Historical and new insights into pathogenesis of type 1 diabetes (2).

In this second and final part of the collection of articles for the Immunology of Diabetes Society review series on insights into pathogenesis of type 1 diabetes, we present two articles. The first of these covers a debate that took place in the Immunology of Diabetes Society meeting in London 2018, in which five investigators presented a case for specific immune cells/targets to be the 'Achilles Heel of type 1 diabetes'. The second article presents further insights into the generation of post-translationally modified peptides. It focuses upon mechanisms and processes that lead to new potentially autoantigenic targets for CD8+ T cells, and complements the review of new hybrid peptide targets for CD4+ T cells in the first part of our series.

Identifying the 'Achilles heel' of type 1 diabetes.

When Thetis dipped her son Achilles into the River Styx to make him immortal, she held him by the heel, which was not submerged, and thus created a weak spot that proved deadly for Achilles. Millennia later, Achilles heel is part of today's lexicon meaning an area of weakness or a vulnerable spot that causes failure. Also implied is that an Achilles heel is often missed, forgotten or under-appreciated until it is under attack, and then failure is fatal. Paris killed Achilles with an arrow 'guided by the Gods'. Understanding the pathogenesis of type 1 diabetes (T1D) in order to direct therapy for prevention and treatment is a major goal of research into T1D. At the International Congress of the Immunology of Diabetes Society, 2018, five leading experts were asked to present the case for a particular cell/element that could represent 'the Achilles heel of T1D'. These included neutrophils, B cells, CD8+ T cells, regulatory CD4+ T cells, and enteroviruses, all of which have been proposed to play an important role in the pathogenesis of type 1 diabetes. Did a single entity emerge as 'the' Achilles heel of T1D? The arguments are summarized here, to make this case.

Historical and new insights into pathogenesis of type 1 diabetes.

In recent years, there have been exciting new insights into pathogenesis of type 1 diabetes in a number of areas of immunology. In this edition, a collection of four review articles are presented, which encompass new findings presented at the Immunology of Diabetes Society meeting in London 2018. The articles are focused particularly in 4 related areas of investigation, which include autoantibodies in type 1 diabetes, new autoantigenic targets for CD4 T cells, trafficking of immune cells to the pancreas and islet-immune interactions in the pancreas.

Association between HbA1c and the development of cystic fibrosis-related diabetes.

AIMS: To examine HbA1c as a predictor of risk for future development of cystic fibrosis-related diabetes and to assess the association with the development of retinopathy in people with cystic fibrosis-related diabetes. METHODS: A 7-year retrospective longitudinal study was conducted in 50 adults with cystic fibrosis, comparing oral glucose tolerance test results with HbA1c values in predicting the development of cystic fibrosis-related diabetes. Retinal screening data were also compared with HbA1c measurements to assess microvascular outcome. RESULTS: An HbA1c value ≥37 mmol/mol (5.5%; hazard ratio 3.49, CI 1.5-8.1) was significantly associated with the development of dysglycaemia, as defined by the oral glucose tolerance test over a 7-year period. Severity of diabetic retinopathy was associated with a higher HbA1c and longer duration of cystic fibrosis-related diabetes. CONCLUSION: There is a link between HbA1c level and the future development of dysglycaemia in cystic fibrosis based on oral glucose tolerance test, as well as microvascular outcomes. Although current guidance does not advocate the use of HbA1c as a diagnostic tool in cystic fibrosis-related diabetes, it may be of clinical use in determining individuals at risk of future development of cystic fibrosis-related diabetes.

Chemical kinetics of silver diammine fluoride in demineralization and remineralization solutions-an in vitro study.

Introduction: Silver Diammine Fluoride (SDF) is a clinical minimal intervention to manage dentin caries. Its chemistry in demineralization conditions has been investigated widely, but far less in remineralization conditions. The aim was to investigate and compare the chemical reactions when SDF is added to remineralization and demineralization solutions. Methods: 0.01 ml SDF (Riva Star) was added to deionized water (DW); demineralization (DS = pH4) and remineralization (RS = pH7.0) solutions. The time sequence of concentrations of NH4+, F-, and Ag+ were measured using ion selective electrodes (ISEs) every 2 min. The pH was also measured. Precipitates were characterized using x-ray Diffraction (XRD) and, 31P and 19F nuclear magnetic resonance spectroscopy (NMR). Results: The concentrations of NH4+ and Ag+ showed decreasing trends in DW (-0.12 and -0.08 mM/h respectively), and in DS (-1.06 and -0.5 mM/h respectively); with corresponding increase in F- concentration (0.04 and 0.7 mM/h respectively). However, in RS, NH4+ concentration showed little change (0.001 mM/h), and Ag+ and F- concentrations were negligible. XRD results showed that precipitates (in RS only) contained AgCl, and metallic Ag. NMR showed that fluorapatite/carbonated fluorapatite (FAP/CFAP) were formed. The pH increased after SDF addition in all three solutions. Discussion: SDF dissolved to release NH4+, F- and Ag + . In DW and DS, NH4+ combined with Ag+ to form diamminesilver, causing an increase of F- and pH. In RS, F- reacted with Ca2+ and (PO)43- to form FAP/CFAP, and Ag+ reacted with Cl- to form AgCl/Ag. These suggests why SDF is effective in managing dentin caries.

Characterization of chemical reactions of silver diammine fluoride and hydroxyapatite under remineralization conditions.

Introduction: Silver Diammine Fluoride (SDF) is a clinically used topical agent to arrest dental caries. However, the kinetics of its chemical interactions with hydroxyapatite (HA), the principal inorganic component of dental enamel, are not known. The aim was to characterize the step-wise chemical interactions between SDF and HA powder during the clinically important process of remineralization. Methods: Two grams of HA powder were immersed in 10 ml acetic acid pH = 4.0 for 2 h to mimic carious demineralization. The powder was then washed and dried for 24 h and mixed with 1.5 ml SDF (Riva Star) for 1 min. The treated powder was then air-dried for 3 min, and 0.2 g was removed and stored in individual tubes each containing 10 ml remineralizing solution. Powder was taken from each tube at various times of exposure to remineralization solution (0 min, 10 min, 2 h, 4 h, 8 h, 24 h, and 10 days), and characterized using Magic Angle Spinning-Nuclear Magnetic Resonance (MAS-NMR) spectroscopy. Results and discussion: 19F MAS-NMR spectra showed that calcium fluoride (CaF2) started to form almost immediately after HA was in contact with SDF. After 24 h, the peak shifted to -104.5 ppm suggesting that fluoride substituted hydroxyapatite (FSHA) was formed with time at the expense of CaF2. The 31P MAS-NMR spectra showed a single peak at 2.7 ppm at all time points showing that the only phosphate species present was crystalline apatite. The 35Cl MAS-NMR spectra showed formation of silver chloride (AgCl) at 24 h. It was observed that after the scan, the whitish HA powder changed to black color. In conclusion, this time sequence study showed that under remineralization conditions, SDF initially reacted with HA to form CaF2 which is then transformed to FSHA over time. In the presence of chloride, AgCl is formed which is subsequently photo-reduced to black metallic silver.

X-ray microtomography study to validate the efficacies of caries removal in primary molars by hand excavation and chemo-mechanical technique.

BACKGROUND/AIMS: Mechanical removal of carious dentine based on perceived hardness is subjective and tends to be excessively destructive; chemo-mechanical techniques have been proposed as being more objective and conservative. The aims of the present study are to use X-ray microtomography (XMT/micro-CT) to determine the three-dimensional mineral concentration distribution in sound, carious and excavated dentine using hand excavation (HE) and a chemo-mechanical, Carisolv (CS), removal technique for primary molars, and to compare the volume of sound dentine removed in order to validate the efficacies of these two techniques. METHODS: Twenty-one primary molars with open carious cavities were hemisected. The carious tissue in one half was then removed by HE and the other by CS. XMT scans were taken before and after caries removal. After alignment, subtracted XMT images from the two scans revealing the tissues removed were generated, from which mineral distributions were determined, and volumes of sound dentine removed by each technique were calculated. RESULTS: It was found that the sound dentine removed by HE and CS techniques accounted for 4.0 and 2.1% of total tissues removed, respectively. The mean cut-off linear attenuation coefficients at 40 keV to which HE and CS excavated to were 1.27 and 1.09 cm(-1), respectively. The corresponding Knoop hardness number for the cut-off for CS was 25 kg · mm(-2). CONCLUSION: It is concluded that using XMT, CS is validated to be more conservative than HE and preserves a layer of partially demineralised dentine with a mineral concentration > 0.97 g · cm(-3).

Best clinical practice guidance for clinicians dealing with children presenting with molar-incisor-hypomineralisation (MIH): an updated European Academy of Paediatric Dentistry policy document.

AIM: To update the existing European Academy of Paediatric Dentistry (EAPD) 2010 policy document on the 'Best Clinical Practice guidance for clinicians dealing with children presenting with Molar-Incisor-Hypomineralisation (MIH).' METHODS: Experts, assigned the EAPD, worked on two different topics: (A) Aetiological factors involved in MIH, and (B) Treatment options for the clinical management of MIH. The group prepared two detailed systematic reviews of the existing literature relevant to the topics and following a consensus process produced the updated EAPD policy document on the 'Best Clinical Practice guidance for clinicians dealing with children presenting with molar-incisor-hypomineralisation (MIH).' The GRADE system was used to assess the quality of evidence regarding aetiology and treatment which was judged as HIGH, MODERATE, LOW or VERY LOW, while the GRADE criteria were used to indicate the strength of recommendation regarding treatment options as STRONG or WEAK/CONDITIONAL. RESULTS: (A) Regarding aetiology, it is confirmed that MIH has a multifactorial aetiology with the duration, strength and timing of occurrence of the aetiological factors being responsible for the variable clinical characteristics of the defect. Perinatal hypoxia, prematurity and other hypoxia related perinatal problems, including caesarean section, appear to increase the risk of having MIH, while certain infant and childhood illnesses are also linked with MIH. In addition, genetic predisposition and the role of epigenetic influences are becoming clearer following twin studies and genome and single-nucleotide polymorphisms analyses in patients and families. Missing genetic information might be the final key to truly understand MIH aetiology. (B) Regarding treatment options, composite restorations, preformed metal crowns and laboratory indirect restorations provide high success rates for the posterior teeth in appropriate cases, while scheduled extractions provide an established alternative option in severe cases. There is great need for further clinical and laboratory studies evaluating new materials and non-invasive/micro-invasive techniques for anterior teeth, especially when aesthetic and oral health related quality of life (OHRQoL) issues are concerned. CONCLUSIONS: MIH has been studied more extensively in the last decade. Its aetiology follows the multifactorial model, involving systemic medical and genetic factors. Further focused laboratory research and prospective clinical studies are needed to elucidate any additional factors and refine the model. Successful preventive and treatment options have been studied and established. The appropriate choice depends on the severity of the defects and the age of the patient. EAPD encourages the use of all available treatment options, whilst in severe cases, scheduled extractions should be considered.

An update of treatment modalities in children and adolescents with teeth affected by molar incisor hypomineralisation (MIH): a systematic review.

PURPOSE: To systematically review the treatment modalities for molar-incisor hypomineralisation for children under the age of 18 years. The research question was, 'What are the treatment options for teeth in children affected by molar incisor hypomineralisation?' METHODS: An electronic search of the following electronic databases was completed MEDLINE, EMBASE, Cochrane Central Register of Controlled Trials, LILACS, Google Scholar and Open Grey identifying studies from 1980 to 2020. The PRISMA guidelines were followed. The studies were screened, data extracted and calibration was completed by two independent reviewers. RESULTS: Of 6220 potential articles, 34 studies were included. Twenty studies investigated management of molars with fissure sealants, glass ionomer cement, polyacid modified resin composite, composite resin, amalgam, preformed metal crowns, laboratory-manufactured crowns and extractions. In four articles management of incisors with microabrasion, resin-infiltration and a combination of approaches was reported. Eight studies looked at strategies to mineralise MIH-affected teeth and/or reduce hypersensitivity. Two studies investigated patient-centred outcomes following treatment. Due to the heterogeneity between the studies, meta-analysis was not performed. CONCLUSION: The use of resin-based fissure sealants, preformed metal crowns, direct composite resin restorations and laboratory-made restorations can be recommended for MIH-affected molars. There is insufficient evidence to support specific approaches for the management of affected incisors. Products containing CPP-ACP may be beneficial for MIH-affected teeth.

Safety of the use of gold nanoparticles conjugated with proinsulin peptide and administered by hollow microneedles as an immunotherapy in type 1 diabetes.

Antigen-specific immunotherapy is an immunomodulatory strategy for autoimmune diseases, such as type 1 diabetes, in which patients are treated with autoantigens to promote immune tolerance, stop autoimmune ß-cell destruction and prevent permanent dependence on exogenous insulin. In this study, human proinsulin peptide C19-A3 (known for its positive safety profile) was conjugated to ultrasmall gold nanoparticles (GNPs), an attractive drug delivery platform due to the potential anti-inflammatory properties of gold. We hypothesised that microneedle intradermal delivery of C19-A3 GNP may improve peptide pharmacokinetics and induce tolerogenic immunomodulation and proceeded to evaluate its safety and feasibility in a first-in-human trial. Allowing for the limitation of the small number of participants, intradermal administration of C19-A3 GNP appears safe and well tolerated in participants with type 1 diabetes. The associated prolonged skin retention of C19-A3 GNP after intradermal administration offers a number of possibilities to enhance its tolerogenic potential, which should be explored in future studies.

An increased frequency of NK cell receptor and HLA-C group 1 combinations in early-onset type 1 diabetes.

AIMS/HYPOTHESIS: Natural killer (NK) cells serve as primary immune surveillance and are partially regulated by combinations of killer immunoglobulin-like receptor (KIR) genes and their HLA class I ligands. Alterations in NK cell activity have been associated with type 1 diabetes. The aim of this study was to determine whether KIR-HLA class I gene frequency: (1) is altered in a current population with type 1 diabetes compared with healthy controls; and (2) has changed over the half century in which the incidence of type 1 diabetes has increased rapidly. METHODS: KIR-HLA class I gene frequencies were compared in 551 individuals diagnosed with type 1 diabetes ≤ 15 years of age (394 in a current cohort and 157 from the historical 'Golden Years' cohort) and 168 healthy controls. The overall balance of activation and inhibition was analysed using KIR-HLA genotype models. RESULTS: Children with type 1 diabetes who were positive for KIR2DS2/KIR2DL2 and KIR2DL3 were more often homozygous for HLA-C group 1 and this effect was strongest in children diagnosed with diabetes before the age of 5 years (p = 0.003, corrected p [p (corr)] = 0.012) and (p = 0.001, p (corr) = 0.004), respectively. Children with type 1 diabetes have fewer inhibitory KIRs with their corresponding ligands compared with healthy controls (p = 1.9 × 10(-4)). This pattern of NK activation has not changed significantly in individuals with type 1 diabetes over the last half century. CONCLUSIONS/INTERPRETATION: Activating combinations of KIR-HLA genes are more frequent in young children with type 1 diabetes diagnosed in the first 5 years of life, suggesting that NK cell responses may be altered in this group.

Isolation and preservation of peripheral blood mononuclear cells for analysis of islet antigen-reactive T cell responses: position statement of the T-Cell Workshop Committee of the Immunology of Diabetes Society.

Autoimmune T cell responses directed against insulin-producing ß cells are central to the pathogenesis of type 1 diabetes (T1D). Detection of such responses is therefore critical to provide novel biomarkers for T1D 'immune staging' and to understand the mechanisms underlying the disease. While different T cell assays are being developed for these purposes, it is important to optimize and standardize methods for processing human blood samples for these assays. To this end, we review data relevant to critical parameters in peripheral blood mononuclear cell (PBMC) isolation, (cryo)preservation, distribution and usage for detecting antigen-specific T cell responses. Based on these data, we propose recommendations on processing blood samples for T cell assays and identify gaps in knowledge that need to be addressed. These recommendations may be relevant not only for the analysis of T cell responses in autoimmune disease, but also in cancer and infectious disease, particularly in the context of clinical trials.

Immunology of Diabetes Society T-Cell Workshop: HLA class I tetramer-directed epitope validation initiative T-Cell Workshop Report-HLA Class I Tetramer Validation Initiative.

BACKGROUND: Identification of T-cell reactivity to ß-cell antigen epitopes is an important goal for studying pathogenesis and for designing and monitoring of immunotherapeutic interventions in type 1 diabetes (T1D). METHODS: We performed a multicentre validation of known human leukocyte antigen (HLA) class I CD8+ T-cell epitopes. To this end, peripheral blood T-cell responses were measured in 35 recently (<2 years) diagnosed HLA-A*02:01+ T1D patients using blind-coded HLA-A2 tetramers (TMrs) and pentamers (PMrs), encompassing two epitopes of preproinsulin (PPI; PPIA12-20 and PPIB10-18) and two epitopes of glutamic acid decarboxylase (GAD; GAD114-122 and GAD536-545). We also compared the readout of TMrs and PMrs with a CD8+ T-cell interferon-γ enzyme-linked immunospot assay. RESULTS: Despite the minute frequencies of autoreactive cells detected by TMrs/PMrs, most (73-77%) T1D patients had responses to one or more of the epitopes used. All four epitopes were recognized by T1D patients, with a prevalence ranging from 5 to 25%. TMrs and PMrs detected more positive responses to the ß-cell epitopes than CD8+ T-cell interferon-γ enzyme-linked immunospot. However, concordance between positive responses to TMrs and PMrs was limited. CONCLUSIONS: Using a multicentre blind-coded setup and three different T-cell assays, we have validated PPI and GAD epitopes as commonly recognized CD8+ T-cell targets in recently diagnosed T1D patients. Both TMrs and PMrs showed higher detection sensitivity than the CD8+ T-cell interferon-γ enzyme-linked immunospot assay. However, there are some important methodological issues that need to be addressed in using these sensitive techniques for detecting low frequency responses.

Identification of an MHC class I-restricted autoantigen in type 1 diabetes by screening an organ-specific cDNA library.

Type 1 diabetes is an autoimmune disease in which the insulin-producing pancreatic beta cells are destroyed at an early age by an immune process that involves both CD4 and CD8 T lymphocytes. The identification of autoantigens in diabetes is very important for the design of antigen-specific immunotherapy. By screening a pancreatic islet cDNA library, we have identified the autoantigen recognized by highly pathogenic CD8 T cells in the non-obese diabetic mouse, one of the best animal models for human diabetes. This is the first identification, to our knowledge, of a CD8 T-cell epitope in an autoimmune disease. The peptide recognized by the cells is in the same region of the insulin B chain as the epitope recognized by previously isolated pathogenic CD4 T cells. This has very important implications for the potential use of insulin in preventative therapy.

A combined UASB-MBR with shortcut nitrification-denitrification for energy reduction in wastewater reclamation.

Shortcut nitrification has been successfully applied in a laboratory scale nitrification-denitrification process consisting of an up-flow anaerobic sludge blanket (UASB) and an aerobic membrane bioreactor (MBR) in treating synthetic and municipal wastewater to simultaneously remove organic carbon and nitrogen. For the treatment of synthetic wastewater, the combined system exhibited a high TOC removal of 98% with a steady ammonia removal efficiency of about 98% in the MBR and a total nitrogen (TN) removal efficiency of 90%. In treating municipal wastewater, due to its low COD concentration, removal efficiencies of TOC, ammonia and TN were 70%, 98% and 60%, respectively. The biogas production was around 76.4 L/m3 wastewater when treating synthetic wastewater. However, little biogas was produced when treating municipal wastewater which was the result of low organic carbon loading to the UASB. Energy analysis has demonstrated that this novel shortcut nitrification process could consume less energy than a conventional process and have the potential of bio-energy generation via biogas production thus helping to achieve a more favorable energy balance.

Referral rates of patients with diabetes to secondary care are inversely related to the prevalence of diabetes in each primary care practice and confidence in treatment, not to HbA1c level.

AIMS: To determine the factors affecting the referral rates of patients with diabetes from primary care to secondary care. METHODS: A study based on 66 GP surgeries in the Cardiff and Vale University Health Board (population: 515,581) was conducted. We included patients who had an established clinical diagnosis of diabetes (type 1 and type 2) from September 2017 to September 2018. HbA1c outcome data of GP surgeries were obtained from the Quality and Outcomes Framework (QOF) database published for 2018. Referral rates were obtained from the electronic referral database of Cardiff and Vale University Health Board over the same period, and this was adjusted according to the number of patients with diabetes in each GP surgery. Confidence level on the treatment of diabetes among GPs was assessed as a sub-study conducted in nine GP surgeries in the same area, using a self-administered questionnaire. Linear regression was undertaken to assess the relationship between adjusted referral rate and key factors which might influence prescribing rate. RESULTS: The average adjusted referral rate to secondary care in one year was 4.23% of patients with diabetes in each GP surgery, with a wide variation of 1.24% to 16.28%. The average percentage of patients with diabetes with HbA1c<59mmol/mol was 63.17% (range: 43.19-76.23%). The average confidence score of GPs in treating diabetes was 67% and ranged from 50-85% in the sub-study. Referral rates correlated inversely with the numbers of patients with diabetes in each practice ß=-0.32; (95% CI -0.57, -0.08) p=0.01, but there was no significant correlation with the HbA1c outcome ß=-0.13; (95% CI -0.39, 0.12); p=0.30. Borderline significant negative correlation was observed between referral rates and overall practice size ß=-0.23; (95% CI -0.48, 0.02) p=0.07. CONCLUSIONS: Referral rates of patients with diabetes to secondary care are determined by the number of patients with diabetes in each practice and confidence level in treatment, not by the overall practice size or HbA1c level. Ensuring quality training in diabetes care for primary care teams as well as the development of integrated diabetes care may be the best way to optimise the volume and appropriateness of referrals to secondary care.

Remineralising fluorine containing bioactive glass composites.

OBJECTIVES: The objective was to investigate the mechanical properties, fluoride release and apatite formation of resin based dental composites based on a fluoride containing Bioactive Glass (BG) with and without a silylating agent. METHODS: A SiO2-P2O5-CaO-SrO-Na2O-CaF2 BG was synthesized by the melt quench route. This glass and a commercially available inert glass (IG) were incorporated into a light cured BisGMA-TEGMA resin. The composite resins were then evaluated in terms of their ability to form apatite by Fourier Transform Infrared spectroscopy (FTIR) and by scanning electron microscopy (SEM) following immersion in artificial saliva at pH 4 (AS4) and pH 7 (AS7). The experiments were performed with and without silylation of the BG. The compressive strength and flexural strength were determined after 1, 28 and 84 days of immersion in the AS4 and AS7 immersion media. RESULTS: The FTIR spectra of the BG composites exhibited split bands at approximately 560 and 600 cm-1 corresponding to a apatite formation in the surface or on the surface under all immersion conditions. SEM showed the presence of a reacted layer of glass particles in the composite surface and the presence of a surface layer of apatite in AS7. The compressive strength and flexural strength were significantly higher for the silylated BG composites. The strengths of both silylated and non silylated BG composites and IG composites decreased upon immersion. SIGNIFICANCE: BG composites exhibit reduced strengths upon immersion but still exhibit strengths comparable to existing composites after 84 days of immersion.

IFN-gamma and IL-10 islet-antigen-specific T cell responses in autoantibody-negative first-degree relatives of patients with type 1 diabetes.

AIMS/HYPOTHESIS: Islet antibody-negative first-degree relatives of type 1 diabetes patients have a very low risk of developing diabetes. We studied the balance between IFN-gamma (proinflammatory) and IL-10 (regulatory) T cell responses in these participants. METHODS: Peripheral blood T cells from adult (18-50 years old, n = 40) DRB1*0401-positive first-degree relatives negative for GAD and tyrosine phosphatase-like insulinoma antigen 2 (IA-2) antibodies were tested for IFN-gamma and IL-10 responses in a sensitive cytokine enzyme-linked immunospot assay against a panel of seven peptide epitopes derived from IA-2 and proinsulin. Comparison was made with HLA-matched newly diagnosed type 1 diabetic patients (n = 42) and healthy controls (n = 39). RESULTS: First-degree relatives and newly diagnosed type 1 diabetic patients displayed a similar frequency of IFN-gamma responses to the peptide panel and both were significantly greater than in healthy controls (relatives 9.6%, patients 11.8%, controls 4.0%, p = 0.003). First-degree relatives and newly diagnosed type 1 diabetic patients also showed similar frequencies of IL-10 responses, which were significantly lower than in healthy controls (relatives 7.1%, patients 9.0%, controls 15.8%, p = 0.003). However, individual IL-10 responses of first-degree relatives were similar in size to those in healthy controls and larger than those in newly diagnosed type 1 diabetic patients (relatives median 29 spot-forming cells/1 x 10(6) peripheral blood mononuclear cells, controls 33, patients 11, p = 0.02). CONCLUSIONS/INTERPRETATION: Taken together, these results suggest that antibody-negative first-degree relatives have a balance of proinflammatory and regulatory T cells, which is intermediate between that of newly diagnosed type 1 diabetic patients and healthy controls. This suggests that even a moderate regulatory response may be sufficient to prevent the development of clinical type 1 diabetes in genetically predisposed individuals.