Siglec-10 expression is up-regulated in activated human CD4+ T cells.

Most sialic acid-binding immunoglobulin-like lectins (Siglecs) suppress immune cell function but are expressed at low levels on human T cells. We found that soluble CD52 inhibited T cell signalling by ligating Siglec-10, but the presence of Siglec-10 on human T cells has been questioned. To address this concern, we examined the expression of Siglec-10 at the RNA and protein level in human CD4+ T cells. Analysis by RNAseq, qPCR and flow cytometry demonstrated that, in contrast to other Siglecs, after activation of CD4+ T cells Siglec-10 was selectively upregulated in a subset of cells also high for CD52 expression. This observation is consistent with a homeostatic role for Siglec-10 in human CD4+ T cells.

Interferon-gamma released from omental adipose tissue of insulin-resistant humans alters adipocyte phenotype and impairs response to insulin and adiponectin release.

BACKGROUND: Inflammatory factors derived from adipose tissue have been implicated in mediating insulin resistance in obesity. We sought to identify these using explanted human adipose tissue exposed to innate and adaptive immune stimuli. METHODS: Subcutaneous and omental adipose tissue from obese, insulin-resistant donors was cultured in the presence of macrophage and T-cell stimuli, and the conditioned medium tested for its ability to inhibit insulin-stimulated glucose uptake into human Simpson-Golabi-Behmel Syndrome (SGBS) adipocytes. The nature of the inhibitory factor in conditioned medium was characterized physicochemically, inferred by gene microarray analysis and confirmed by antibody neutralization. RESULTS: Conditioned medium from omental adipose tissue exposed to a combination of macrophage- and T-cell stimuli inhibited insulin action and adiponectin secretion in SGBS adipocytes. This effect was associated with a pronounced change in adipocyte morphology, characterized by a decreased number of lipid droplets of increased size. The bioactivity of conditioned medium was abolished by trypsin treatment and had a molecular weight of 46 kDa by gel filtration. SGBS adipocytes exposed to a bioactive medium expressed multiple gene transcripts regulated by interferon-gamma (IFN-γ). Recombinant human IFN-γ recapitulated the effects of the bioactive medium and neutralizing antibody against IFN-γ but not other candidate factors abrogated medium bioactivity. CONCLUSIONS: IFN-γ released from inflamed omental adipose tissue may contribute to the metabolic abnormalities seen in human obesity.

A fatal case of transplantation-mediated alloimmune thrombocytopenia following liver transplantation.

OBJECTIVE AND IMPORTANCE: Transplantation-mediated alloimmune thrombocytopenia (TMAT) occurs when leukocytes transferred in a donor organ from a patient with immune thrombocytopenia (ITP), mount a response against recipient platelets. We present the first fatal case of TMAT following liver transplantation and review its aetiology and treatment. CLINICAL PRESENTATION: The liver donor had ITP and died from an intracranial haemorrhage. The recipient platelet count fell to 2 × 109/l on post-operative day 2. Treatment refractory thrombocytopenia resulted in pulmonary haemorrhage and death. TMAT did not occur in a kidney recipient from the same ITP donor. INTERVENTION: Extramedullary haematopoiesis was identified in the donor liver biopsy. Antibodies against platelet GPIb/IX were demonstrated in both donor and recipient. The thrombocytopenia was refractory to platelet transfusions, intravenous immunoglobulin, methylprednisolone, rituximab, romiplostim, plasmapheresis, vincristine and splenic artery embolization. On review of the literature, severe thrombocytopenia (<10 × 109/l) has started within 3 days of transplantation in all reported TMAT cases. Serious non-fatal bleeding was observed in 3/5 previously reported cases. The optimal treatment is unclear. TMAT should resolve as donor lymphocytes are eliminated but re-transplantation may be required in severe refractory cases. TMAT has been reported in recipients of a liver but not kidney or heart transplant from ITP donors, probably because of the greater burden of co-transplanted lymphoid tissue. CONCLUSION: Before using the liver of an ITP donor, the recipient's fully informed consent is required. However, the risk of TMAT from an ITP donor is currently unknown and systematic review of donor registries is needed.

GM3 ganglioside and phosphatidylethanolamine-containing lipids are adipose tissue markers of insulin resistance in obese women.

AIMS: The association between central obesity and insulin resistance reflects the properties of visceral adipose tissue. Our aim was to gain further insight into this association by analysing the lipid composition of subcutaneous and omental adipose tissue in obese women with and without insulin resistance. METHODS: Subcutaneous and omental adipose tissue and serum were obtained from 29 obese non-diabetic women, 13 of whom were hyperinsulinemic. Histology, lipid and gene profiling were performed. RESULTS: In omental adipose tissue of obese, insulin-resistant women, adipocyte hypertrophy and macrophage infiltration were accompanied by an increase in GM3 ganglioside and its synthesis enzyme ST3GAL5; in addition, phosphatidylethanolamine (PE) lipids were increased and their degradation enzyme, phosphatidylethanolamine methyl transferase (PEMT), decreased. ST3GAL5 was expressed predominantly in adipose stromovascular cells and PEMT in adipocytes. Insulin resistance was also associated with an increase in PE lipids in serum. INTERPRETATION: The relevance of these findings to insulin resistance in humans is supported by published mouse studies, in which adipocyte GM3 ganglioside, increased by the inflammatory cytokine tumour necrosis factor-α, impaired insulin action and PEMT was required for adipocyte lipid storage. Thus in visceral adipose tissue of obese humans, an increase in GM3 ganglioside secondary to inflammation may contribute to insulin resistance and a decrease in PEMT may be a compensatory response to adipocyte hypertrophy.

The Great Pretender: early syphilis mimicking acute sclerosing cholangitis.

A 36-year old man with known HIV infection presented to an outpatient genitourinary service with jaundice, rash and sore throat. Investigations revealed marked biochemical abnormalities, including alkaline phosphatase and alanine transaminase >10 times the upper limit of normal. Liver ultrasound was normal, but stricturing and beading of the intrahepatic biliary tree was seen on magnetic resonance cholangiopancreatography (MRCP), similar to changes associated with sclerosing cholangitis. Serological syphilis antibodies were detected with a positive immunoglobulin M (IgM) and rapid plasma reagin of 1:128, in keeping with early infection. Liver biopsy showed large bile duct obstruction with portal oedema, bilirubinostasis and neutrophil polymorph infiltration around proliferating ductules; specific stains for spirochaetes were negative. Symptoms and biochemical markers improved rapidly after treatment for secondary syphilis with oral steroids and intramuscular benzathine penicillin. A repeat MRCP 18 months post syphilis treatment showed resolution. This case illustrates syphilis presenting as acute sclerosing cholangitis.

Maturity-onset diabetes of the young type 5 in a family with diabetes and mild kidney disease diagnosed by whole exome sequencing.

Exome sequencing is being increasingly used to identify disease-associated gene mutations. We used whole exome sequencing to determine the genetic basis of a syndrome of diabetes and renal disease affecting a mother and her son. We identified a mutation in the hepatocyte nuclear factor 1-b (HNF1B) gene that encoded a methionine to valine amino acid change (M160V) in the HNF1B protein. This leads us to the previously unappreciated diagnosis of maturity-onset diabetes of the young type 5 and provided a basis for genetic counselling of other family members.

Higher body mass index in adults at diagnosis of the slowly progressive form of type 1 diabetes mellitus is associated with lower risk HLA genes.

We hypothesised that higher body weight, a proposed risk factor for type 1 diabetes mellitus, would be associated with increased penetrance of lower risk genes. In adults at diagnosis of the slowly progressive form of type 1 diabetes mellitus we found that higher body mass index was associated with the absence of the highest risk HLA genes.

Ex-vivo analysis of human natural killer T cells demonstrates heterogeneity between tissues and within established CD4(+) and CD4(-) subsets.

Our understanding of human type 1 natural killer T (NKT) cells has been heavily dependent on studies of cells from peripheral blood. These have identified two functionally distinct subsets defined by expression of CD4, although it is widely believed that this underestimates the true number of subsets. Two recent studies supporting this view have provided more detail about diversity of the human NKT cells, but relied on analysis of NKT cells from human blood that had been expanded in vitro prior to analysis. In this study we extend those findings by assessing the heterogeneity of CD4(+) and CD4(-) human NKT cell subsets from peripheral blood, cord blood, thymus and spleen without prior expansion ex vivo, and identifying for the first time cytokines expressed by human NKT cells from spleen and thymus. Our comparative analysis reveals highly heterogeneous expression of surface antigens by CD4(+) and CD4(-) NKT cell subsets and identifies several antigens whose differential expression correlates with the cytokine response. Collectively, our findings reveal that the common classification of NKT cells into CD4(+) and CD4(-) subsets fails to reflect the diversity of this lineage, and that more studies are needed to establish the functional significance of the antigen expression patterns and tissue residency of human NKT cells.

Diffusion tensor imaging of acute inflammatory lesion evolution in multiple sclerosis.

The initiating events in multiple sclerosis (MS) plaque formation are poorly understood. Retrospective analysis of serial imaging data can improve the understanding of tissue changes characterising acute MS lesion evolution. This study aimed to assess lesion evolution using diffusion tensor imaging data from serially acquired scans from 22 patients with MS. Mean diffusivity (MD) and fractional anisotropy (FA) were measured from 13 suitable plaques from five patients and carefully matched regions of contralateral normal-appearing white matter. Measurement times were on average: 5 months and 1 month prior to, during, and 1 month and 2 months post gadolinium-enhancement. A significant increase in MD (7.25%) but no change in FA was observed in white matter areas that exhibited enhancement 5 months later. The pre-lesional MD increase was significantly correlated with the MD increase 2 months subsequent to enhancement (R=0.73, p=0.04) but not to the MD increase during enhancement (R=0.11). These results suggest that MD is sensitive to tissue changes that precede blood-brain barrier (BBB) breakdown by at least 5 months and that MD assessments may predict injury following BBB restoration.

Fulminant type 1 diabetes in Australia in the absence of humoral and cellular immune responses to pancreatic islet autoantigens.

We document the first reported case of fulminant diabetes, without evidence of pancreatic islet autoimmunity, in Australia in a woman of Chinese Vietnamese background. Fulminant diabetes occurs primarily in Asia, particularly in Japan, but with widespread population migration clinicians need to be aware of this form of diabetes.

INS(GFP/w) human embryonic stem cells facilitate isolation of in vitro derived insulin-producing cells.

AIMS/HYPOTHESIS: We aimed to generate human embryonic stem cell (hESC) reporter lines that would facilitate the characterisation of insulin-producing (INS⁺) cells derived in vitro. METHODS: Homologous recombination was used to insert sequences encoding green fluorescent protein (GFP) into the INS locus, to create reporter cell lines enabling the prospective isolation of viable INS⁺ cells. RESULTS: Differentiation of INS(GFP/w) hESCs using published protocols demonstrated that all GFP⁺ cells co-produced insulin, confirming the fidelity of the reporter gene. INS-GFP⁺ cells often co-produced glucagon and somatostatin, confirming conclusions from previous studies that early hESC-derived insulin-producing cells were polyhormonal. INS(GFP/w) hESCs were used to develop a 96-well format spin embryoid body (EB) differentiation protocol that used the recombinant protein-based, fully defined medium, APEL. Like INS-GFP⁺ cells generated with other methods, those derived using the spin EB protocol expressed a suite of pancreatic-related transcription factor genes including ISL1, PAX6 and NKX2.2. However, in contrast with previous methods, the spin EB protocol yielded INS-GFP⁺ cells that also co-expressed the beta cell transcription factor gene, NKX6.1, and comprised a substantial proportion of monohormonal INS⁺ cells. CONCLUSIONS/INTERPRETATION: INS(GFP/w) hESCs are a valuable tool for investigating the nature of early INS⁺ progenitors in beta cell ontogeny and will facilitate the development of novel protocols for generating INS⁺ cells from differentiating hESCs.

A randomized trial of high-dose vitamin D2 in relapsing-remitting multiple sclerosis.

OBJECTIVE: Higher latitude, lower ultraviolet exposure, and lower serum 25-hydroxyvitamin D (25OHD) correlate with higher multiple sclerosis (MS) prevalence, relapse rate, and mortality. We therefore evaluated the effects of high-dose vitamin D2 (D2) in MS. METHODS: Adults with clinically active relapsing-remitting MS (RRMS) were randomized to 6 months' double-blind placebo-controlled high-dose vitamin D2, 6,000 IU capsules, dose adjusted empirically aiming for a serum 25OHD 130-175 nM. All received daily low-dose (1,000 IU) D2 to prevent deficiency. Brain MRIs were performed at baseline, 4, 5, and 6 months. Primary endpoints were the cumulative number of new gadolinium-enhancing lesions and change in the total volume of T2 lesions. Secondary endpoints were Expanded Disability Status Scale (EDSS) score and relapses. RESULTS: Twenty-three people were randomized, of whom 19 were on established interferon or glatiramer acetate (Copaxone) treatment. Median 25OHD rose from 54 to 69 nM (low-dose D2) vs 59 to 120 nM (high-dose D2) (p = 0.002). No significant treatment differences were detected in the primary MRI endpoints. Exit EDSS, after adjustment for entry EDSS, was higher following high-dose D2 than following low-dose D2 (p = 0.05). There were 4 relapses with high-dose D2 vs none with low-dose D2 (p = 0.04). CONCLUSION: We did not find a therapeutic advantage in RRMS for high-dose D2 over low-dose D2 supplementation. CLASSIFICATION OF EVIDENCE: This study provides Class I evidence that high-dose vitamin D2 (targeting 25OHD 130-175 nM), compared to low-dose supplementation (1,000 IU/d), was not effective in reducing MRI lesions in patients with RRMS.

The A-chain of insulin is a hot-spot for CD4+ T cell epitopes in human type 1 diabetes.

Type 1 diabetes (T1D) is caused by T cell-mediated destruction of the pancreatic insulin-producing beta 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.

Immune characterization of an individual with an exceptionally high natural killer T cell frequency and her immediate family.

Natural killer T cells (NKT) are a regulatory subset of T lymphocytes whose frequency in peripheral blood is highly variable within the human population. Lower than normal NKT frequencies are associated with increased predisposition to a number of diseases, including type 1 diabetes and some forms of cancer, raising the possibility that an increased frequency may be protective. However, there is little or no understanding of how high NKT frequencies arise or, most importantly, whether the potential exists to boost and maintain NKT levels for therapeutic advantage. Here, we provide a detailed functional and phenotypic characterization of the NKT compartment of a human donor with NKT levels approximately 50 times greater than normal, including an analysis of NKT in her immediate family members. The study focuses upon the characteristics of this donor and her family, but demonstrates more broadly that the size and flexibility of the NKT niche is far greater than envisioned previously. This has important implications for understanding how the human NKT compartment is regulated, and supports the concept that the human NKT compartment might be expanded successfully for therapeutic benefit.

Vascularized tissue-engineered chambers promote survival and function of transplanted islets and improve glycemic control.

We have developed a chamber model of islet engraftment that optimizes islet survival by rapidly restoring islet-extracellular matrix relationships and vascularization. Our aim was to assess the ability of syngeneic adult islets seeded into blood vessel-containing chambers to correct streptozotocin-induced diabetes in mice. Approximately 350 syngeneic islets suspended in Matrigel extracellular matrix were inserted into chambers based on either the splenic or groin (epigastric) vascular beds, or, in the standard approach, injected under the renal capsule. Blood glucose was monitored weekly for 7 weeks, and an intraperitoneal glucose tolerance test performed at 6 weeks in the presence of the islet grafts. Relative to untreated diabetic animals, glycemic control significantly improved in all islet transplant groups, strongly correlating with islet counts in the graft (P<0.01), and with best results in the splenic chamber group. Glycemic control deteriorated after chambers were surgically removed at week 8. Immunohistochemistry revealed islets with abundant insulin content in grafts from all groups, but with significantly more islets in splenic chamber grafts than the other treatment groups (P<0.05). It is concluded that hyperglycemia in experimental type 1 diabetes can be effectively treated by islets seeded into a vascularized chamber functioning as a "pancreatic organoid."

Latent autoimmune diabetes in adults (LADA) should be less latent.

'Latent autoimmune diabetes in adults' (LADA) is the term coined to describe adults who have a slowly progressive form of autoimmune or type 1 diabetes that can be treated initially without insulin injections. The diagnosis of LADA is currently based on three clinical criteria: (1) adult age at onset of diabetes; (2) the presence of circulating islet autoantibodies, which distinguishes LADA from type 2 diabetes; and (3) insulin independence at diagnosis, which distinguishes LADA from classic type 1 diabetes. The prevalence of LADA in adults presenting with non-insulin-requiring diabetes is approximately 10%. Recognition of LADA expands the concept and prevalence of autoimmune diabetes, but LADA remains poorly understood at both a clinical and research level. In this perspective, we review the nomenclature, diagnostic criteria, genetics, pathology and therapy of LADA, to arrive at recommendations that might advance knowledge and management of this form of diabetes.

Insulin resistance is a risk factor for progression to type 1 diabetes.

AIMS/HYPOTHESIS: Glucose homeostasis is determined by an interplay between insulin secretion and insulin action. In type 1 diabetes, autoimmune destruction of pancreatic beta cells leads to impaired insulin secretion. However, the contribution of impaired insulin action (insulin resistance) to the development of type 1 diabetes has received little attention. We investigated whether insulin resistance was a risk factor for progression to type 1 diabetes. METHODS: Islet-antibody-positive first-degree relatives of type 1 diabetes probands were followed for 4.0 years (median). Insulin secretion was measured as first-phase insulin response (FPIR) to intravenous glucose. Insulin resistance was estimated by homeostasis model assessment of insulin resistance (HOMA-R). We compared subjects who progressed (n=43) and subjects who did not progress (n=61) to diabetes, including 21 pairs matched for age, sex, islet antibodies and FPIR. RESULTS: Progressors had higher insulin resistance relative to insulin secretion at baseline (median HOMA-R : FPIR 0.033 vs 0.013, p<0.0001). According to Cox proportional hazards analysis, islet antibody number, FPIR, fasting plasma glucose, fasting serum insulin, HOMA-R and log(HOMA-R : FPIR) were each predictive of progression to diabetes. However, log(HOMA-R : FPIR) (hazard ratio 2.57 per doubling, p<0.001) was the only metabolic variable independently associated with progression. In the matched comparison, progressors had higher fasting glucose, fasting insulin, HOMA-R and HOMA-R : FPIR, both at baseline and during the follow-up pre-clinical phase. CONCLUSIONS/INTERPRETATION: Relatives positive for islet antibodies who progress most rapidly to diabetes have a subtle disturbance of insulin-glucose homeostasis years before the onset of symptoms, distinguished by greater insulin resistance for their level of insulin secretion. Taking steps to reduce this insulin resistance could therefore delay the development of type 1 diabetes.

Fas ligand down-regulates cytokine-induced Fas receptor expression on insulinoma (NIT-1), but not islet cells, from autoimmune nonobese diabetic mice.

In the pathogenesis of autoimmune type 1 diabetes, the apoptosis receptor Fas appears de novo on the surface of insulin-producing beta-cells. Fas expression is thought to be induced by proinflammatory cytokines, such as IL-1beta, interferon-gamma (IFNgamma), and TNFalpha, released by islet-infiltrating mononuclear cells. To determine whether beta-cells can modulate their sensitivity to apoptosis at the level of Fas, we investigated the effect of Fas ligand (FasL) on surface expression of Fas in NIT-1 insulinoma cells from nonobese diabetic (NOD) mice prone to autoimmune diabetes and islet cells from NOD and nonautoimmune BALB/c mice. In NIT-1 insulinoma cells, Fas expression induced by the cytokine combination IL-1beta and IFNgamma was reduced in the presence of FasL, whereas in islet cells Fas expression was unaffected by FasL. The effect of FasL on NIT-1 cells was evident during and after the induction of Fas expression by IL-1beta and IFNgamma. Thus, FasL down-regulates cytokine-induced Fas expression in NOD mouse-derived NIT-1 cells, but not in NOD or BALB/c mouse islets. The ability of NIT-1 cells to down-regulate Fas receptor in response to ligation is similar to that of a variety of tumor cells, which may use this mechanism to escape destruction by cytotoxic T cells. Islets apparently cannot protect themselves against FasL-induced apoptosis by down-regulating the Fas receptor. Understanding how NIT-1 insulinoma cells down-regulate Fas receptor in response to ligation by FasL has therapeutic implications for protecting normal beta-cells in autoimmune type 1 diabetes.