP) cell-depleted Xn-Treg counterparts, HLA-DR+CD27+ DP-enriched Xn-Tregs expressed upregulated Treg function markers CD95 and ICOS with enhanced suppression of xenogeneic but not polyclonal mixed lymphocyte reaction. They also had less Treg-specific demethylation in the region of FOXP3 and were more resistant to conversion to effector cells under inflammatory conditions. Adoptive transfer of porcine islet recipient NOD/SCID IL2 receptor γ-/- mice with HLA-DR+CD27+ DP-enriched Xn-Tregs in a humanized mouse model inhibited porcine islet graft rejection mediated by 25-fold more human effector cells. The prolonged graft survival was associated with enhanced accumulation of FOXP3+ Tregs and upregulated expression of Treg functional genes, IL10 and cytotoxic T lymphocyte antigen 4, but downregulated expression of effector Th1, Th2, and Th17 cytokine genes, within surviving grafts. Collectively, human HLA-DR+CD27+ DP-enriched Xn-Tregs expressed a specific regulatory signature that enabled identification and isolation of antigen-specific and functionally stable Tregs with potential as a Treg-based therapy.

Type 1 diabetes in children is heralded by a preclinical phase defined by circulating autoantibodies to pancreatic islet antigens. How islet autoimmunity is initiated and then progresses to clinical diabetes remains poorly understood. Only one study has reported gene expression in specific immune cells of children at risk associated with progression to islet autoimmunity. We analyzed gene expression with RNA sequencing in CD4+ and CD8+ T cells, natural killer (NK) cells, and B cells, and chromatin accessibility by assay for transposase-accessible chromatin sequencing (ATAC-seq) in CD4+ T cells, in five genetically at risk children with islet autoantibodies who progressed to diabetes over a median of 3 years ("progressors") compared with five children matched for sex, age, and HLA-DR who had not progressed ("nonprogressors"). In progressors, differentially expressed genes (DEGs) were largely confined to CD4+ T cells and enriched for cytotoxicity-related genes/pathways. Several top-ranked DEGs were validated in a semi-independent cohort of 13 progressors and 11 nonprogressors. Flow cytometry confirmed that progression was associated with expansion of CD4+ cells with a cytotoxic phenotype. By ATAC-seq, progression was associated with reconfiguration of regulatory chromatin regions in CD4+ cells, some linked to differentially expressed cytotoxicity-related genes. Our findings suggest that cytotoxic CD4+ T cells play a role in promoting progression to type 1 diabetes., (© 2022 by the American Diabetes Association.)

Aims: Studies of the gut microbiome have focused on its bacterial composition. We aimed to characterize the gut fungal microbiome (mycobiome) across pregnancy in women with and without type 1 diabetes., Methods: Faecal samples (n = 162) were collected from 70 pregnant women (45 with and 25 without type 1 diabetes) across all trimesters. Fungi were analysed by internal transcribed spacer 1 amplicon sequencing. Markers of intestinal inflammation (faecal calprotectin) and intestinal epithelial integrity (serum intestinal fatty acid binding protein; I-FABP), and serum antibodies to Saccharomyces cerevisiae (ASCA) were measured., Results: Women with type 1 diabetes had decreased fungal alpha diversity by the third trimester, associated with an increased abundance of Saccharomyces cerevisiae that was inversely related to the abundance of the anti-inflammatory butyrate-producing bacterium Faecalibacterium prausnitzii. Women with type 1 diabetes had higher concentrations of calprotectin, I-FABP and ASCA., Conclusions: Women with type 1 diabetes exhibit a shift in the gut mycobiome across pregnancy associated with evidence of gut inflammation and impaired intestinal barrier function. The relevance of these findings to the higher rate of pregnancy complications in type 1 diabetes warrants further study., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

The proximity pattern and radial distribution of chromosome territories within spherical nuclei are random and non-random, respectively. Whether this distribution pattern is conserved in the partitioned or lobed nuclei of polymorphonuclear cells is unclear. Here we use chromosome paint technology to examine the chromosome territories of all 46 chromosomes in hundreds of single human neutrophils - an abundant and famously polymorphonuclear immune cell. By comparing the distribution of chromosomes to randomly shuffled controls and validating with orthogonal chromosome conformation capture technology, we show for the first time that human chromosomes randomly distribute to neutrophil nuclear lobes, while maintaining a non-random radial distribution within these lobes. Furthermore, we demonstrate that chromosome length correlates with three-dimensional volume not only in neutrophils but other human immune cells. This work demonstrates that chromosomes are largely passive passengers during the neutrophil lobing process but are able to subsequently maintain their macro-level organization within lobes., Competing Interests: The authors declare no competing interests., (© 2021 The Author(s).)

Remodelling of chromatin architecture is known to regulate gene expression and has been well characterized in cell lineage development but less so in response to cell perturbation. Activation of T cells, which triggers extensive changes in transcriptional programs, serves as an instructive model to elucidate how changes in chromatin architecture orchestrate gene expression in response to cell perturbation. To characterize coordinate changes at different levels of chromatin architecture, we analyzed chromatin accessibility, chromosome conformation and gene expression in activated human T cells. T cell activation was characterized by widespread changes in chromatin accessibility and interactions that were shared between activated CD4 + and CD8 + T cells, and with the formation of active regulatory regions associated with transcription factors relevant to T cell biology. Chromatin interactions that increased and decreased were coupled, respectively, with up- and down-regulation of corresponding target genes. Furthermore, activation was associated with disruption of long-range chromatin interactions and with partitioning of topologically associating domains (TADs) and remodelling of their TAD boundaries. Newly formed/strengthened TAD boundaries were associated with higher nucleosome occupancy and lower accessibility, linking changes in lower and higher order chromatin architecture. T cell activation exemplifies coordinate multi-level remodelling of chromatin underlying gene transcription.

Loss of heterochromatin has been proposed as a universal mechanism of aging across different species and cell types. However, a comprehensive analysis of hematopoietic changes caused by heterochromatin loss is lacking. Moreover, there is conflict in the literature around the role of the major heterochromatic histone methyltransferase Suv39h1 in the aging process. Here, we use individual and dual deletion of Suv39h1 and Suv39h2 enzymes to examine the causal role of heterochromatin loss in hematopoietic cell development. Loss of neither Suv39h1 nor Suv39h2 individually had any effect on hematopoietic stem cell function or the development of mature lymphoid or myeloid lineages. However, deletion of both enzymes resulted in characteristic changes associated with aging such as reduced hematopoietic stem cell function, thymic involution and decreased lymphoid output with a skewing toward myeloid development, and increased memory T cells at the expense of naive T cells. These cellular changes were accompanied by molecular changes consistent with aging, including alterations in nuclear shape and increased nucleolar size. Together, our results indicate that the hematopoietic system has a remarkable tolerance for major disruptions in chromatin structure and reveal a role for Suv39h2 in depositing sufficient H3K9me3 to protect the entire hematopoietic system from changes associated with premature aging., (© 2020 by The American Society of Hematology.)

CD52, a glycophosphatidylinositol (GPI)-anchored glycoprotein, is released in a soluble form following T cell activation and binds to the Siglec (sialic acid-binding Ig-like lectin)-10 receptor on T cells to suppress their function. We show that binding of CD52-Fc to Siglec-10 and T cell suppression requires the damage-associated molecular pattern (DAMP) protein, high-mobility group box 1 (HMGB1). CD52-Fc bound specifically to the proinflammatory Box B domain of HMGB1, and this in turn promoted binding of the CD52 N-linked glycan, in α-2,3 sialic acid linkage with galactose, to Siglec-10. Suppression of T cell function was blocked by anti-HMGB1 antibody or the antiinflammatory Box A domain of HMGB1. CD52-Fc induced tyrosine phosphorylation of Siglec-10 and was recovered from T cells complexed with HMGB1 and Siglec-10 in association with SHP1 phosphatase and the T cell receptor (TCR). Thus, soluble CD52 exerts a concerted immunosuppressive effect by first sequestering HMGB1 to nullify its proinflammatory Box B, followed by binding to the inhibitory Siglec-10 receptor, triggering recruitment of SHP1 to the intracellular immunoreceptor tyrosine-based inhibitory motif of Siglec-10 and its interaction with the TCR. This mechanism may contribute to immune-inflammatory homeostasis in pathophysiologic states and underscores the potential of soluble CD52 as a therapeutic agent., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)

It has been proposed that interactions between mammalian chromosomes, or transchromosomal interactions (also known as kissing chromosomes), regulate gene expression and cell fate determination. Here we aimed to identify novel transchromosomal interactions in immune cells by high-resolution genome-wide chromosome conformation capture. Although we readily identified stable interactions in cis, and also between centromeres and telomeres on different chromosomes, surprisingly we identified no gene regulatory transchromosomal interactions in either mouse or human cells, including previously described interactions. We suggest that advances in the chromosome conformation capture technique and the unbiased nature of this approach allow more reliable capture of interactions between chromosomes than previous methods. Overall our findings suggest that stable transchromosomal interactions that regulate gene expression are not present in mammalian immune cells and that lineage identity is governed by cis, not trans chromosomal interactions., Competing Interests: The authors have declared that no competing interests exist.

How T cells differentiate in the neonate may critically determine the ability of the infant to cope with infections, respond to vaccines and avert allergies. Previously, we found that naïve cord blood CD4 + T cells differentiated toward an IL-4-expressing phenotype when activated in the presence of TGF-β and monocyte-derived inflammatory cytokines, the latter are more highly secreted by infants who developed food allergy. Here, we show that in the absence of IL-2 or IL-12, naïve cord blood CD8 + T cells have a natural propensity to differentiate into IL-4-producing non-classic T C 2 cells when they are activated alone, or in the presence of TGF-β and/or inflammatory cytokines. Mechanistically, non-classic T C 2 development is associated with decreased expression of IL-2 receptor alpha (CD25) and glycolysis, and increased fatty acid metabolism and caspase-dependent cell death. Consequently, the short chain fatty acid, sodium propionate (NaPo), enhanced IL-4 expression, but exogenous IL-2 or pan-caspase inhibition prevented IL-4 expression. In children with endoscopically and histologically confirmed non-inflammatory bowel disease and non-infectious pediatric idiopathic colitis, the presence of TGF-β, NaPo, and IL-1β or TNF-α promoted T C 2 differentiation in vitro . In vivo , colonic mucosa of children with colitis had significantly increased expression of IL-4 in CD8 + T cells compared with controls. In addition, activated caspase-3 and IL-4 were co-expressed in CD8 + T cells in the colonic mucosa of children with colitis. Thus, in the context of colonic inflammation and limited IL-2 signaling, CD8 + T cells differentiate into non-classic T C 2 that may contribute to the pathology of inflammatory/allergic diseases in children.

Objectives: The side population (SP) contains cells with stem cell/progenitor properties. Previously, we observed that the mouse pancreas SP expanded after pancreatic injury. We aimed to characterize the SP in human pancreas as a potential source of stem cells., Methods: Human organ donor pancreata were fractionated into islets and exocrine tissue, enriched by tissue culture and dispersed into single cells. Cells were phenotyped by flow cytometry, and the SP was defined by efflux of fluorescent dye Hoechst 33342 visualized by ultraviolet excitation. Cells were flow sorted, and their colony-forming potential measured on feeder cells in culture., Results: An SP was identified in islet and exocrine cells from human organ donors: 2 with type 1 diabetes, 3 with type 2 diabetes, and 28 without diabetes. Phenotyping revealed that exocrine SP cells had an epithelial origin, were enriched for carbohydrate antigen 19-9 ductal cells expressing stem cell markers CD133 and CD26, and had greater colony-forming potential than non-SP cells. The exocrine SP was increased in a young adult with type 1 diabetes and ongoing islet autoimmunity., Conclusions: The pancreatic exocrine SP is a potential reservoir of adult stem/progenitor cells, consistent with previous evidence that such cells are duct-derived and express CD133.

MicroRNAs (miRNAs) regulate T cell development and function and the disruption of miRNAs in natural regulatory CD4(+) FOXP3(+) T cells (nTreg) leads to autoimmune disease in mice. To investigate miRNA expression in relation to autoimmune disease risk in humans we sequenced them in purified CD4(+) T cell subsets from individuals at high risk of type 1 diabetes (pre-T1D), as well as other healthy individuals. Differences in miRNA expression patterns were observed between specific T cell subsets and, within subsets, between pre-T1D and healthy individuals. Compared to healthy, naive CD4(+) T cells in pre-T1D displayed 32 differentially expressed miRNAs, potentially a template for altered miRNA expression in effector memory T cells in T1D. Naive nTreg in pre-T1D displayed two differentially expressed miRNAs, Let-7c and miR-15a. In contrast, nTreg activated in vivo displayed a large number of differentially expressed miRNAs, revealing a pro-inflammatory and FOXP3-repressive signature. Differential expression of specific miRNAs was also a signpost to altered T cell function. For example, in pre-T1D, increased expression of miR-26a in nTreg activated in vivo or in vitro was associated with decreased expression of its target, the histone methyltransferase EZH2. Chemical inhibition of EZH2 decreased the number of activated naïve nTreg and their expression of nTreg signature genes FOXP3 and TIGIT. Our findings demonstrate that miRNAs differentially expressed in CD4(+) T cell subsets are markers of risk and T cell dysfunction in T1D., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Food allergy is a major health burden in early childhood. Infants who develop food allergy display a proinflammatory immune profile in cord blood, but how this is related to interleukin-4 (IL-4)/T helper 2 (T(H)2)-type immunity characteristic of allergy is unknown. In a general population-derived birth cohort, we found that in infants who developed food allergy, cord blood displayed a higher monocyte to CD4(+) T cell ratio and a lower proportion of natural regulatory T cell (nT(reg)) in relation to duration of labor. CD14(+) monocytes of food-allergic infants secreted higher amounts of inflammatory cytokines (IL-1β, IL-6, and tumor necrosis factor-α) in response to lipopolysaccharide. In the presence of the mucosal cytokine transforming growth factor-β, these inflammatory cytokines suppressed IL-2 expression by CD4(+) T cells. In the absence of IL-2, inflammatory cytokines decreased the number of activated nT(reg) and diverted the differentiation of both nT(reg) and naïve CD4(+) T cells toward an IL-4-expressing nonclassical TH2 phenotype. These findings provide a mechanistic explanation for susceptibility to food allergy in infants and suggest anti-inflammatory approaches to its prevention., (Copyright © 2016, American Association for the Advancement of Science.)

Aim: DPP-4/CD26 degrades the incretins GLP-1 and GIP. The localization of DPP-4 within the human pancreas is not well documented but is likely to be relevant for understanding incretin function. We aimed to define the cellular localization of DPP-4 in the human pancreas from cadaveric organ donors with and without diabetes., Methods: Pancreas was snap-frozen and immunoreactive DPP-4 detected in cryosections using the APAAP technique. For co-localization studies, pancreas sections were double-stained for DPP-4 and proinsulin or glucagon and scanned by confocal microscopy. Pancreata were digested and cells in islets and in islet-depleted, duct-enriched digests analyzed for expression of DPP-4 and other markers by flow cytometry., Results: DPP-4 was expressed by pancreatic duct and islet cells. In pancreata from donors without diabetes or with type 2 diabetes, DPP-4-positive cells in islets had the same location and morphology as glucagon-positive cells, and the expression of DPP-4 and glucagon overlapped. In donors with type 1 diabetes, the majority of residual cells in islets were DPP-4-positive., Conclusion: In the human pancreas, DPP-4 expression is localized to duct and alpha cells. This finding is consistent with the view that DPP-4 regulates exposure to incretins of duct cells directly and of beta cells indirectly in a paracrine manner., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Differentiation of naïve CD4(+) T cells into effector (Th1, Th2, and Th17) and induced regulatory (iTreg) T cells requires lineage-specifying transcription factors and epigenetic modifications that allow appropriate repression or activation of gene transcription. The epigenetic silencing of cytokine genes is associated with the repressive H3K27 trimethylation mark, mediated by the Ezh2 or Ezh1 methyltransferase components of the polycomb repressive complex 2 (PRC2). Here we show that silencing of the Ifng, Gata3, and Il10 loci in naïve CD4(+) T cells is dependent on Ezh2. Naïve CD4(+) T cells lacking Ezh2 were epigenetically primed for overproduction of IFN-γ in Th2 and iTreg and IL-10 in Th2 cells. In addition, deficiency of Ezh2 accelerated effector Th cell death via death receptor-mediated extrinsic and intrinsic apoptotic pathways, confirmed in vivo for Ezh2-null IFN-γ-producing CD4(+) and CD8(+) T cells responding to Listeria monocytogenes infection. These findings demonstrate the key role of PRC2/Ezh2 in differentiation and survival of peripheral T cells and reveal potential immunotherapeutic targets., (© 2014 by The American Society of Hematology.)

Regulatory T cells (Treg) prevent the emergence of autoimmune disease. Prototypic natural Treg (nTreg) can be reliably identified by demethylation at the Forkhead-box P3 (FOXP3) locus. To explore the methylation landscape of nTreg, we analyzed genome-wide methylation in human naive nTreg (rTreg) and conventional naive CD4(+) T cells (Naive). We detected 2315 differentially methylated cytosine-guanosine dinucleotides (CpGs) between these 2 cell types, many of which clustered into 127 regions of differential methylation (RDMs). Activation changed the methylation status of 466 CpGs and 18 RDMs in Naive but did not alter DNA methylation in rTreg. Gene-set testing of the 127 RDMs showed that promoter methylation and gene expression were reciprocally related. RDMs were enriched for putative FOXP3-binding motifs. Moreover, CpGs within known FOXP3-binding regions in the genome were hypomethylated. In support of the view that methylation limits access of FOXP3 to its DNA targets, we showed that increased expression of the immune suppressive receptor T-cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain (TIGIT), which delineated Treg from activated effector T cells, was associated with hypomethylation and FOXP3 binding at the TIGIT locus. Differential methylation analysis provides insight into previously undefined human Treg signature genes and their mode of regulation.

Insulin-dependent or type 1 diabetes (T1D) is a paradigm for prevention of autoimmune disease: Pancreatic β-cell autoantigens are defined, at-risk individuals can be identified before the onset of symptoms, and autoimmune diabetes is preventable in rodent models. Intervention in asymptomatic individuals before or after the onset of subclinical islet autoimmunity places a premium on safety, a requirement met only by lifestyle-dietary approaches or autoantigen-based vaccination to induce protective immune tolerance. Insulin is the key driver of autoimmune β-cell destruction in the nonobese diabetic (NOD) mouse model of T1D and is an early autoimmune target in children at risk for T1D. In the NOD mouse, mucosal administration of insulin induces regulatory T cells that protect against diabetes. The promise of autoantigen-specific vaccination in humans has yet to be realized, but recent trials of oral and nasal insulin vaccination in at-risk humans provide grounds for cautious optimism.

The homeobox gene Pdx1 is a key regulator of pancreas and foregut development. Loss of Pdx1 expression results in pancreas agenesis and impaired development of the gastro-duodenal domain including Brunner’s glands. We previously demonstrated a key role for Pdx1 in maintaining the integrity and function of insulin-secreting beta cells in the adult pancreas. In the present study, we aimed to determine if expression of Pdx1 is required to maintain the cellular identity of the gastro-duodenal domain in adult mice. Immunohistological studies were performed in a mouse model in which expression of Pdx1 was conditionally repressed with the doxycycline-responsive tetracycline transactivator system. Mice in which Pdx1 was chronically repressed developed hamartomas in the gastro-duodenal domain. These lesions appeared to arise from ectopic foci of anteriorized cells, consistent with a localised anterior homeotic shift. They emerge with the intercalation of tissue between the anteriorized and normal domains and appear strikingly similar to lesions in the colon of mice heterozygous for another Parahox gene, Cdx2. Continuing expression of Pdx1 into adult life is required to maintain regional cellular identity in the adult foregut, specifically at the gastro-duodenal boundary. Loss of Pdx1 expression leads to anterior transformation and intercalary regeneration of ectopic tissue. We propose a model in which the posterior dominance of classical Hox genes is mirrored by the Parahox genes, providing further evidence of the functional conservation of the Parahox genes. These findings may have implications for further understanding the molecular basis of gastro-duodenal metaplasia and gastro-intestinal transformations such as Barrett’s esophagus.

Pancreas stem cells are a potential source of insulin-producing β cells for the therapy of diabetes. In adult tissues the 'side population' (SP) of cells that effluxes the DNA binding dye Hoechst 33342 through ATP-binding cassette transporters has stem cell properties. We hypothesised therefore that the SP would expand in response to β cell injury and give rise to functional β cells. SP cells were flow sorted from dissociated pancreas cells of adult mice, analysed for phenotype and cultured with growth promoting and differentiation factors before analysis for hormone expression and glucose-stimulated insulin secretion. SP cell number and colony forming potential (CFP) increased significantly in models of type diabetes, and after partial pancreatectomy, in the absence of hyperglycaemia. SP cells, ∼1% of total pancreas cells at 1 week of age, were enriched >10-fold for CFP compared to non-SP cells. Freshly isolated SP cells contained no insulin protein or RNA but expressed the homeobox transcription factor Pdx1 required for pancreas development and β cell function. Pdx1, along with surface expression of CD326 (Ep-Cam), was a marker of the colony forming and proliferation potential of SP cells. In serum-free medium with defined factors, SP cells proliferated and differentiated into islet hormone-expressing cells that secreted insulin in response to glucose. Insulin expression was maintained when tissue was transplanted within vascularised chambers into diabetic mice. SP cells in the adult pancreas expand in response to β cell injury and are a source of β cell progenitors with potential for the treatment of diabetes.

Detection of human Ag-specific T cells is limited by sensitivity and blood requirements. As dendritic cells (DCs) can potently stimulate T cells, we hypothesized that their induction in PBMCs in situ could link Ag processing and presentation to Ag-specific T-cell activation. To this end, unfractionated PBMCs (fresh or frozen) or whole blood were incubated for 48 hours with protein or peptide Ag together with different DC-activating agents to rapidly and sequentially induce, pulse, and mature DCs. DC activation was therefore lined up with Ag recognition by neighboring T cells, thus telescoping the sequential steps of T-cell activation. Efficient processing of protein Ags made prior knowledge of epitopes and HLA restrictions dispensable. While reducing stimulation time, manipulation and blood requirements, in situ DC induction specifically amplified Ag-specific T-cell responses (cytokine secretion, proliferation, CD137/CD154 up-regulation, and binding of peptide-HLA multimers). IL-1β, although released by DCs, was also secreted in an Ag-specific fashion, thus providing an indirect biomarker of T-cell responses. These accelerated cocultured DC (acDC) assays offered a sensitive means with which to evaluate T-cell responses to viral and melanoma Ag vaccination, and may therefore find application for immune monitoring in viral, tumor, autoimmune, and transplantation settings.

Steroid hormones induce changes in gene expression by binding to intracellular receptors that then translocate to the nucleus. Steroids have also been shown to rapidly modify cell function by binding to surface membrane receptors. We identified a candidate steroid membrane receptor, the progestin and adipoQ receptor (PAQR) 10, a member of the PAQR family, in a screen for genes differentially expressed in mouse pancreatic beta-cells. PAQR10 gene expression was tissue restricted compared with other PAQRs. In the mouse embryonic pancreas, PAQR10 expression mirrored development of the endocrine lineage, with PAQR10 protein expression confined to endocrine islet-duct structures in the late embryo and neonate. In the adult mouse pancreas, PAQR10 was expressed exclusively in islet cells except for its reappearance in ducts of maternal islets during pregnancy. PAQR10 has a predicted molecular mass of 29 kDa, comprises seven transmembrane domains, and, like other PAQRs, is predicted to have an intracellular N-terminus and an extracellular C-terminus. In silico analysis indicated that three members of the PAQR family, PAQRs 9, 10, and 11, have a candidate mitochondrial localization signal (MLS) at the N-terminus. We showed that PAQR10 has a functional N-terminal MLS and that the native protein localizes to mitochondria. PAQR10 is structurally related to some bacterial hemolysins, pore-forming virulence factors that target mitochondria and regulate apoptosis. We propose that PAQR10 may act at the level of the mitochondrion to regulate pancreatic endocrine cell development/survival.

Increased plasma levels of several acute phase proteins, such as C-reactive protein (CRP), have been documented among different patients with chronic renal failure (CRF). The aim of the present study was to determine whether pentraxin-3 (PTX3) is a reliable marker of inflammation in CRF. Plasma samples and monocytes were taken from 43 patients before and after undergoing haemodialysis (HD), from 45 uraemic patients (UR) without HD treatment and from 25 healthy controls. Plasma and monocyte samples were analyzed by ELISA for levels of PTX3, tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta) and interleukin-6 (IL-6); all of these protein levels were higher in CRF patients with respect to the controls. After HD, plasma PTX3 and cytokine levels increased. Inter- and intra-individual variations in CRP were observed in HD patients, while PTX3 plasma levels were stable. Release of PTX3, TNF-alpha, IL-1beta and IL-6 by unstimulated monocytes from patients, before and after HD, was higher with respect to UR patients and controls. After lipopolysaccharide stimulation, all values were higher in patients before HD than those in UR patients, but lower when compared to those in the controls. In contrast, no changes were observed after HD. A significant correlation among plasma PTX3 versus fibrinogen, TNF-alpha and IL-1beta was observed in HD and UR patients. Collectively, these data suggest that PTX3 protein may represent an additional and stable marker of inflammation in CRF.

The homeodomain transcription factor Pdx1 is essential for pancreas development. To investigate the role of Pdx1 in the adult pancreas, we employed a mouse model in which transcription of Pdx1 could be reversibly repressed by administration of doxycycline. Repression of Pdx1 in adult mice impaired expression of insulin and glucagon, leading to diabetes within 14 days. Pdx1 repression was associated with increased cell proliferation predominantly in the exocrine pancreas and upregulation of genes implicated in pancreas regeneration. Following withdrawal of doxycycline and derepression of Pdx1, normoglycemia was restored within 28 days; during this period, Pdx1(+)/Ins(+) and Pdx(+)/Ins(-) cells were observed in association with the duct epithelia. These findings confirm that Pdx1 is required for beta-cell function in the adult pancreas and indicate that in the absence of Pdx1 expression, a regenerative program is initiated with the potential for Pdx1-dependent beta-cell neogenesis.

Mutations in the triple PDZ domain-containing protein harmonin have been identified as the cause of Usher deafness syndrome type 1C. Independently, we identified harmonin in a screen for genes expressed in pancreatic beta cells. Using a yeast two-hybrid assay, we show that the first PDZ domain of harmonin interacts with a novel protein, designated harp for harmonin-interacting, ankyrin repeat-containing protein. This interaction was confirmed in an over-expression system and in mammalian cells, and shown to be mediated by the three C-terminal amino acids of harp. Harp is expressed in many of the same epithelia as harmonin and co-localization of native harp and harmonin was demonstrated by confocal microscopy in pancreatic duct epithelium and in a pancreatic beta-cell line. Harp, predicted molecular mass 48 kDa, has a domain structure which includes three ankyrin repeats and a sterile alpha motif. Human harp maps to chromosome 16, and its mouse homologue to chromosome 7. Sequences with similarity to harp include the sans gene, mutations of which are responsible for deafness in the Jackson shaker 2 (js) mutant mouse and in human Usher syndrome type 1G. The functional domain structures of harp and harmonin, their interaction under native conditions and their co-localization suggest they constitute a scaffolding complex to facilitate signal transduction in epithelia.

Tissue function is regulated by the extracellular microenvironment including cell basement membranes, in which laminins are a major component. Previously, we found that laminin-1 promotes differentiation and survival of pancreatic islet cells. Here we characterize the expression pattern of laminins and their integrin receptors in adult pancreas. Although they are expressed in the basement membrane of acinar cells and duct epithelium, no laminin chains examined were detected extracellularly in the pancreatic islets. In contrast to laminin beta(1)- and gamma(1)-chains, the alpha(1)-chain, unique to laminin-1, was not detected. Laminin-10 (alpha(5)beta(1)gamma(1)) was expressed in acinar tissue, whereas laminins-2 (alpha(2)beta(1)gamma(1)) and -10 were expressed in the blood vessels. The laminin connector molecule, nidogen-1, had a distribution similar to that of laminin beta(1) and gamma(1), whereas fibulin-1 and -2, which compete with nidogen-1, were mostly confined to blood vessels. Integrin subunits alpha(6) and alpha(3) were detected in acinar cells and duct epithelial cells, but alpha(6) was absent in islet cells. Integrin alpha(6)beta(4) was detected only in duct cells, alpha(6)beta(1) in both acinar and ductal cells, and alpha(3)beta(1) in acinar, duct, and islet cells. These findings are a basis for further investigation of the role of extracellular matrix molecules and their receptors in pancreas function.

Cytokine receptors from the IL-6 receptor family are comprised of ligand specific alpha chains and a common signalling chain, gp-130, which is also required for high affinity binding. A cDNA library generated from the beta-TC3 SV40 T-antigen transformed insulinoma cell line was screened for members of this receptor family potentially relevant to both beta cell development and autoimmunity. Degenerate oligonucleotide primers to a consensus region of these receptors were used and the IL-11 receptor alpha chain was identified. Despite confirmation of IL-11 receptor mRNA expression, iodinated bioactive IL-11 did not bind specifically to beta-TC3 cells and gp-130-dependent cytokines did not elicit signalling events in beta cell lines. This was explained by absence of gp-130 protein or mRNA in the beta cell lines tested and in primary islets. We conclude from these results that the previously recognised effects of IL-6 family member cytokines on pancreatic islets must be indirect via other non-beta cells within the islet, rather than due to direct effects on beta cells themselves.

We have cloned a member of the STE20/SPS1 protein kinase family from a transformed rat pancreatic beta cell line. SPAK (STE20/SPS1-related, proline alanine-rich kinase) belongs to the SPS1 subfamily of STE20 kinases and is highly conserved between species. SPAK is expressed ubiquitously, although preferentially in brain and pancreas. Biochemical characterization of SPAK catalytic activity demonstrates that is a serine/threonine kinase that can phosphorylate itself and an exogenous substrate in vitro. SPAK is immunoprecipitated from transfected mammalian cells as a complex with another, as yet uncharacterized, serine/threonine kinase which is capable of phosphorylating catalytically-inactive SPAK and myelin basic protein in an in vitro kinase assay. SPAK specifically activates the p38 pathway in cotransfection assays. Like MST1 and MST2, SPAK contains a putative caspase cleavage site at the junction of the catalytic domain and the C-terminal region. Full-length SPAK is expressed in the cytoplasm in transfected cells, while a mutant corresponding to caspase-cleaved SPAK is expressed predominantly in the nucleus. The similarity of SPAK to other SPS1 family members, its ability to activate the p38 pathway, in addition to its putative caspase cleavage site, provide evidence that SPAK may act as a novel mediator of stress-activated signals. Oncogene (2000) 19, 4290 - 4297

Purpose: To report the use of an aortic endograft to treat a ruptured false aneurysm at the anastomosis of an aortofemoral bypass graft., Methods and Results: A 68-year-old man with a 30-year-old aorto-right femoral bypass and multiple comorbidities was admitted to the hospital complaining of acute abdominal pain. Imaging identified a 60-mm ruptured aortic false aneurysm with associated retroperitoneal hematoma, a 9-cm right femoral false aneurysm, and a calcified 23-mm left common iliac aneurysm. Two slightly overlapping Vanguard straight stent-grafts were implanted in the aorta and left common iliac artery in an emergency procedure owing to the patient's high surgical risk. The anastomotic false aneurysm and the bypass were excluded. A left-to-right femorofemoral bypass was performed to re-establish flow to the right femoral artery with ligation of the external iliac artery. The patient recovered uneventfully. He remained well with a successful repair until his death of a myocardial infarction 6 months after the procedure., Conclusions: Endovascular grafting can be used successfully for the urgent treatment of aortic false aneurysm rupture.

Events controlling differentiation to insulin-secreting beta-cells in the pancreas are not well understood, although beta-cells are thought to arise from pluripotent ductal precursor cells. To search for signaling proteins that might be involved in beta-cell maturation, we analyzed protein kinase expression in two developmentally and functionally distinct pancreatic beta-cell lines, RIN-5AH and RIN-A12, by reverse transcriptase polymerase chain reaction. A number of tyrosine and serine/threonine kinases were identified in both lines. One protein kinase, mixed lineage kinase-1 (MLK-1), was expressed at both the RNA and protein levels in RIN-5AH cells, which display an immature beta-cell phenotype, but was not detected in the more mature RIN-A12 cells. Furthermore, levels of MLK-1 mRNA and protein were increased after brief stimulation of RIN-5AH cells with either the differentiation inducer, sodium butyrate, or with serum after serum starvation. These increases in expression were independent of phenotypic markers such as insulin secretion or surface expression of major histocompatibility class I- and A2B5-reactive ganglioside. In addition, increases in MLK-1 expression in the stimulated RIN-5AH cells were accompanied by phosphorylation of MLK-1 on serine but not tyrosine. Antisense oligonucleotides to two distinct regions of MLK-1 caused RIN-5AH cells, but not RIN-A12 cells, to adopt a highly undifferentiated morphology, with a reduction in DNA synthesis and MLK-1 protein levels and elevated glucagon mRNA levels, but with no effect on insulin mRNA. In an immunohistochemical survey of embryonic mouse tissues, we found that temporal expression of MLK-1 was regulated in a tissue-specific manner. In the embryonic pancreas, MLK-1 expression was evident in ductal cells from day 13 to 16 but was not detected in late stage gestation or neonatal pancreas. These data suggest that MLK-1 is regulated in immature pancreatic beta-cells and their ductal precursors at the level of functional maturity and may therefore play a role in beta-cell development.

To obtain a better understanding of the immune response to Epstein-Barr virus (EBV), we measured the cytokines tumour necrosis factor (TNF)-alpha/beta, interleukin-2 (IL-2), interferon-gamma (IFN-gamma), IL-6 and granulocyte-macrophage colony-stimulating factor (GM-CSF) in the conditioned medium of peripheral blood mononuclear cells from 10 healthy adults before and at 48 h and at 1, 2, 3 and 4 weeks following infection in vitro with EBV. Cultures were examined for regression of outgrowths of nascent virus-transformed B cells, and populations of cells in the cultures were analysed by flow cytometry. TNF-alpha/beta was not detected in infected or non-infected cultures. In infected cultures assayed at the nominated times, the highest levels of IL-2 were detected at 48 hours, IFN-gamma at 1 week, IL-6 at 2 weeks and GM-CSF between 2 and 4 weeks. IL-6 and GM-CSF, but not IL-2 or IFN-gamma, were detected in non-infected cultures but at lower levels than in infected cultures. Nine of the 10 healthy adults showed regression of outgrowths of virus-transformed B cells and, of these, seven had antibodies to the EBV capsid antigen (VCA). Strong regression was associated with sequential increases in IL-2, IFN-gamma, and low levels of IL-6 and GM-CSF. Absent or weak regression was associated with an undetectable level of IL-2, a low level of IFN-gamma, high levels of IL-6 and GM-CSF and an increased frequency of cells bearing the phenotype CD20 and HLA-DR in the final weeks of culture.(ABSTRACT TRUNCATED AT 250 WORDS)

Between May 1978 and March 1990, 700 patients were operated on with direct coronary surgery without extracorporeal circulation (ECC): 529 (76 percent) were male and 171 (24 percent) were female. The average age was 64 years (range, 35 to 86 years), 454 (65 percent) had unstable angina, 163 (23 percent) had stable angina, 51 (7 percent) had postmyocardial infarction angina, and 32 (5 percent) had acute myocardial infarction at the moment of the operation. In this series of patients, all branches of the coronary arteries were bypassed; the mammary artery was used in 40 percent of the cases, the average bypass per patient was 2.2 (range, 1 to 5), and 26 percent had associated disease of high risk to undergo ECC. The morbidity was 4 percent and the mortality for this series of patients was 1 percent; the probability of survival at seven years was 90 percent. This experience shows us that this surgery is an alternative in the treatment of coronary disease, especially for aged patients with associated disease, in some cases of acute transmural infarction, and also for patients who need coronary angioplasty. Also, it can improve the relation cost/benefit in coronary surgery.

We describe a new surgical technique for the treatment of fibrosed interventricular septum, with or without left ventricular aneurysm. It is designed for patients whose ventriculograms revealed important septal dysfunction. Eleven patients ranging from 37 to 66 years of age were operated upon between 1984 and 1988. The left ventricle was opened through the aneurysm or the anterior wall when only anterior septal fibrosis was present. In patients with large aneurysms, two purse-string sutures were placed on the inside surface of the intact ventricle, around its limits, for approximation. The fibrosed septum was thus excluded from the new ventricular cavity. A patch was placed between the fibrosed and the healthy septum, reaching to the ventricular wall, all around the transitional edge. Both, ventricular geometry and function were improved. All patients were asymptomatic after one year follow-up.

Sera from 120 patients with suspected autoimmune rheumatic disease and antinuclear antibodies of anti-SS-B/La specificity were examined by Western blotting for reactivity with the SS-B/La polypeptide of HeLa cells and recombinant SS-B/La derived from a 1.4 kilobase (kb) cDNA encoding approximately 90% of the SS-B/La molecule. All sera reacted with the HeLa cell and the recombinant SS-B/La. One hundred and fourteen (95%) reacted with a set of three Staph. aureus V8 protease-resistant peptides of Mr 30,000, 29,00 and 28,000 from a methionine-rich region of HeLa cell SS-B/La designated the X domain, and 98 (82%) reacted with another set of two protease-resistant peptides of Mr 24,000 and 23,000 from a phosphorylated region of HeLa cell La designated the Y domain. One reacted weakly with the Y domain only. All sera that reacted with X and Y reacted more strongly with X, suggesting that X was the major epitope. Antibodies affinity purified from the X domain reacted strongly with the X peptides but not with the Y peptides and conversely, antibodies affinity purified from the Y domain reacted with the Y peptides but not with the X peptides. Both antibodies reacted with a fusion protein comprising 102 amino acids at the carboxyl terminus of the SS-B/La molecule. This protein contained no methionine, demonstrating that methionines were not involved in the antibody-binding site. Over 80% of patients whose only criteria for selection was the presence of anti-SS-B/La had the clinical, histologic, serologic and phenotypic features of Sjögren's syndrome whilst the remaining 20% had at least two of the features.(ABSTRACT TRUNCATED AT 250 WORDS)

Human recombinant La nucleoprotein was purified from cultures of Escherichia coli containing a vector with a 1.4 kilobase cDNA encoding La; the nucleoprotein was used to test for antinuclear antibodies (ANA) to La. Serum samples from 260 patients with autoimmune diseases associated with ANA and 100 healthy subjects were tested by an enzyme-linked immunosorbent assay (ELISA). Samples from 47 (94%) of 50 patients with primary Sjögren's syndrome and 1 (7%) of 14 patients with secondary Sjögren's syndrome reacted with the recombinant La. No reactivity was demonstrated in 196 patients with other ANA-associated autoimmune diseases or in 100 healthy subjects. The study confirms the high correlation between ANA, anti-La, and primary Sjögren's syndrome and shows how gene cloning can provide large quantities of human autoantigens for use in highly specific and sensitive diagnostic assays.