Long-term treatment with insulin and retinoic acid increased glucose utilization in L6 muscle cells via glycogenesis.

The skeletal muscle regulates glucose homeostasis. Here, the effects of vitamin A metabolites including retinoic acid (RA) alone, and in combination with insulin, on glucose utilization were investigated in rat L6 muscle cells during the differentiation process. L6 cells were treated with differentiation medium containing retinol, retinal, RA, and (or) insulin. The glucose levels and pH values in the medium were measured every 2 days. The expression levels of insulin signaling and glycogen synthesis proteins, as well as glycogen content were determined. Retinal and RA reduced the glucose content and pH levels in the medium of the L6 cells. RA acted synergistically with insulin to reduce glucose and pH levels in the medium. The RA- and insulin-mediated reduction of glucose in the medium only occurred when glucose levels were at or above 15 mmol/L. Insulin-induced phosphorylation of Akt Thr308 was further enhanced by RA treatment through the activation of retinoic acid receptor. RA acted synergistically with insulin to phosphorylate glycogen synthase kinase 3ß, and dephosphorylate glycogen synthase (GS), which was associated with increases in the protein and mRNA levels of GS. Increases in glycogen content were induced by insulin, and was further enhanced in the presence of RA. We conclude that activation of the RA signaling pathway enhanced insulin-induced glucose utilization in differentiating L6 cells through increases in glycogenesis.

Structural Insights into Reovirus σ1 Interactions with Two Neutralizing Antibodies.

Reovirus attachment protein σ1 engages glycan receptors and junctional adhesion molecule-A (JAM-A) and is thought to undergo a conformational change during the proteolytic disassembly of virions to infectious subvirion particles (ISVPs) that accompanies cell entry. The σ1 protein is also the primary target of neutralizing antibodies. Here, we present a structural and functional characterization of two neutralizing antibodies that target σ1 of serotype 1 (T1) and serotype 3 (T3) reoviruses. The crystal structures revealed that each antibody engages its cognate σ1 protein within the head domain via epitopes distinct from the JAM-A-binding site. Surface plasmon resonance and cell-binding assays indicated that both antibodies likely interfere with JAM-A engagement by steric hindrance. To define the interplay between the carbohydrate receptor and antibody binding, we conducted hemagglutination inhibition assays using virions and ISVPs. The glycan-binding site of T1 σ1 is located in the head domain and is partly occluded by the bound Fab in the crystal structure. The T1-specific antibody inhibited hemagglutination by virions and ISVPs, probably via direct interference with glycan engagement. In contrast to T1 σ1, the carbohydrate-binding site of T3 σ1 is located in the tail domain, distal to the antibody epitope. The T3-specific antibody inhibited hemagglutination by T3 virions but not ISVPs, indicating that the antibody- and glycan-binding sites in σ1 are in closer spatial proximity on virions than on ISVPs. Our results provide direct evidence for a structural rearrangement of σ1 during virion-to-ISVP conversion and contribute new information about the mechanisms of antibody-mediated neutralization of reovirus. IMPORTANCE: Virus attachment proteins mediate binding to host cell receptors, serve critical functions in cell and tissue tropism, and are often targeted by the neutralizing antibody response. The structural investigation of antibody-antigen complexes can provide valuable information for understanding the molecular basis of virus neutralization. Studies with enveloped viruses, such as HIV and influenza virus, have helped to define sites of vulnerability and guide vaccination strategies. By comparison, less is known about antibody binding to nonenveloped viruses. Here, we structurally investigated two neutralizing antibodies that bind the attachment protein σ1 of reovirus. Furthermore, we characterized the neutralization efficiency, the binding affinity for σ1, and the effect of the antibodies on reovirus receptor engagement. Our analysis defines reovirus interactions with two neutralizing antibodies, allows us to propose a mechanism by which they block virus infection, and provides evidence for a conformational change in the σ1 protein during viral cell entry.

RADVAN: a randomised phase 2 trial of WBRT plus vandetanib for melanoma brain metastases - results and lessons learnt.

BACKGROUND: Brain metastases occur in up to 75% of patients with advanced melanoma. Most are treated with whole-brain radiotherapy (WBRT), with limited effectiveness. Vandetanib, an inhibitor of vascular endothelial growth factor receptor, epidermal growth factor receptor and rearranged during transfection tyrosine kinases, is a potent radiosensitiser in xenograft models. We compared WBRT with WBRT plus vandetanib in the treatment of patients with melanoma brain metastases. METHODS: In this double-blind, multi-centre, phase 2 trial patients with melanoma brain metastases were randomised to receive WBRT (30 Gy in 10 fractions) plus 3 weeks of concurrent vandetanib 100 mg once daily or placebo. The primary endpoint was progression-free survival in brain (PFS brain). The main study was preceded by a safety run-in phase to confirm tolerability of the combination. A post-hoc analysis and literature review considered barriers to recruiting patients with melanoma brain metastases to clinical trials. RESULTS: Twenty-four patients were recruited, six to the safety phase and 18 to the randomised phase. The study closed early due to poor recruitment. Median PFS brain was 3.3 months (90% confidence interval (CI): 1.6-5.6) in the vandetanib group and 2.5 months (90% CI: 0.2-4.8) in the placebo group (P=0.34). Median overall survival (OS) was 4.6 months (90% CI: 1.6-6.3) and 2.5 months (90% CI: 0.2-7.2), respectively (P=0.54). The most frequent adverse events were fatigue, alopecia, confusion and nausea. The most common barrier to study recruitment was availability of alternative treatments. CONCLUSIONS: The combination of WBRT plus vandetanib was well tolerated. Compared with WBRT alone, there was no significant improvement in PFS brain or OS, although we are unable to provide a definitive result due to poor accrual. A review of barriers to trial accrual identified several factors that affect study recruitment in this difficult disease area.

Synergistic expression of the CXCL10 gene in response to IL-1ß and IFN-γ involves NF-κB, phosphorylation of STAT1 at Tyr701, and acetylation of histones H3 and H4.

The CXCL10 gene encodes a peptide that chemoattracts a variety of leukocytes associated with type 1 and type 2 diabetes. The present study was undertaken to determine the molecular mechanisms required for expression of the CXCL10 gene in response to IL-1ß and IFN-γ using rat islets and ß cell lines. IL-1ß induced the expression of the CXCL10 gene and promoter activity, whereas the combination of IL-1ß plus IFN-γ was synergistic. Small interfering RNA-mediated suppression of NF-κB p65 markedly inhibited the ability of cytokines to induce the expression of the CXCL10 gene, whereas targeting STAT1 only diminished the synergy provided by IFN-γ. Furthermore, we found that a JAK1 inhibitor dose dependently reduced IFN-γ-controlled CXCL10 gene expression and promoter activity, concomitant with a decrease in STAT1 phosphorylation at Tyr(701). We further discovered that, although the Tyr(701) phosphorylation site is inducible (within 15 min of IFN-γ exposure), the Ser(727) site within STAT1 is constitutively phosphorylated. Thus, we generated single-mutant STAT1 Y701F and double-mutant STAT1 Y701F/S727A adenoviruses. Using these recombinant adenoviruses, we determined that overexpression of either the single- or double-mutant STAT1 decreased the IFN-γ-mediated potentiation of CXCL10 gene expression, promoter activity, and secretion of protein. Moreover, the Ser(727) phosphorylation was neither contingent on a functional Y701 site in ß cells nor was it required for cytokine-mediated expression of the CXCL10 gene. We conclude that the synergism of IL-1ß and IFN-γ to induce expression of the CXCL10 gene requires NF-κB, STAT1 phosphorylated at Tyr(701), recruitment of coactivators, and acetylation of histones H3 and H4.

NF-κB and STAT1 control CXCL1 and CXCL2 gene transcription.

Diabetes mellitus results from immune cell invasion into pancreatic islets of Langerhans, eventually leading to selective destruction of the insulin-producing ß-cells. How this process is initiated is not well understood. In this study, we investigated the regulation of the CXCL1 and CXCL2 genes, which encode proteins that promote migration of CXCR2(+) cells, such as neutrophils, toward secreting tissue. Herein, we found that IL-1ß markedly enhanced the expression of the CXCL1 and CXCL2 genes in rat islets and ß-cell lines, which resulted in increased secretion of each of these proteins. CXCL1 and CXCL2 also stimulated the expression of specific integrin proteins on the surface of human neutrophils. Mutation of a consensus NF-κB genomic sequence present in both gene promoters reduced the ability of IL-1ß to promote transcription. In addition, IL-1ß induced binding of the p65 and p50 subunits of NF-κB to these consensus κB regulatory elements as well as to additional κB sites located near the core promoter regions of each gene. Additionally, serine-phosphorylated STAT1 bound to the promoters of the CXCL1 and CXCL2 genes. We further found that IL-1ß induced specific posttranslational modifications to histone H3 in a time frame congruent with transcription factor binding and transcript accumulation. We conclude that IL-1ß-mediated regulation of the CXCL1 and CXCL2 genes in pancreatic ß-cells requires stimulus-induced changes in histone chemical modifications, recruitment of the NF-κB and STAT1 transcription factors to genomic regulatory sequences within the proximal gene promoters, and increases in phosphorylated forms of RNA polymerase II.

Accelerated atherosclerosis in Apoe-/- mice heterozygous for the insulin receptor and the insulin receptor substrate-1.

OBJECTIVE: Prediabetic states are associated with accelerated atherosclerosis, but the availability of mouse models to study connections between these diseases has been limited. The aim of this study was to test the selective role of impaired insulin receptor/insulin receptor substrate-1 signaling on atherogenesis. METHODS AND RESULTS: To address the effects of impaired insulin signaling associated with hyperinsulinemia on atherosclerosis in the absence of obesity and hyperglycemia, we generated insulin receptor (Insr)/insulin receptor substrate-1 (Insr1) double heterozygous apolipoprotein (Apoe)-knockout mice (Insr(+/-)Irs1(+/-)Apoe(-/-)) mice. Insr(+/-)Irs1(+/-)Apoe(-/-) mice fed a Western diet for 15 weeks showed elevated levels of fasting insulin compared to Insr(+/+)Irs1(+/+)Apoe(-/-) mice. There were no significant differences in glucose, triglyceride, HDL, VLDL, cholesterol levels or free fatty acid in the plasma of Insr(+/-)Irs1(+/-)Apoe(-/-) and Insr(+/+)Irs1(+/+)Apoe(-/-) mice. Atherosclerotic lesions were increased in male (brachiocephalic artery) and female (aortic tree) Insr(+/-)Irs1(+/-)Apoe(-/-) compared to Insr(+/+)Irs1(+/+)Apoe(-/-) mice. Bone marrow transfer experiments demonstrated that nonhematopoietic cells have to be Insr(+/-)Irs1(+/-) to accelerate atherosclerosis. Impaired insulin signaling resulted in decreased levels of vascular phospho-eNOS, attenuated endothelium-dependent vasorelaxation and elevated VCAM-1 expression in aortas of Insr(+/-)Irs1(+/-)Apoe(-/-) mice. In addition, phospho-ERK and vascular smooth muscle cell proliferation were significantly elevated in aortas of Insr(+/-)Irs1(+/-)Apoe(-/-) mice. CONCLUSIONS: These results demonstrate that defective insulin signaling is involved in accelerated atherosclerosis in Insr(+/-)Irs1(+/-)Apoe(-/-) mice by promoting vascular dysfunction and inflammation.

Anti-influenza H7 human antibody targets antigenic site in hemagglutinin head domain interface.

Although broadly protective, stem-targeted Abs against the influenza A virus hemagglutinin (HA) have been well studied, very limited information is available on Abs that broadly recognize the head domain. We determined the crystal structure of the HA protein of the avian H7N9 influenza virus in complex with a pan-H7, non-neutralizing, protective human Ab. The structure revealed a B cell epitope in the HA head domain trimer interface (TI). This discovery of a second major protective TI epitope supports a model in which uncleaved HA trimers exist on the surface of infected cells in a highly dynamic state that exposes hidden HA head domain features.

Dual Erb B Inhibition in Oesophago-gastric Cancer (DEBIOC): A phase I dose escalating safety study and randomised dose expansion of AZD8931 in combination with oxaliplatin and capecitabine chemotherapy in patients with oesophagogastric adenocarcinoma.

BACKGROUND: AZD8931 has equipotent activity against epidermal growth factor receptor, erbB2, and erbB3. Primary objectives were to determine the recommended phase II dose (RP2D) of AZD8931 + chemotherapy, and subsequently assess safety/preliminary clinical activity in patients with operable oesophagogastric cancer (OGC). METHODS: AZD8931 (20 mg, 40 mg or 60 mg bd) was given with Xelox (oxaliplatin + capecitabine) for eight 21-day cycles, continuously or with intermittent schedule (4 days on/3 off every week; 14 days on/7 off, per cycle) in a rolling-six design. Subsequently, patients with OGC were randomised 2:1 to AZD8931 + Xelox at RP2D or Xelox only for two cycles, followed by radical oesophagogastric surgery. Secondary outcomes were safety, complete resection (R0) rate, six-month progression-free survival (PFS) and overall survival. RESULTS: During escalation, four dose-limiting toxicities were observed among 24 patients: skin rash (1) and failure to deliver 100% of Xelox because of treatment-associated grade III-IV adverse events (AEs) (3: diarrhoea and vomiting; vomiting; fatigue). Serious adverse events (SAE) occurred in 15 of 24 (63%) patients. RP2D was 20-mg bd with the 4/3 schedule. In the expansion phase, 2 of 20 (10%) patients in the Xelox + AZD8931 group and 5/10 (50%) patients in the Xelox group had grade III-IV AEs. Six-month PFS was 85% (90% CI: 66%-94%) in Xelox + AZD8931 and 100% in Xelox alone. Seven deaths (35%) occurred with Xelox + AZD8931 and one (10%) with Xelox. R0 rate was 45% (9/20) with Xelox + AZD8931 and 90% (9/10) with Xelox-alone (P = 0.024). CONCLUSION: Xelox + AZD8931 (20 mg bd 4/3 days) has an acceptable safety profile administered as neoadjuvant therapy in operable patients with OGC. (Trial registration: EudraCT 2011-003169-13, ISRCTN-68093791).

Developmental exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin alters postnatal T cell phenotypes and T cell function and exacerbates autoimmune lupus in 24-week-old SNF1 mice.

BACKGROUND: Untreated, more than 95% of female SWR x NZB: F(1) (SNF(1)) mice spontaneously develop a fatal lupus-like glomerulonephritis by 8 months-of-age, while disease onset in males is much slower. METHODS: : Timed-pregnant SNF(1) mice (10 per treatment) were exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on gestational day (GD) 12 by oral maternal gavage with 0, 40, or 80 microg/kg TCDD. RESULTS: Offspring of the TCDD-exposed dams showed numerous alterations in T lineage cells at 24 weeks-of-age. Females but not males showed decreased CD4(+)8(+) and increased CD4(-)8(-) thymocytes. Females also showed increased autoreactive CD4(+)Vbeta17(a+) axillary and inguinal lymph node T cells. Concanavalin A-stimulated splenocytes from prenatal TCDD-treated mice produced decreased interleukin 17 (IL-17) in the females while males showed increased IL-2 and IFN-gamma, and diminished IL-4. Mitogen-stimulated pan-lymphoproliferative responses were significantly increased across sex by TCDD. Anti-IgG and anti-C3 immune complex deposition in kidneys was present in the males after TCDD, and visibly worsened in females. CONCLUSIONS: Developmental TCDD exposure can permanently alter T lymphopoiesis in autoimmune-prone SNF1 mice. The alteration profile is beyond the classic immune suppression response, to also include exacerbation and induction of a lupuslike autoimmune disease.

An in vivo model of a mechanically-induced bone marrow lesion.

Bone marrow lesions (BMLs) are radiologic abnormalities in magnetic resonance images of subchondral bone that are correlated with osteoarthritis. Little is known about the physiologic processes within a BML, although BMLs are associated with mechanical stress, bone tissue microdamage and increased bone remodeling. Here we establish a rabbit model to study the pathophysiology of BMLs. We hypothesized that in vivo loads that generate microdamage in cancellous bone would also create BMLs and increase bone remodeling. In vivo cyclic loading (0.2-2.0 MPa in compression for 10,000 cycles at 2 Hz) was applied to epiphyseal cancellous bone in the distal femurs of New Zealand white rabbits (n=3, right limb loaded, left limb controls experienced surgery but no loading). Magnetic resonance images were collected using short tau inversion recovery (STIR) and T1 weighted sequences at 1 and 2 weeks after surgery/loading and histological analysis of the BML was performed after euthanasia to examine tissue microdamage and remodeling. Loaded limbs displayed BMLs while control limbs showed only a small BML-like signal caused by surgery. Histological analysis of the BML at 2 weeks after loading showed increased tissue microdamage (p=0.03) and bone resorption (p=0.01) as compared to controls. The model described here displays the hallmarks of load-induced BMLs, supporting the use of the model to examine changes in bone during the development, progression and treatment of BMLs.

Eating Frequency, Food Intake, and Weight: A Systematic Review of Human and Animal Experimental Studies.

Eating frequently during the day, or "grazing," has been proposed to assist with managing food intake and weight. This systematic review assessed the effect of greater eating frequency (EF) on intake and anthropometrics in human and animal experimental studies. Studies were identified through the PubMed electronic database. To be included, studies needed to be conducted in controlled settings or use methods that carefully monitored food intake, and measure food intake or anthropometrics. Studies using human or animal models of disease states (i.e., conditions influencing glucose or lipid metabolism), aside from being overweight or obese, were not included. The 25 reviewed studies (15 human and 10 animal studies) contained varying study designs, EF manipulations (1-24 eating occasions per day), lengths of experimentation (230 min to 28 weeks), and sample sizes (3-56 participants/animals per condition). Studies were organized into four categories for reporting results: (1) human studies conducted in laboratory/metabolic ward settings; (2) human studies conducted in field settings; (3) animal studies with experimental periods <1 month; and (4) animal studies with experimental periods >1 month. Out of the 13 studies reporting on consumption, 8 (61.5%) found no significant effect of EF. Seventeen studies reported on anthropometrics, with 11 studies (64.7%) finding no significant effect of EF. Future, adequately powered, studies should examine if other factors (i.e., disease states, physical activity, energy balance and weight status, long-term increased EF) influence the relationship between increased EF and intake and/or anthropometrics.

Higher protein kinase C ζ in fatty rat liver and its effect on insulin actions in primary hepatocytes.

We previously showed the impairment of insulin-regulated gene expression in the primary hepatocytes from Zucker fatty (ZF) rats, and its association with alterations of hepatic glucose and lipid metabolism. However, the molecular mechanism is unknown. A preliminary experiment shows that the expression level of protein kinase C ζ (PKCζ), a member of atypical PKC family, is higher in the liver and hepatocytes of ZF rats than that of Zucker lean (ZL) rats. Herein, we intend to investigate the roles of atypical protein kinase C in the regulation of hepatic gene expression. The insulin-regulated hepatic gene expression was evaluated in ZL primary hepatocytes treated with atypical PKC recombinant adenoviruses. Recombinant adenovirus-mediated overexpression of PKCζ, or the other atypical PKC member PKCι/λ, alters the basal and impairs the insulin-regulated expressions of glucokinase, sterol regulatory element-binding protein 1c, the cytosolic form of phosphoenolpyruvate carboxykinase, the catalytic subunit of glucose 6-phosphatase, and insulin like growth factor-binding protein 1 in ZL primary hepatocytes. PKCζ or PKCι/λ overexpression also reduces the protein level of insulin receptor substrate 1, and the insulin-induced phosphorylation of AKT at Ser473 and Thr308. Additionally, PKCι/λ overexpression impairs the insulin-induced Prckz expression, indicating the crosstalk between PKCζ and PKCι/λ. We conclude that the PKCζ expression is elevated in hepatocytes of insulin resistant ZF rats. Overexpressions of aPKCs in primary hepatocytes impair insulin signal transduction, and in turn, the down-stream insulin-regulated gene expression. These data suggest that elevation of aPKC expression may contribute to the hepatic insulin resistance at gene expression level.

Regulation of iNOS gene transcription by IL-1ß and IFN-γ requires a coactivator exchange mechanism.

The proinflammatory cytokines IL-1ß and IFN-γ decrease functional islet ß-cell mass in part through the increased expression of specific genes, such as inducible nitric oxide synthase (iNOS). Dysregulated iNOS protein accumulation leads to overproduction of nitric oxide, which induces DNA damage, impairs ß-cell function, and ultimately diminishes cellular viability. However, the transcriptional mechanisms underlying cytokine-mediated expression of the iNOS gene are not completely understood. Herein, we demonstrated that individual mutations within the proximal and distal nuclear factor-κB sites impaired cytokine-mediated transcriptional activation. Surprisingly, mutating IFN-γ-activated site (GAS) elements in the iNOS gene promoter, which are classically responsive to IFN-γ, modulated transcriptional sensitivity to IL-1ß. Transcriptional sensitivity to IL-1ß was increased by generation of a consensus GAS element and decreased correspondingly with 1 or 2 nucleotide divergences from the consensus sequence. The nuclear factor-κB subunits p65 and p50 bound to the κB response elements in an IL-1ß-dependent manner. IL-1ß also promoted binding of serine-phosphorylated signal transducer and activator of transcription-1 (STAT1) (Ser727) but not tyrosine-phosphorylated STAT1 (Tyr701) to GAS elements. However, phosphorylation at Tyr701 was required for IFN-γ to potentiate the IL-1ß response. Furthermore, coactivator p300 and coactivator arginine methyltransferase were recruited to the iNOS gene promoter with concomitant displacement of the coactivator CREB-binding protein in cells exposed to IL-1ß. Moreover, these coordinated changes in factor recruitment were associated with alterations in acetylation, methylation, and phosphorylation of histone proteins. We conclude that p65 and STAT1 cooperate to control iNOS gene transcription in response to proinflammatory cytokines by a coactivator exchange mechanism. This increase in transcription is also associated with signal-specific chromatin remodeling that leads to RNA polymerase II recruitment and phosphorylation.

Monoclonal antibodies to DIPA: a novel binding partner of p120-catenin isoform 1.

The coiled-coil domain-containing delta-interacting protein A (DIPA) is a transcription factor implicated in developmental regulation. DIPA is the first protein discovered to selectively interact with the p120-catenin (p120) isoform 1, an alternatively spliced form of p120 expressed preferentially in mesenchymal cells. Although a small fraction of p120 can be observed in the nucleus under certain circumstances, the vast majority of it associates with classical cadherins at adherens junctions. We observed for the first time that a discrete fraction of DIPA exists at cell-cell junctions, in addition to its predominantly nuclear localization. Thus, the p120-DIPA interaction may regulate cell signaling and/or transcriptional events, as has been described previously for ß-catenin and the LEF/TCF transcription factor family. To facilitate further study of DIPA and to determine the physiological relevance of its interaction with p120, we have generated and characterized a panel of five DIPA-specific monoclonal antibodies (MAbs) that function in immunoblotting, immunoprecipitation, and immunofluorescence assays.

Regulation of the CCL2 gene in pancreatic ß-cells by IL-1ß and glucocorticoids: role of MKP-1.

Release of pro-inflammatory cytokines from both resident and invading leukocytes within the pancreatic islets impacts the development of Type 1 diabetes mellitus. Synthesis and secretion of the chemokine CCL2 from pancreatic ß-cells in response to pro-inflammatory signaling pathways influences immune cell recruitment into the pancreatic islets. Therefore, we investigated the positive and negative regulatory components controlling expression of the CCL2 gene using isolated rat islets and INS-1-derived ß-cell lines. We discovered that activation of the CCL2 gene by IL-1ß required the p65 subunit of NF-κB and was dependent on genomic response elements located in the -3.6 kb region of the proximal gene promoter. CCL2 gene transcription in response to IL-1ß was blocked by pharmacological inhibition of the IKKß and p38 MAPK pathways. The IL-1ß-mediated increase in CCL2 secretion was also impaired by p38 MAPK inhibition and by glucocorticoids. Moreover, multiple synthetic glucocorticoids inhibited the IL-1ß-stimulated induction of the CCL2 gene. Induction of the MAP Kinase Phosphatase-1 (MKP-1) gene by glucocorticoids or by adenoviral-mediated overexpression decreased p38 MAPK phosphorylation, which diminished CCL2 gene expression, promoter activity, and release of CCL2 protein. We conclude that glucocorticoid-mediated repression of IL-1ß-induced CCL2 gene transcription and protein secretion occurs in part through the upregulation of the MKP-1 gene and subsequent deactivation of the p38 MAPK. Furthermore, the anti-inflammatory actions observed with MKP-1 overexpression were obtained without suppressing glucose-stimulated insulin secretion. Thus, MKP-1 is a possible target for anti-inflammatory therapeutic intervention with preservation of ß-cell function.