Conversion of human cardiac progenitor cells into cardiac pacemaker-like cells.

We used a screening strategy to test for reprogramming factors for the conversion of human cardiac progenitor cells (CPCs) into Pacemaker-like cells. Human transcription factors SHOX2, TBX3, TBX5, TBX18, and the channel protein HCN2, were transiently induced as single factors and in trio combinations into CPCs, first transduced with the connexin 30.2 (CX30.2) mCherry reporter. Following screens for reporter CX30.2 mCherry gene activation and FACS enrichment, we observed the definitive expression of many pacemaker specific genes; including, CX30.2, KCNN4, HCN4, HCN3, HCN1, and SCN3b. These findings suggest that the SHOX2, HCN2, and TBX5 (SHT5) combination of transcription factors is a much better candidate in driving the CPCs into Pacemaker-like cells than other combinations and single transcription factors. Additionally, single-cell RNA sequencing of SHT5 mCherry+ cells revealed cellular enrichment of pacemaker specific genes including TBX3, KCNN4, CX30.2, and BMP2, as well as pacemaker specific potassium and calcium channels (KCND2, KCNK2, and CACNB1). In addition, similar to human and mouse sinoatrial node (SAN) studies, we also observed the down-regulation of NKX2.5. Patch-clamp recordings of the converted Pacemaker-like cells exhibited HCN currents demonstrated the functional characteristic of pacemaker cells. These studies will facilitate the development of an optimal Pacemaker-like cell-based therapy within failing hearts through the recovery of SAN dysfunction.

Arginine Metabolism Is Altered in Adults with A-ß + Ketosis-Prone Diabetes.

Background: A-ß + ketosis-prone diabetes (KPD) is a subset of type 2 diabetes in which patients have severe but reversible ß cell dysfunction of unknown etiology. Plasma metabolomic analysis indicates that abnormal arginine metabolism may be involved. Objective: The objective of this study was to determine the relation between gut microbiome and arginine metabolism and the relation between arginine availability and ß cell function in KPD patients compared with control participants. Methods: Kinetics of arginine and related metabolites were measured with stable isotope tracers, and insulin secretory responses to arginine and glucose were determined under euglycemic and hyperglycemic conditions in 6 KPD patients and 6 age-, gender-, and body mass index-matched control participants. Glucose potentiation of arginine-induced insulin secretion was performed in a different set of 6 KPD and 3 control participants. Results: Arginine availability was higher in KPD patients during euglycemia [53.5 ± 4.3 (mean ± SEM) compared with 40.3 ± 2.4 µmol · kg lean body mass (LBM)-1 · h-1, P = 0.03] but declined more in response to hyperglycemia (Δ 10.15 ± 2.6 compared with Δ 3.20 ± 1.3 µmol · kg LBM-1 · h-1, P = 0.041). During hyperglycemia, ornithine flux was not different between groups but after an arginine bolus, plasma ornithine AUC trended higher in KPD patients (3360 ± 294 compared with 2584 ± 259 min · µmol · L-1, P = 0.08). In both euglycemia and hyperglycemia, the first-phase insulin responses to glucose stimulation were lower in KPD patients (euglycemic insulin AUC 282 ± 108 compared with 926 ± 257 min · µU · mL-1, P = 0.02; hyperglycemic insulin AUC 358 ± 79 compared with 866 ± 292 min · µU · mL-1, P = 0.05), but exogenous arginine restored first-phase insulin secretion in KPD patients to the level of control participants. Conclusion: Compared with control participants, KPD patients have increased arginine availability in the euglycemic state, indicating a higher requirement. This is compromised during hyperglycemia, with an inadequate supply of arginine to sustain metabolic functions such as insulin secretion. Exogenous arginine administration restores a normal insulin secretory response.

Transcription factors ETS2 and MESP1 transdifferentiate human dermal fibroblasts into cardiac progenitors.

Unique insights for the reprograming of cell lineages have come from embryonic development in the ascidian Ciona, which is dependent upon the transcription factors Ci-ets1/2 and Ci-mesp to generate cardiac progenitors. We tested the idea that mammalian v-ets erythroblastosis virus E26 oncogene homolog 2 (ETS2) and mesoderm posterior (MESP) homolog may be used to convert human dermal fibroblasts into cardiac progenitors. Here we show that murine ETS2 has a critical role in directing cardiac progenitors during cardiopoiesis in embryonic stem cells. We then use lentivirus-mediated forced expression of human ETS2 to convert normal human dermal fibroblasts into replicative cells expressing the cardiac mesoderm marker KDR(+). However, although neither ETS2 nor the purported cardiac master regulator MESP1 can by themselves generate cardiac progenitors de novo from fibroblasts, forced coexpression of ETS2 and MESP1 or cell treatment with purified proteins reprograms fibroblasts into cardiac progenitors, as shown by the de novo appearance of core cardiac transcription factors, Ca(2+) transients, and sarcomeres. Our data indicate that ETS2 and MESP1 play important roles in a genetic network that governs cardiopoiesis.

Impaired lipoprotein processing in HIV patients on antiretroviral therapy: aberrant high-density lipoprotein lipids, stability, and function.

OBJECTIVE: HIV patients on antiretroviral therapy (HIV/ART) exhibit a unique atherogenic dyslipidemic profile with hypertriglyceridemia (HTG) and low plasma concentrations of high-density lipoprotein (HDL) cholesterol. In the Heart Positive Study of HIV/ART patients, a hypolipidemic therapy of fenofibrate, niacin, diet, and exercise reduced HTG and plasma non-HDL cholesterol concentrations and raised plasma HDL cholesterol and adiponectin concentrations. We tested the hypothesis that HIV/ART HDL have abnormal structures and properties and are dysfunctional. APPROACH AND RESULTS: Hypolipidemic therapy reduced the TG contents of low-density lipoprotein and HDL. At baseline, HIV/ART low-density lipoproteins were more triglyceride (TG)-rich and HDL were more TG- and cholesteryl ester-rich than the corresponding lipoproteins from normolipidemic (NL) subjects. Very-low-density lipoproteins, low-density lipoprotein, and HDL were larger than the corresponding lipoproteins from NL subjects; HIV/ART HDL were less stable than NL HDL. HDL-[(3)H]cholesteryl ester uptake by Huh7 hepatocytes was used to assess HDL functionality. HIV/ART plasma were found to contain significantly less competitive inhibition activity for hepatocyte HDL-cholesteryl ester uptake than NL plasma were found to contain (P<0.001). CONCLUSIONS: Compared with NL subjects, lipoproteins from HIV/ART patients are larger and more neutral lipid-rich, and their HDL are less stable and less receptor-competent. On the basis of this work and previous studies of lipase activity in HIV, we present a model in which plasma lipolytic activities or hepatic cholesteryl ester uptake are impaired in HIV/ART patients. These findings provide a rationale to determine whether the distinctive lipoprotein structure, properties, and function of HIV/ART HDL predict atherosclerosis as assessed by carotid artery intimal medial thickness.

STEMIN and YAP5SA synthetic modified mRNAs regenerate and repair infarcted mouse hearts.

Introduction: The adult heart lacks the regenerative capacity to self-repair. Serum response factor (SRF) is essential for heart organogenesis, sarcomerogenesis, and contractility. SRF interacts with co-factors, such as NKX2.5 and GATA4, required for cardiac specified gene activity. ETS factors such as ELK1 interact with SRF and drive cell replication. To weaken SRF interactions with NKX2.5 and GATA4, one mutant, SRF153(A3) named STEMIN, did not bind CArG boxes, yet induced stem cell factors such as NANOG and OCT4, cardiomyocyte dedifferentiation, and cell cycle reentry. The mutant YAP5SA of the Hippo pathway also promotes cardiomyocyte proliferation and growth. Aim: Infarcted adult mouse hearts were injected with translatable STEMIN and YAP5SA mmRNA to evaluate their clinical potential. Methods and Results: Mice were pulsed one day later with alpha-EDU and then heart sections were DAPI stained. Replicating cells were identified by immuno-staining against members of the DNA replisome pathway that mark entry to S phase of the cell cycle. Echocardiography was used to determine cardiac function following infarcts and mRNA treatment. To monitor cardiac wall repair, microscopic analysis was performed, and the extent of myocardial fibrosis was analyzed for immune cell infiltration. Injections of STEMIN and YAP5SA mmRNA into the left ventricles of infarcted adult mice promoted a greater than 17-fold increase in the DAPI stained and alpha-EDU marked cardiomyocyte nuclei, within a day. We observed de novo expression of phospho-histone H3, ORC2, MCM2, and CLASPIN. Cardiac function was significantly improved by four weeks post-infarct, and fibrosis and immune cell infiltration were diminished in hearts treated with STEMIN and YAP5SA mmRNA than each alone. Conclusion: STEMIN and YAP5SA mmRNA improved cardiac function and myocardial fibrosis in left ventricles of infarcted adult mice. The combinatorial use of mmRNA encoding STEMIN and YAP5SA has the potential to become a powerful clinical strategy to treat human heart disease.

Serum response factor orchestrates nascent sarcomerogenesis and silences the biomineralization gene program in the heart.

Our conditional serum response factor (SRF) knockout, Srf (Cko), in the heart-forming region blocked the appearance of rhythmic beating myocytes, one of the earliest cardiac defects caused by the ablation of a cardiac-enriched transcription factor. The appearance of Hand1 and Smyd1, transcription and chromatin remodeling factors; Acta1, Acta2, Myl3, and Myom1, myofibril proteins; and calcium-activated potassium-channel gene activity (KCNMB1), the channel protein, were powerfully attenuated in the Srf(CKO) mutant hearts. A requisite role for combinatorial cofactor interactions with SRF, as a major determinant for regulating the appearance of organized sarcomeres, was shown by viral rescue of SRF-null ES cells with SRF point mutants that block cofactor interactions. In the absence of SRF genes associated with biomineralization, GATA-6, bone morphogenetic protein 4 (BMP4), and periostin were strongly up-regulated, coinciding with the down regulation of many SRF dependent microRNA, including miR1, which exerted robust silencer activity over the induction of GATA-6 leading to the down regulation of BMP4 and periostin.

HIV-1 Viral Protein R Couples Metabolic Inflexibility With White Adipose Tissue Thermogenesis.

Persons living with HIV (PLWH) manifest chronic disorders of brown and white adipose tissues that lead to diabetes and metabolic syndrome. The mechanisms that link viral factors to defective adipose tissue function and abnormal energy balance in PLWH remain incompletely understood. Here, we explored how the HIV accessory protein viral protein R (Vpr) contributes to adaptive thermogenesis in two mouse models and human adipose tissues. Uncoupling protein 1 (UCP1) gene expression was strongly increased in subcutaneous white adipose tissue (WAT) biopsy specimens from PLWH and in subcutaneous WAT of the Vpr mice, with nearly equivalent mRNA copy number. Histology and functional studies confirmed beige transformation in subcutaneous but not visceral WAT in the Vpr mice. Measurements of energy balance indicated Vpr mice displayed metabolic inflexibility and could not shift efficiently from carbohydrate to fat metabolism during day-night cycles. Furthermore, Vpr mice showed a marked inability to defend body temperature when exposed to 4°C. Importantly, Vpr couples higher tissue catecholamine levels with UCP1 expression independent of ß-adrenergic receptors. Our data reveal surprising deficits of adaptive thermogenesis that drive metabolic inefficiency in HIV-1 Vpr mouse models, providing an expanded role for viral factors in the pathogenesis of metabolic disorders in PLWH.

ß-Adrenergic stimuli and rotating suspension culture enhance conversion of human adipogenic mesenchymal stem cells into highly conductive cardiac progenitors.

Clinical trials using human adipogenic mesenchymal stem cells (hAdMSCs) for the treatment of cardiac diseases have shown improvement in cardiac function and were proven safe. However, hAdMSCs do not convert efficiently into cardiomyocytes (CMs) or vasculature. Thus, reprogramming hAdMSCs into myocyte progenitors may fare better in future investigations. To reprogramme hAdMSCs into electrically conductive cardiac progenitor cells, we pioneered a three-step reprogramming strategy that uses proven MESP1/ETS2 transcription factors, ß-adrenergic and hypoxic signalling induced in three-dimensional (3D) cardiospheres. In Stage 1, ETS2 and MESP1 activated NNKX2.5, TBX5, MEF2C, dHAND, and GATA4 during the conversion of hAdMSCs into cardiac progenitor cells. Next, in Stage 2, ß2AR activation repositioned cardiac progenitors into de novo immature conductive cardiac cells, along with the appearance of RYR2, CAV2.1, CAV3.1, NAV1.5, SERCA2, and CX45 gene transcripts and displayed action potentials. In Stage 3, electrical conduction that was fostered by 3D cardiospheres formed in a Synthecon®, Inc. rotating bioreactor induced the appearance of hypoxic genes: HIF-1α/ß, PCG 1α/ß, and NOS2, which coincided with the robust activation of adult contractile genes including MLC2v, TNNT2, and TNNI3, ion channel genes, and the appearance of hyperpolarization-activated and cyclic nucleotide-gated channels (HCN1-4). Conduction velocities doubled to ~200 mm/s after hypoxia and doubled yet again after dissociation of the 3D cell clusters to ~400 mm/s. By comparison, normal conduction velocities within working ventricular myocytes in the whole heart range from 0.5 to 1 m/s. Epinephrine stimulation of stage 3 cardiac cells in patches resulted in an increase in amplitude of the electrical wave, indicative of conductive cardiac cells. Our efficient protocol that converted hAdMSCs into highly conductive cardiac progenitors demonstrated the potential utilization of stage 3 cells for tissue engineering applications for cardiac repair.

Cysteine-rich LIM-only proteins CRP1 and CRP2 are potent smooth muscle differentiation cofactors.

Cysteine-rich LIM-only proteins, CRP1 and CRP2, expressed during cardiovascular development act as bridging molecules that associate with serum response factor and GATA proteins. SRF-CRP-GATA complexes strongly activated smooth muscle gene targets. CRP2 was found in the nucleus during early stages of coronary smooth muscle differentiation from proepicardial cells. A dominant-negative CRP2 mutant blocked proepicardial cells from differentiating into smooth muscle cells. Together with SRF and GATA proteins, CRP1 and CRP2 converted pluripotent 10T1/2 fibroblasts into smooth muscle cells, while muscle LIM protein CRP3 inhibited the conversion. Thus, LIM-only proteins of the CRP family play important roles in organizing multiprotein complexes, both in the cytoplasm, where they participate in cytoskeletal remodeling, and in the nucleus, where they strongly facilitate smooth muscle differentiation.

Association of amino-terminal-specific antiglutamate decarboxylase (GAD65) autoantibodies with beta-cell functional reserve and a milder clinical phenotype in patients with GAD65 antibodies and ketosis-prone diabetes mellitus.

CONTEXT: We previously characterized patients presenting with diabetic ketoacidosis prospectively into four subgroups of ketosis-prone diabetes mellitus (KPDM), based on the presence or absence of beta-cell autoimmunity (A+ or A-) and beta-cell functional reserve (B+ or B-). The A+B- KPDM subgroup comprises patients with classic, autoimmune type 1 diabetes, whereas the A+B+ KPDM subgroup has only partial beta-cell loss and a distinct clinical phenotype. OBJECTIVE: We hypothesized that epitope specificity of autoantibodies directed against the 65-kDa isoform of glutamate decarboxylase (GAD65) reflects differences in beta-cell destruction. DESIGN: Sera of sequential GAD65Ab-positive KPDM patients admitted for diabetic ketoacidosis (n = 36) were analyzed for their epitope recognition using five GAD65-specific recombinant Fab and their ability to inhibit GAD65 enzymatic activity. All patients were followed longitudinally to assess beta-cell functional reserve and insulin dependence. RESULTS: Binding to an amino-terminal epitope defined by monoclonal antibody DPD correlated positively with fasting serum C-peptide levels at baseline (P = 0.0008) and after 1 yr (P = 0.007). Binding to the DPD-defined epitope also correlated positively with area under the curve for C-peptide after glucagon stimulation (P = 0.007) and with homeostasis model assessment percent B at 1 yr (P = 0.03). Binding to the DPD-defined epitope was significantly stronger in A+B+ than in A+B- patients (P = 0.001). Sera of 16 patients (44%) significantly inhibited GAD65 enzymatic activity, but this did not correlate with beta-cell function. CONCLUSION: DPD-defined epitope specificity is correlated directly with preserved beta-cell functional reserve in GAD65Ab-positive patients and is associated with the milder clinical phenotype of A+B+ KPDM.

HIV-1 viral protein R (Vpr) induces fatty liver in mice via LXRα and PPARα dysregulation: implications for HIV-specific pathogenesis of NAFLD.

HIV patients develop hepatic steatosis. We investigated hepatic steatosis in transgenic mice expressing the HIV-1 accessory protein Vpr (Vpr-Tg) in liver and adipose tissues, and WT mice infused with synthetic Vpr. Vpr-Tg mice developed increased liver triglyceride content and elevated ALT, bilirubin and alkaline phosphatase due to three hepatic defects: 1.6-fold accelerated de novo lipogenesis (DNL), 45% slower fatty acid ß-oxidation, and 40% decreased VLDL-triglyceride export. Accelerated hepatic DNL was due to coactivation by Vpr of liver X receptor-α (LXRα) with increased expression of its lipogenic targets Srebp1c, Chrebp, Lpk, Dgat, Fasn and Scd1, and intranuclear SREBP1c and ChREBP. Vpr enhanced association of LXRα with Lxrα and Srebp1c promoters, increased LXRE-LXRα binding, and broadly altered hepatic expression of LXRα-regulated lipid metabolic genes. Diminished hepatic fatty acid ß-oxidation was associated with decreased mRNA expression of Pparα and its targets Cpt1, Aox, Lcad, Ehhadh, Hsd10 and Acaa2, and blunted VLDL export with decreased expression of Mttp and its product microsomal triglyceride transfer protein. With our previous findings that Vpr circulates in HIV patients (including those with undetectable plasma HIV-1 RNA), co-regulates the glucocorticoid receptor and PPARγ and transduces hepatocytes, these data indicate a potential role for Vpr in HIV-associated fatty liver disease.

Severely dysregulated disposal of postprandial triacylglycerols exacerbates hypertriacylglycerolemia in HIV lipodystrophy syndrome.

BACKGROUND: The pathogenesis of hypertriacylglycerolemia, a characteristic feature of HIV lipodystrophy syndrome (HLS), is incompletely understood. One mechanism is accelerated lipolysis in the fasted state, but the severity of the hypertriacylglycerolemia suggests that additional underlying abnormalities may exist in the disposal of dietary fat. OBJECTIVE: Our objective was to investigate abnormalities in dietary fat disposal in the pathogenesis of hypertriacylglycerolemia in HLS. DESIGN: We studied 6 nondiabetic men with HLS and 6 men without HIV matched for age and body mass index as control subjects for 8 h after consumption of an isocaloric meal containing 2 g labeled [(13)C(3)]tripalmitin. Chylomicron-triacylglycerol disposal was estimated from labeled [(13)C(1)]palmitate in the plasma chylomicron fraction, and [(13)C(1)]palmitate oxidation was estimated from the (13)CO(2) enrichment in the breath and CO(2) production, over 8 h after the meal. RESULTS: HLS patients had significantly elevated concentrations of fasting plasma triacylglycerols in both chylomicron (x + SE: 100.3 +/- 49.5 compared with 29.2 +/- 2.2 mg/dL; P < 0.01) and VLDL (82.4 +/- 39.0 compared with 10.8 +/- 2.8 mg/dL; P < 0.01) fractions. Chylomicron-triacylglycerol-derived [(13)C(1)]palmitate disposal was markedly lower in the HLS patients (3.09 +/- 0.41 compared with 6.42 +/- 0.18 mmol [(13)C(1)]palmitate/8 h; P < 0.001) in the 8-h postmeal period. Further, HLS patients had lowered storage of chylomicron-triacylglycerols (0.74 +/- 0.38 compared with 5.05 +/- 0.16 mmol; P < 0.0001) and elevated plasma [(13)C(1)]palmitate concentrations (2.01 +/- 0.27 compared with 1.18 +/- 0.16 mmol; P < 0.05) 8 h after the meal. CONCLUSIONS: Patients with HLS have key defects that markedly impair postprandial disposal and storage of chylomicron-triacylglycerols. These defects contribute significantly to hypertriacylglycerolemia in HLS.

Factors associated with early relapse to insulin dependence in unprovoked A-ß+ ketosis-prone diabetes.

OBJECTIVE: Unprovoked "A-ß+" Ketosis-Prone Diabetes (KPD), a unique diabetic syndrome of adult-onset, obesity and proneness to ketoacidosis, is associated with rapid recovery of ß cell function and insulin-independence. Whereas most patients experience prolonged remission, a subset relapses early to insulin dependence. We sought to define factors associated with early relapse. METHODS: We utilized a prospective, longitudinal database to analyze 50 unprovoked A-ß+ KPD patients with >2 measurements of ß cell function and glycemia following baseline assessment. RESULTS: 19 patients (38%) relapsed to insulin dependence <1 year after the index DKA episode, while 31 (62%) remained insulin-independent for >1 year (median 4.2 years). Younger age at baseline (OR=0.947, P=0.033), and lower HOMA2-%ß (OR=0.982, P=0.001), lower HOMA2-IR (OR=0.582, P=0.046) and higher HbA1c at 1 year (OR=1.71, P=0.002) were associated with early relapse. A multivariate model with these variables and the interaction of HOMA2-%ß and HbA1c at 1 year provided a good fit (P<0.05). CONCLUSIONS: Relapse to insulin dependence in unprovoked A-ß+ KPD patients is associated with younger age and, after 1 year, lack of robust increase in ß cell functional reserve, and suboptimal glycemic control. Measurements of these parameters 1 year after the index DKA episode can be used to assess the need for insulin therapy.

Human adipose tissue as a reservoir for memory CD4+ T cells and HIV.

OBJECTIVE: The objective of this study is to determine whether adipose tissue functions as a reservoir for HIV-1. DESIGN: We examined memory CD4(+) T cells and HIV DNA in adipose tissue-stromal vascular fraction (AT-SVF) of five patients [four antiretroviral therapy (ART)-treated and one untreated]. To determine whether adipocytes stimulate CD4(+) T cells and regulate HIV production, primary human adipose cells were cocultured with HIV-infected CD4(+) T cells. METHODS: AT-SVF T cells were studied by flow cytometry, and AT-SVF HIV DNA (Gag and Env) was examined by nested PCR and sequence analyses. CD4(+) T-cell activation and HIV production were measured by flow cytometry and ELISA. RESULTS: AT-SVF CD3(+) T cells were activated (>60% CD69(+)) memory CD4(+) and CD8(+) T cells in uninfected and HIV-infected persons, but the AT-SVF CD4(+)/CD8(+) ratio was lower in HIV patients. HIV DNA (Gag and Env) was detected in AT-SVF of all five patients examined by nested PCR, comparably to other tissues [peripheral blood mononuclear cell (PBMC), lymph node or thymus]. In coculture experiments, adipocytes increased CD4(+) T-cell activation and HIV production approximately two to three-fold in synergy with gamma-chain cytokines interleukin (IL)-2, IL7 or IL15. These effects were mitigated by neutralizing antibodies against IL6 and integrin-α1ß1. Adipocytes also enhanced T-cell viability. CONCLUSION: Adipose tissues of ART-treated patients harbour activated memory CD4(+) T cells and HIV DNA. Adipocytes promote CD4(+) T-cell activation and HIV production in concert with intrinsic adipose factors. Adipose tissue may be an important reservoir for HIV.

Ketosis-prone diabetes: dissection of a heterogeneous syndrome using an immunogenetic and beta-cell functional classification, prospective analysis, and clinical outcomes.

Ketosis-prone diabetes is heterogeneous. Its causes could include novel beta-cell functional defects. To characterize such defects, 103 patients with diabetic ketoacidosis were evaluated for beta-cell autoimmunity and human leukocyte antigen (HLA) class II alleles, with longitudinal measurements of beta-cell function and biochemical and clinical parameters. They were classified into four A beta groups, based on the presence of glutamic acid decarboxylase (GAD)65, GAD67, or IA-2 autoantibodies (A+ or A-) and beta-cell functional reserve (beta+ or beta-). The group distribution was: 18 A+beta-, 23 A-beta-, 11 A+beta+, and 51 A-beta+. Collectively, the two beta- groups differed from the two beta+ groups in earlier onset and longer duration of diabetes, lower body mass index, less glycemic improvement, and persistent insulin requirement. HLA class II genotyping showed that the A-beta- group differed from the A+beta- group in having lower frequencies of two alleles strongly associated with autoimmune type 1 diabetes susceptibility: DQA*03 and DQB1*02. Similarly, the A-beta+ group differed from the A+beta+ group in having a lower frequency of DQB1*02. Ketosis-prone diabetes comprises at least four etiologically distinct syndromes separable by autoantibody status, HLA genotype, and beta-cell functional reserve. Novel, nonautoimmune causes of beta-cell dysfunction are likely to underlie the A-beta+ and A-beta- syndromes.

Developmental expression of serum response factor in the rat central nervous system.

Serum response factor (SRF), a transcription factor known to be essential for early embryonic development as well as post-natal regulation of both cellular proliferation and myogenic differentiation, is expressed broadly in neurons within the adult mammalian central nervous system (CNS). The function of SRF within the developing CNS is not well established, but it is likely to play an important role in neuraxial development and neuronal function, since many of its known target genes (e.g., c-fos) and transcriptional partners (e.g., Elk-1) are also highly expressed in neurons. Immunohistochemical survey of the post-natal developing rat brain revealed a progressive increase in SRF immunoreactivity in neurons of the cerebral and cerebellar cortices, and in selective subcortical regions from birth (P0) through post-natal day 28 (P28). SRF immunoreactivity stabilized from P28 into adulthood. A few loci, such as the nucleus of cranial nerve VII, showed the reverse expression pattern (strong immunoreactivity at P0-P7, declining by P28). The developmental expression pattern of SRF overlaps significantly with that of myotonic dystrophy protein kinase, a potential upstream regulator, and of the LIM-only genes Lmo1, Lmo2 and Lmo3, whose products belong to a family of proteins known to be strong positive regulators of SRF's transcriptional activity. These data suggest that SRF has a significant function in the early post-natal development of the CNS.

Improved outcomes in indigent patients with ketosis-prone diabetes: effect of a dedicated diabetes treatment unit.

OBJECTIVE: To investigate whether a specialized intervention program could improve diabetes-related health outcomes in indigent patients with type 1 diabetes who were prone to occurrence of diabetic ketoacidosis (DKA). METHODS: Patients with type 1 diabetes mellitus admitted because of DKA during a 24-month period were invited to receive outpatient care in a diabetes treatment unit (DTU). We compared DKA-related readmission rates, change in hemoglobin A1c (HbA1c) values, and diabetes-related medical costs in patients who participated in the DTU program (+DTU) and in those who did not (-DTU). RESULTS: During the study period, 115 patients underwent assessment. Of this overall group, 57 patients (49.6%) consented to participate in the DTU program, and 58 (50.4%) did not. After the follow-up period (median duration, 657 days), the following significant improvements were observed in the +DTU group (in comparison with the -DTU group): lower frequency of readmission for DKA (16% versus 43%; P = 0.001), lower number of readmissions for DKA per patient (0.22 +/- 0.6 versus 1.17 +/- 2.2 [mean +/- standard deviation]; P = 0.003), lower HbA1c level (10.4 +/- 2.3% versus 13.5 +/- 2.3%; P<0.0001), and lower cost of diabetes-related medical care (3,427.20 US dollars +/- 6,275.60 versus 10,119.10 US dollars +/- 19,688.10; P = 0.01). Multivariate analysis revealed that participation in the DTU program was the only factor associated with a significantly decreased risk of DKA-related readmission. CONCLUSION: Low-cost intervention by a dedicated outpatient DTU resulted in significant decreases in DKA-associated readmissions, in HbA1c values, and in costs of diabetes care in a multiethnic, indigent, ketosis-prone patient population.

Masked and overt autoantibodies specific to the DPD epitope of 65-kDa glutamate decarboxylase (GAD65-DPD) are associated with preserved ß-cell functional reserve in ketosis-prone diabetes.

CONTEXT: Ketosis-prone diabetes (KPD), defined by presentation with diabetic ketoacidosis (DKA), comprises 4 subgroups based on the presence or absence of islet cell autoantibodies (A(-) or A(+)) and ß-cell functional reserve (ß(-) or ß(+)). Among A(+) KPD, autoantibody epitope reactivity to 65-kDa glutamate decarboxylase (GAD65), defined by monoclonal GAD65Ab(DPD), was associated with greater ß-cell functional reserve. In a majority of healthy individuals, GAD65Ab are present in the sera but are masked by anti-idiotypic antibodies; in contrast, overtly GAD65Ab-positive patients with autoimmune type 1 diabetes patients lack these anti-idiotypic antibodies. OBJECTIVE: Our objective was to determine the presence of masked and overt GAD65Ab(DPD) in relation to ß-cell function and genetic risk factors in KPD patients. DESIGN: We investigated the associations of masked and overt GAD65Ab(DPD) with ß-cell functional reserve, and their relationship with human leukocyte antigen (HLA) class II haplotypes linked to autoimmune diabetes susceptibility or resistance, in a large KPD cohort. PATIENTS: Adult KPD patients (n = 384) were followed longitudinally in a research clinic. MAIN OUTCOME MEASURES: ß-Cell function, autoantibody status, GAD65Ab epitopes, and HLA class II haplotypes were evaluated. RESULTS: Overall, KPD patients with ß-cell functional reserve (ß(+) subgroups) showed significantly higher frequency of masked GAD65Ab(DPD) than patients without ß-cell functional reserve (ß(-) subgroups): 112 of 144 (79%) compared with 59 of 100 (59%), respectively (P = .002). Masked or overt GAD65Ab(DPD) were also more frequent among autoantibody-positive patients with preserved ß-cell functional reserve (A(+)ß(+) KPD) than those lacking ß-cell function (A(+)ß(-) KPD): 77% compared with 55% (P = .01). The susceptibility HLA haplotypes DQA1*0301/DQB1*0302 and DQA1*0301/DQB1*0201 were associated with absence of overt or masked GAD65Ab(DPD) (odds Ratios 2.3 and 2.2, respectively). CONCLUSIONS: Masked GAD65Ab(DPD) are strongly associated with preserved ß-cell functional reserve among patients with KPD. Absence of GAD65Ab(DPD) reactivity is associated with 2 HLA class II susceptibility haplotypes for autoimmune type 1 diabetes.

Association of TCF7L2 variation with single islet autoantibody expression in children with type 1 diabetes.

BACKGROUND: The transcription factor 7-like 2 (TCF7L2) gene has the strongest genetic association with type 2 diabetes. TCF7L2 also associates with latent autoimmune diabetes in adults, which often presents with a single islet autoantibody, but not with classical type 1 diabetes. METHODS: We aimed to test if TCF7L2 is associated with single islet autoantibody expression in pediatric type 1 diabetes. We studied 71 prospectively recruited children who had newly diagnosed type 1 diabetes and evidence of islet autoimmunity, that is, expressed ≥1 islet autoantibody to insulin, glutamic acid decarboxylase 65, islet cell autoantigen 512, or zinc transporter 8. TCF7L2 rs7903146 alleles were identified. Data at diagnosis were cross-sectionally analyzed. RESULTS: We found that 21.1% of the children with autoimmune type 1 diabetes expressed a single islet autoantibody. The distribution of TCF7L2 rs7903146 genotypes in children with a single autoantibody (n=15) was 40% CC, 26.7% CT and 33.3% TT, compared with children with ≥2 islet autoantibodies (50% CC, 42.9% CT and 7.1% TT, p=0.024). Furthermore, compared with children with ≥2 autoantibodies, single-autoantibody children had characteristics reflecting milder autoimmune destruction of ß-cells. Restricting to lean children (body mass index<85th centile; n=36), 45.5% of those expressing a single autoantibody were rs7903146 TT homozygotes, compared with 0% of those with ≥2 autoantibodies (p<0.0001). CONCLUSION: These results suggest that, in children with only mild islet autoimmunity, mechanisms associated with TCF7L2 genetic variation contribute to diabetogenesis, and this contribution is larger in the absence of obesity.

Islet-specific T-cell responses and proinflammatory monocytes define subtypes of autoantibody-negative ketosis-prone diabetes.

OBJECTIVE: Ketosis-prone diabetes (KPD) is characterized by diabetic ketoacidosis (DKA) in patients lacking typical features of type 1 diabetes. A validated classification scheme for KPD includes two autoantibody-negative ("A-") phenotypic forms: "A-ß-" (lean, early onset, lacking ß-cell functional reserve) and "A-ß+" (obese, late onset, with substantial ß-cell functional reserve after the index episode of DKA). Recent longitudinal analysis of a large KPD cohort revealed that the A-ß+ phenotype includes two distinct subtypes distinguished by the index DKA episode having a defined precipitant ("provoked," with progressive ß-cell function loss over time) or no precipitant ("unprovoked," with sustained ß-cell functional reserve). These three A- KPD subtypes are characterized by absence of humoral islet autoimmune markers, but a role for cellular islet autoimmunity is unknown. RESEARCH DESIGN AND METHODS: Islet-specific T-cell responses and the percentage of proinflammatory (CD14+CD16+) blood monocytes were measured in A-ß- (n = 7), provoked A-ß+ (n = 15), and unprovoked A-ß+ (n = 13) KPD patients. Genotyping was performed for type 1 diabetes-associated HLA class II alleles. RESULTS: Provoked A-ß+ and A-ß- KPD patients manifested stronger islet-specific T-cell responses (P < 0.03) and higher percentages of proinflammatory CD14+CD16+ monocytes (P < 0.01) than unprovoked A-ß+ KPD patients. A significant relationship between type 1 diabetes HLA class II protective alleles and negative T-cell responses was observed. CONCLUSIONS: Provoked A-ß+ KPD and A-ß- KPD are associated with a high frequency of cellular islet autoimmunity and proinflammatory monocyte populations. In contrast, unprovoked A-ß+ KPD lacks both humoral and cellular islet autoimmunity.