Coxsackievirus infection induces direct pancreatic ß cell killing but poor antiviral CD8+ T cell responses.

Coxsackievirus B (CVB) infection of pancreatic ß cells is associated with ß cell autoimmunity and type 1 diabetes. We investigated how CVB affects human ß cells and anti-CVB T cell responses. ß cells were efficiently infected by CVB in vitro, down-regulated human leukocyte antigen (HLA) class I, and presented few, selected HLA-bound viral peptides. Circulating CD8+ T cells from CVB-seropositive individuals recognized a fraction of these peptides; only another subfraction was targeted by effector/memory T cells that expressed exhaustion marker PD-1. T cells recognizing a CVB epitope cross-reacted with ß cell antigen GAD. Infected ß cells, which formed filopodia to propagate infection, were more efficiently killed by CVB than by CVB-reactive T cells. Our in vitro and ex vivo data highlight limited CD8+ T cell responses to CVB, supporting the rationale for CVB vaccination trials for type 1 diabetes prevention. CD8+ T cells recognizing structural and nonstructural CVB epitopes provide biomarkers to differentially follow response to infection and vaccination.

Coxsackievirus infection induces direct pancreatic ß-cell killing but poor anti-viral CD8+ T-cell responses.

Coxsackievirus B (CVB) infection of pancreatic ß cells is associated with ß-cell autoimmunity. We investigated how CVB impacts human ß cells and anti-CVB T-cell responses. ß cells were efficiently infected by CVB in vitro, downregulated HLA Class I and presented few, selected HLA-bound viral peptides. Circulating CD8+ T cells from CVB-seropositive individuals recognized only a fraction of these peptides, and only another sub-fraction was targeted by effector/memory T cells that expressed the exhaustion marker PD-1. T cells recognizing a CVB epitope cross-reacted with the ß-cell antigen GAD. Infected ß cells, which formed filopodia to propagate infection, were more efficiently killed by CVB than by CVB-reactive T cells. Thus, our in-vitro and ex-vivo data highlight limited T-cell responses to CVB, supporting the rationale for CVB vaccination trials for type 1 diabetes prevention. CD8+ T cells recognizing structural and non-structural CVB epitopes provide biomarkers to differentially follow response to infection and vaccination.

Coxsackievirus and Type 1 Diabetes: Diabetogenic Mechanisms and Implications for Prevention.

The evidence for an association between coxsackievirus B (CVB) infection, pancreatic islet autoimmunity, and clinical type 1 diabetes is increasing. Results from prospective cohorts and pancreas histopathology studies have provided a compelling case. However, the demonstration of a causal relationship is missing, and is likely to remain elusive until tested in humans by avoiding exposure to this candidate viral trigger. To this end, CVB vaccines have been developed and are entering clinical trials. However, the progress made in understanding the biology of the virus and in providing tools to address the long-standing question of causality contrasts with the scarcity of information about the antiviral immune responses triggered by infection. Beta-cell death may be primarily induced by CVB itself, possibly in the context of poor immune protection, or secondarily provoked by T-cell responses against CVB-infected beta cells. The possible involvement of epitope mimicry mechanisms skewing the physiological antiviral response toward autoimmunity has also been suggested. We here review the available evidence for each of these 3 non-mutually exclusive scenarios. Understanding which ones are at play is critical to maximize the odds of success of CVB vaccination, and to develop suitable tools to monitor the efficacy of immunization and its intermingling with autoimmune onset or prevention.

The type 1 diabetes gene TYK2 regulates ß-cell development and its responses to interferon-α.

Type 1 diabetes (T1D) is an autoimmune disease that results in the destruction of insulin producing pancreatic ß-cells. One of the genes associated with T1D is TYK2, which encodes a Janus kinase with critical roles in type-Ι interferon (IFN-Ι) mediated intracellular signalling. To study the role of TYK2 in ß-cell development and response to IFNα, we generated TYK2 knockout human iPSCs and directed them into the pancreatic endocrine lineage. Here we show that loss of TYK2 compromises the emergence of endocrine precursors by regulating KRAS expression, while mature stem cell-islets (SC-islets) function is not affected. In the SC-islets, the loss or inhibition of TYK2 prevents IFNα-induced antigen processing and presentation, including MHC Class Ι and Class ΙΙ expression, enhancing their survival against CD8+ T-cell cytotoxicity. These results identify an unsuspected role for TYK2 in ß-cell development and support TYK2 inhibition in adult ß-cells as a potent therapeutic target to halt T1D progression.

Circulating platelet-neutrophil aggregates characterize the development of type 1 diabetes in humans and NOD mice.

Platelet-neutrophil aggregates (PNAs) facilitate neutrophil activation and migration and could underpin the recruitment of neutrophils to the pancreas during type 1 diabetes (T1D) pathogenesis. PNAs, measured by flow cytometry, were significantly elevated in the circulation of autoantibody-positive (Aab+) children and new-onset T1D children, as well as in pre-T1D (at 4 weeks and 10-12 weeks) and T1D-onset NOD mice, compared with relevant controls, and PNAs were characterized by activated P-selectin+ platelets. PNAs were similarly increased in pre-T1D and T1D-onset NOD isolated islets/insulitis, and immunofluorescence staining revealed increased islet-associated neutrophil extracellular trap (NET) products (myeloperoxidase [MPO] and citrullinated histones [CitH3]) in NOD pancreata. In vitro, cell-free histones and NETs induced islet cell damage, which was prevented by the small polyanionic drug methyl cellobiose sulfate (mCBS) that binds to histones and neutralizes their pathological effects. Elevated circulating PNAs could, therefore, act as an innate immune and pathogenic biomarker of T1D autoimmunity. Platelet hyperreactivity within PNAs appears to represent a previously unrecognized hematological abnormality that precedes T1D onset. In summary, PNAs could contribute to the pathogenesis of T1D and potentially function as a pre-T1D diagnostic.

Exocrine pancreas function is impaired in adult relatives of patients with type 1 diabetes.

AIMS: Alterations of the exocrine pancreas have been reported in type 1 diabetes, but their contribution to the pathogenesis of the disease is poorly understood. Here, we investigated markers of exocrine pancreas dysfunction in individuals at-risk of developing type 1 diabetes. METHODS: Serum P-amylase and lipase levels were assessed in samples obtained from healthy controls, patients with new onset type 1 diabetes, relatives participating to the TrialNet Pathway to Prevention who were, at blood collection, autoantibody negative or positive for a single autoantibody (low-risk individuals), and positive for multiple autoantibodies (high-risk individuals). Linear mixed models were adopted to estimate variation of pancreatic enzymes among the groups and to evaluate the influence of high-risk HLA genotypes and residual beta cell function on exocrine pancreas function. RESULTS: In adults, but not children, reduced levels of P-amylase and lipase were shown in at-risk individuals, including (for P-amylase levels only) those at low-risk, and in T1Dnew. Furthermore, while high-risk HLA genotypes negatively affected P-amylase levels in autoantibody negative adult individuals, fasting C-peptide levels did not correlate with pancreatic enzyme levels. CONCLUSIONS: Exocrine pancreas dysfunction precedes the onset of type 1 diabetes in adult at-risk individuals and may be unrelated to fasting C-peptide levels.

New Evidence of Exocrine Pancreatopathy in Pre-symptomatic and Symptomatic Type 1 Diabetes.

PURPOSE OF REVIEW: Type 1 diabetes (T1D) is one of the most frequent chronic autoimmune diseases in humans, characterized by the lack of insulin production resulting in high blood glucose levels and lifelong requirement of exogenous insulin administration for survival. It is now recognized that the autoimmune process begins years before the clinical onset, in a stage called pre-symptomatic T1D, in which the presence of ß-cell-specific autoantibodies is detectable. Our aim is to review evidence for T1D as a "whole-pancreas disease," featured by both endocrine and exocrine pancreas alterations already at early disease stages. RECENT FINDINGS: In this review, we discuss a series of recent observations indicating that in genetically predisposed individuals, structural and functional abnormalities as well as immune cell infiltration of the exocrine pancreas are already present in the pre-symptomatic stages of the disease. Despite T1D being considered a ß-cell-specific disease, numerous reports point to the presence of exocrine pancreas subclinical abnormalities occurring during disease development. These observations challenge the long-standing idea that T1D exocrine damage exists as a mere consequence of disease progression and provide further explanation of mechanisms underlying T1D pathogenesis.

Neutrophils and type 1 diabetes: current knowledge and suggested future directions.

PURPOSE OF REVIEW: Purpose of this review is to describe the most recent human studies on neutrophils in type 1 diabetes (T1D) and to focus on the key questions that still need to be addressed. RECENT FINDINGS: Recent evidences demonstrate that neutrophils have marked abnormalities in phenotype and function and play a central role in initiation and perpetuation of aberrant immune responses and organ damage in various systemic autoimmune diseases such as lupus erythematosus and rheumatoid arthritis. In T1D, we have recently demonstrated that reduced circulating neutrophil numbers precede and accompany the disease and that neutrophils infiltrate the pancreas and extrude neutrophil extracellular traps already before the onset of clinical symptoms. However, few other evidences of alterations in neutrophil phenotype and function have been reported in humans, especially in the T1D presymptomatic phases. SUMMARY: Dissecting the pathogenic role of these cells in human T1D is crucial for a better understanding of the disease and to open new therapeutic opportunities.

Abnormal neutrophil signature in the blood and pancreas of presymptomatic and symptomatic type 1 diabetes.

BACKGROUND: Neutrophils and their inflammatory mediators are key pathogenic components in multiple autoimmune diseases, while their role in human type 1 diabetes (T1D), a disease that progresses sequentially through identifiable stages prior to the clinical onset, is not well understood. We previously reported that the number of circulating neutrophils is reduced in patients with T1D and in presymptomatic at-risk subjects. The aim of the present work was to identify possible changes in circulating and pancreas-residing neutrophils throughout the disease course to better elucidate neutrophil involvement in human T1D. METHODS: Data collected from 389 subjects at risk of developing T1D, and enrolled in 4 distinct studies performed by TrialNet, were analyzed with comprehensive statistical approaches to determine whether the number of circulating neutrophils correlates with pancreas function. To obtain a broad analysis of pancreas-infiltrating neutrophils throughout all disease stages, pancreas sections collected worldwide from 4 different cohorts (i.e., nPOD, DiViD, Siena, and Exeter) were analyzed by immunohistochemistry and immunofluorescence. Finally, circulating neutrophils were purified from unrelated nondiabetic subjects and donors at various T1D stages and their transcriptomic signature was determined by RNA sequencing. RESULTS: Here, we show that the decline in ß cell function is greatest in individuals with the lowest peripheral neutrophil numbers. Neutrophils infiltrate the pancreas prior to the onset of symptoms and they continue to do so as the disease progresses. Of interest, a fraction of these pancreas-infiltrating neutrophils also extrudes neutrophil extracellular traps (NETs), suggesting a tissue-specific pathogenic role. Whole-transcriptome analysis of purified blood neutrophils revealed a unique molecular signature that is distinguished by an overabundance of IFN-associated genes; despite being healthy, said signature is already present in T1D-autoantibody-negative at-risk subjects. CONCLUSIONS: These results reveal an unexpected abnormality in neutrophil disposition both in the circulation and in the pancreas of presymptomatic and symptomatic T1D subjects, implying that targeting neutrophils might represent a previously unrecognized therapeutic modality. FUNDING: Juvenile Diabetes Research Foundation (JDRF), NIH, Diabetes UK.

APOA-1Milano muteins, orally delivered via genetically modified rice, show anti-atherogenic and anti-inflammatory properties in vitro and in Apoe-/- atherosclerotic mice.

BACKGROUND: Atherosclerosis is a slowly progressing, chronic multifactorial disease characterized by the accumulation of lipids, inflammatory cells, and fibrous tissue that drives to the formation of asymmetric focal thickenings in the tunica intima of large and mid-sized arteries. Despite the high therapeutic potential of ApoA-1 proteins, the purification and delivery into the disordered organisms of these drugs is still limited by low efficiency in these processes. METHODS AND RESULTS: We report here a novel production and delivery system of anti-atherogenic APOA-1Milano muteins (APOA-1M) by means of genetically modified rice plants. APOA-1M, delivered as protein extracts from transgenic rice seeds, significantly reduced macrophage activation and foam cell formation in vitro in oxLDL-loaded THP-1 model. The APOA-1M delivery method and therapeutic efficacy was tested in healthy mice and in Apoe-/- mice fed with high cholesterol diet (Western Diet, WD). APOA-1M rice milk significantly reduced atherosclerotic plaque size and lipids composition in aortic sinus and aortic arch of WD-fed Apoe-/- mice as compared to wild type rice milk-treated, WD-fed Apoe-/- mice. APOA-1M rice milk also significantly reduced macrophage number in liver of WD-fed Apoe-/- mice as compared to WT rice milk treated mice. TRANSLATIONAL IMPACT: The delivery of therapeutic APOA-1M full length proteins via oral administration of rice seeds protein extracts (the 'rice milk') to the disordered organism, without any need of purification, might overcome the main APOA1-based therapies' limitations and improve the use of this molecules as therapeutic agents for cardiovascular patients.

Use of high-dose acyclovir in pityriasis rosea.

BACKGROUND: The association of human herpesvirus 6 (HHV-6) and HHV-7 with pityriasis rosea suggests that systemic drugs directed against HHV may hasten recovery of patients with pityriasis rosea. OBJECTIVE: The purpose of this study was to verify the efficacy of oral acyclovir in the treatment of pityriasis rosea. METHODS: Eighty-seven consecutive patients were treated for 1 week with either oral acyclovir (800 mg 5 times daily) or placebo. In all patients, the time of lesion clearing and the number of new lesions appearing during treatment were recorded. RESULTS: On the 14th day of treatment, 79% of treated patients fully regressed compared with 4% of the placebo group. The lesions cleared in 18.5 days in treated patients and in 37.9 days in the placebo group. Clearance was achieved in 17.2 days in patients treated in the first week from onset and in 19.7 days in the patients treated later. On the 7th day, there were significantly fewer new lesions in patients treated in the first week than in those treated later. LIMITATIONS: This trial was neither randomized nor double blind. Objectivity was achieved by counting the lesions. CONCLUSION: Acyclovir may be effective in the treatment of pityriasis rosea, especially in patients treated in the first week from onset, when replicative viral activity of HHV is probably very high.

Distinguishing androgenetic alopecia from chronic telogen effluvium when associated in the same patient: a simple noninvasive method.

BACKGROUND: Distinguishing chronic telogen effluvium (CTE) from androgenetic alopecia (AGA) may be difficult especially when associated in the same patient. OBSERVATIONS: One hundred consecutive patients with hair loss who were clinically diagnosed as having CTE, AGA, AGA + CTE, or remitting CTE. Patients washed their hair in the sink in a standardized way. All shed hairs were counted and divided "blindly" into 5 cm or longer, intermediate length (>3 to <5 cm), and 3 cm or shorter. The latter were considered telogen vellus hairs, and patients having at least 10% of them were classified as having AGA. We assumed that patients shedding 200 hairs or more had CTE. The kappa statistic revealed, however, that the best concordance between clinical and numerical diagnosis (kappa = 0.527) was obtained by setting the cutoff shedding value at 100 hairs or more. Of the 100 patients, 18 with 10% or more of hairs that were 3 cm or shorter and who shed fewer than 100 hairs were diagnosed as having AGA; 34 with fewer than 10% of hairs that were 3 cm or shorter and who shed at least 100 hairs were diagnosed as having CTE; 34 with 10% or more of hairs that were 3 cm or shorter and who shed at least 100 hairs were diagnosed as having AGA + CTE; and 14 with fewer than 10% of hairs that were 3 cm or shorter and who shed fewer than 100 hairs were diagnosed as having CTE in remission. CONCLUSION: This method is simple, noninvasive, and suitable for office evaluation.