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The notion that mimicry between a self and a microbial peptide antigen can trigger or aggravate autoimmune pathology remains a popular hypothesis in autoimmunity research. Tremendous recent progress in our understanding of the interface between the T cell receptor (TCR) and peptide/MHC complexes has revealed a vast potential for degenerate recognition of numerous structurally similar pMHC complexes by each T lymphocyte. Moreover, functional and structural studies have confirmed that structural similarity between unrelated pMHC complexes is frequently sufficient for recognition by a single TCR. However, despite clear evidence that vaccination with mimetic microbial antigens has the potential to activate autoreactive T cells, crucial evidence for triggering of autoimmunity by mimetic sequences in natural pathogens is wanting. Antigen spreading, i.e. the fact that the number of self antigens targeted by a chronic autoimmune response tends to increase with its duration, does not facilitate the task of proving initial triggering, or subsequent acceleration, of autoimmune conditions by mimetic microbial antigens. Moreover, considering that activation rather than presence of autoreactive T cells is the hallmark of autoimmune disease, the creation of an environment resulting in failure of tolerance and regulatory mechanisms, rather than emergence of novel microbial antigenic determinants, may well be at the root of autoimmunity. Based on these considerations, we contend that the mimicry concept remains largely hypothetical, and that novel carefully designed animal models are needed to make a convincing case for a role of mimicry in autoimmune diseases. [Copyright &y& Elsevier]

Diabetes constitutes a major public health problem, with dramatic consequences for patients. Both genetic and environmental factors were shown to contribute to the different forms of the disease. The monogenic forms, found both in humans and in animal models, specially help to decipher the role of key genes in the physiopathology of the disease. Here, we describe the phenotype of early diabetes in a colony of NOD mice, with spontaneous invalidation of Akt2, that we called HYP. The HYP mice were characterised by a strong and chronic hyperglycaemia, beginning around the age of one month, especially in male mice. The phenotype was not the consequence of the acceleration of the autoimmune response, inherent to the NOD background. Interestingly, in HYP mice, we observed hyperinsulinemia before hyperglycaemia occurred. We did not find any difference in the pancreas' architecture of the NOD and HYP mice (islets' size and staining for insulin and glucagon) but we detected a lower insulin content in the pancreas of HYP mice compared to NOD mice. These results give new insights about the role played by Akt2 in glucose homeostasis and argue for the ß cell failure being the primary event in the course of diabetes. [ABSTRACT FROM AUTHOR]

Background: Multiple antigenic stimulations are crucial to immune system training during early post-natal life. These stimulations can be either due to commensals, which accounts for the acquisition and maintenance of tolerance, or to pathogens, which triggers immunity. In pig, only few works previously explored the influence of natural exposition to pathogens upon immune competence. We propose herein the results of a multicentric, field study, conducted on 265 piglets exposed to contrasted pathogen levels in their living environment. Piglets were housed in 15 different commercial farms, sorted in two groups, low (HSLOW)- and high (HSHIGH)-health status farms, depending on their recurrent exposition to five common swine pathogens. Results: Using animal-based measures, we compared the immune competence and growth performances of HSLOW and HSHIGH pigs around weaning. As expected, we observed a rise in the number of circulating leucocytes with age, which affected different cell populations. Monocyte, antigen-experienced and cytotoxic lymphocyte subpopulation counts were higher in piglets reared in HSLOW farms as compared to their HSHIGH homologs. Also, the age-dependent evolution in γδ T cell and neutrophil counts was significantly affected by the health status. With age, circulating IFNα level decreased and IgM level increased while being greater in HSLOW piglets at any time. After weaning, LPS-stimulated blood cells derived from HSLOW piglets were more prone to secrete IL-8 than those derived from HSHIGH pigs did. Monocytes and granulocytes issued from HSLOW pigs also exhibited comparable phagocytosis capacity. Altogether our data emphasize the more robust immunophenotype of HSLOW piglets. Finally, piglets raised under higher pathogen pressure grew less than HSHIGH piglets did and exhibited a different metabolic profile. The higher cost of the immune responses associated with the low farm health status may account for lower HSLOW piglet performances. Conclusions: Altogether, our data, obtained in field conditions, provide evidence that early exposure to pathogens shapes the immune competence of piglets. They also document the negative impact of an overstimulation of the immune system on piglets' growth. [ABSTRACT FROM AUTHOR]

A bioartificial pancreas (BAP) encapsulating high pancreatic islets concentration is a promising alternative for type 1 diabetes therapy. However, the main limitation of this approach is O2 supply, especially until graft neovascularization. Here, we described a methodology to design an optimal O2-balanced BAP using statistical design of experiment (DoE). A full factorial DoE was first performed to screen two O2-technologies on their ability to preserve pseudo-islet viability and function under hypoxia and normoxia. Then, response surface methodology was used to define the optimal O2-carrier and islet seeding concentrations to maximize the number of viable pseudo-islets in the BAP containing an O2-generator under hypoxia. Monitoring of viability, function and maturation of neonatal pig islets for 15 days in vitro demonstrated the efficiency of the optimal O2-balanced BAP. The findings should allow the design of a more realistic BAP for humans with high islets concentration by maintaining the O2 balance in the device. [ABSTRACT FROM AUTHOR]

CD11b+Gr-1+ inhibitory macrophages (iMacs) were implicated in profound depression of T cell functions sometimes observed during cyclophosphamide treatments and overwhelming infections, through a secretion of nitric oxide (NO). Myeloid origin and maturation stages of iMacs are still unknown. As tumor necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ) contributed crucially to the activation of inducible NO synthase (iNOS) gene transcription and to the differentiation of macrophages, we tested their roles in the induction of iMacs differentiation from bone marrow hematopoietic progenitor cells (HPC) of uncompromised mice. Lineage phenotypes-negative (lin−) c-kit+ cells of Balb/c mice were cultured 6 days with granulocyte-macrophage colony-stimulating factor (GM-CSF), stem cell factor (SCF, c-kit ligand) in presence or not of TNF-α or IFN-γ. CD11b+Gr-1+ cells only derived in presence of {GM-CSF + SCF + TNF-α} or {GM-CSF + SCF + IFN-γ} could express iNOS upon in vitro stimulation with {IFN-γ + TNF-α} or {IFN-γ + LPS} known to boost iNOS expression in murine macrophages. However, whereas {GM-CSF + SCF + TNF-α} induced only weakly iMacs generation and contributed also to the differentiation of CD11b+Gr-1−CD11c+ myeloid dendritic cells, {GM-CSF + SCF + IFN-γ} induced exclusively and importantly iMacs differentiation. Moreover {GM-CSF + SCF + IFN-γ}-generated iMacs were more mature than {GM-CSF + SCF + TNF-α}-derived iMacs since IFN-γ increased more strongly CD11b+Gr-1+ cells expressing Ly-6C and generated lesser cells expressing MHC class II and CD86 molecules. Finally {GM-CSF + SCF + IFN-γ}-generated CD11b+ cells showing a powerful suppressive activity on T cell proliferations, correlated with NO secretion. In conclusion, our study showed, for the first time, that IFN-γ induced very efficiently the differentiation of functional iMacs from lin−c-kit+ murine HPC in vitro, and indicated clearly that iMacs progenitors may be present in bone marrow of naı¨ve mice. [Copyright &y& Elsevier]

The cross-talk between sympatho-adreno-medullar axis and innate immunity players was mainly studied in rodents. In intensive husbandry, pigs are exposed to multiple stressors inducing repeated releases of catecholamines that bind to adrenoreceptors (AR) on target cells. Among adrenoreceptors, the β2-AR is largely expressed by immune cells including macrophages. We report herein on the effects of catecholamines, through β2-AR stimulation, on pig macrophage functions activated by LPS. β2-AR stimulation of porcine macrophages prevented the LPS-induced increase in TNFα and IL-8 secretion while increasing IL-10 secretion. In contrast, treatment with a β2-agonist had no effect on anti-microbial functions. Lastly, β2-AR stimulation of macrophages reduced the expression of genes up-regulated by LPS. Altogether, we demonstrated that β2-AR stimulation of porcine macrophages prevented polarization towards a pro-inflammatory phenotype. Since porcine macrophages are a suitable model for human macrophages, our results might be relevant to appreciate catecholamine effects on human macrophages. [ABSTRACT FROM AUTHOR]

Pig husbandry is known as an intensive breeding system, piglets being submitted to multiple stressful events such as early weaning, successive mixing, crowding and shipping. These stressors are thought to impair immune defences and might contribute, at least partly, to the prophylactic use of antibiotics. Robustness was recently defined as the ability of an individual to express a high-production potential in a wide variety of environmental conditions. Increasing robustness thus appears as a valuable option to improve resilience to stressors and could be obtained by selecting piglets upon their adrenocortical activity. In this study, we aimed at depicting the consequences of an acute social stress on the immune capacity of piglets genetically selected upon divergent hypothalamic-pituitary-adrenocortical (HPA) axis activity. For this purpose, we monitored neuroendocrine and immune parameters, in high- (HPA hi ) and low- (HPA lo ) responders to ACTH, just before and immediately after a one-hour mixing with unfamiliar conspecifics. As expected, stressed piglets displayed higher levels of circulating cortisol and norepinephrine. Blood cell count analysis combined to flow cytometry revealed a stress-induced leukocyte mobilization in the bloodstream with a specific recruitment of CD8α + lymphocytes. Besides, one-hour mixing decreased LPS-induced IL-8 and TNFα secretions in whole-blood assays (WBA) and reduced mononuclear cell phagocytosis. Altogether, our data demonstrate that acute social stress alters immune competence of piglets from both groups, and bring new insights in favour of good farming practices. While for most parameters high- and low-responders to ACTH behaved similarly, HPA hi piglets displayed higher number of CD4 + CD8α − T cells, as well as increased cytokine production in WBA (LPS-induced TNFα and PIL-induced IL-8), which could confer them increased resistance to pathogens. Finally, a principal component analysis including all parameters highlighted that overall stress effects were less pronounced on piglets with a strong HPA axis. Thus, selection upon adrenocortical axis activity seems to reduce the magnitude of response to stress and appears as a good tool to increase piglet robustness. [ABSTRACT FROM AUTHOR]

Polyclonal xenogenic IgGs, although having been used in the prevention and cure of severe infectious diseases, are highly immunogenic, which may restrict their usage in new applications such as Ebola hemorrhagic fever. IgG glycans display powerful xenogeneic antigens in humans, for example α1–3 Galactose and the glycolyl form of neuraminic acid Neu5Gc, and IgGs deprived of these key sugar epitopes may represent an advantage for passive immunotherapy. In this paper, we explored whether low immunogenicity IgGs had a protective effect on a guinea pig model of Ebola virus (EBOV) infection. For this purpose, a double knock-out pig lacking α1–3 Galactose and Neu5Gc was immunized against virus-like particles displaying surface EBOV glycoprotein GP. Following purification from serum, hyper-immune polyclonal IgGs were obtained, exhibiting an anti-EBOV GP titer of 1:100,000 and a virus neutralizing titer of 1:100. Guinea pigs were injected intramuscularly with purified IgGs on day 0 and day 3 post-EBOV infection. Compared to control animals treated with IgGs from non-immunized double KO pigs, the anti-EBOV IgGs-treated animals exhibited a significantly prolonged survival and a decreased virus load in blood on day 3. The data obtained indicated that IgGs lacking α1–3 Galactose and Neu5Gc, two highly immunogenic epitopes in humans, have a protective effect upon EBOV infection. [ABSTRACT FROM AUTHOR]

Background: Rabbit-generated antithymocyte globulins (ATGs), which target human T cells, are widely used as immunosuppressive agents during treatment of kidney allograft recipients. However, ATGs can induce immune complex diseases, including serum sickness disease (SSD). Rabbit and human IgGs have various antigenic differences, including expression of the sialic acid Neu5Gc and α-1-3-Gal (Gal), which are not synthesized by human beings. Moreover, anti-Neu5Gc antibodies have been shown to preexist and be elicited by immunization in human subjects. This study aimed to assess the effect of SSD on long-term kidney allograft outcome and to compare the immunization status of grafted patients presenting with SSD following ATG induction treatment.Methods: We analyzed data from a cohort of 889 first kidney graft recipients with ATG induction (86 with SSD [SSD(+)] and 803 without SSD [SSD(-)]) from the Données Informatisées et Validées en Transplantation data bank. Two subgroups of SSD(+) and SSD(-) patients that had received ATG induction treatment were then assessed for total anti-ATG, anti-Neu5Gc, and anti-Gal antibodies using ELISA assays on sera before and after transplantation.Results: SSD was significantly associated with long-term graft loss (>10 years, P = 0.02). Moreover, SSD(+) patients exhibited significantly elevated titers of anti-ATG (P = 0.043) and anti-Neu5Gc (P = 0.007) IgGs in late post-graft samples compared with SSD(-) recipients.Conclusion: In conclusion, our data indicate that SSD is a major contributing factor of late graft loss following ATG induction and that anti-Neu5Gc antibodies increase over time in SSD(+) patients.Funding: This study was funded by Société d'Accélération du Transfert de Technologies Ouest Valorisation, the European FP7 "Translink" research program, the French National Agency of Research, Labex Transplantex, the Natural Science and Engineering Research Council of Canada, and the Canadian Foundation for Innovation. [ABSTRACT FROM AUTHOR]

In humans, CXCR1 and CXCR2 are two homologous proteins that bind ELR+ chemokines. Both receptors play fundamental roles in neutrophil functions such as migration and reactive oxygen species production. Mouse Cxcr1 and Cxcr2 genes are located in an insulin-dependent diabetes genetic susceptibility locus. The non obese diabetic (NOD) mouse is a spontaneous well-described animal model for insulin-dependent type 1 diabetes. In this disease, insulin deficiency results from the destruction of insulin-producing beta cells by autoreactive T lymphocytes. This slow-progressing disease is dependent on both environmental and genetic factors. Here, we report descriptive data about the Cxcr1 gene in NOD mice. We demonstrate decreased expression of mRNA for Cxcr1 in neutrophils and CD4+ lymphocytes isolated from NOD mice compared to other strains, related to reduced NOD Cxcr1 gene promoter activity. Looking for Cxcr1 protein, we next analyze the membrane proteome of murine neutrophils by mass spectrometry. Although Cxcr2 protein is clearly found in murine neutrophils, we did not find evidence of Cxcr1 peptides using this method. Nevertheless, in view of recently-published experimental data obtained in NOD mice, we argue for possible Cxcr1 involvement in type 1 diabetes pathogenesis. [ABSTRACT FROM AUTHOR]

In addition to important regulatory roles in gene expression through RNA interference, it has recently been shown that microRNAs display immune stimulatory effects through direct interaction with receptors of innate immunity of the Toll-like receptor family, aggravating neuronal damage and tumour growth. Yet no evidence exists on consequences of microRNA immune stimulatory actions in the context of an autoimmune disease. Using microRNA analogues, we here show that pancreatic beta cell-derived microRNA sequences induce pro-inflammatory (TNFa, IFNa, IL-12, IL-6) or suppressive (IL-10) cytokine secretion by primary mouse dendritic cells in a sequence-dependent manner. For miR-29b, immune stimulation in RAW264.7 macrophages involved the endosomal Toll-like receptor-7, independently of the canonical RNA interference pathway. In vivo, the systemic delivery of miR-29b activates CD11b+B220− myeloid and CD11b-B220+ plasmacytoid dendritic cells and induces IFNa, TNFa and IL-6 production in the serum of recipient mice. Strikingly, in a murine model of adoptive transfer of autoimmune diabetes, miR-29b reduces the cytolytic activity of transferred effector CD8+ T-cells, insulitis and disease incidence in a single standalone intervention. Endogenous miR-29b, spontaneously released from beta-cells within exosomes, stimulates TNFa secretion from spleen cells isolated from diabetes-prone NOD mice in vitro. Hence, microRNA sequences modulate innate and ongoing adaptive immune responses raising the question of their potential role in the breakdown of tolerance and opening up new applications for microRNA-based immune therapy. [ABSTRACT FROM AUTHOR]

Despite widespread usage of β-adrenergic receptor (AR) agonists and antagonists in current clinical practice, our understanding of their interactions with the immune system is surprisingly sparse. Among the AR expressed by dendritic cells (DC), β2-AR can modify in vitro cytokine release upon stimulation. Because DC play a pivotal role in CD8+ T cell immune responses, we examined the effects of β2-AR stimulation on MHC class I exogenous peptide presentation and cross-presentation capacities. We demonstrate that β2-AR agonist-exposed mature DC display a reduced ability to cross-present protein Ags while retaining their exogenous peptide presentation capability. This effect is mediated through the nonclassical inhibitory G (Gai/0) protein. Moreover, inhibition of cross-presentation is neither due to reduced costimulatory molecule expression nor Ag uptake, but rather to impaired phagosomal Ag degradation. We observed a crosstalk between the TLR4 and β2-AR transduction pathways at the NF-κB level. In vivo, β2-AR agonist treatment of mice inhibits Ag protein cross-presentation to CD8+ T cells but preserves their exogenous MHC class I peptide presentation capability. These findings may explain some side effects on the immune system associated with stress or β-agonist treatment and pave the way for the development of new immunomodulatory strategies. [ABSTRACT FROM AUTHOR]

Abstract: Cyclophosphamide (CTX) was previously shown to induce the recruitment of immunosuppressive myeloid cells in mouse. In the non-obese diabetic (NOD) mouse, which develops spontaneously type I diabetes, CTX is widely known to accelerate the autoimmune process. Our data demonstrated that CTX actually did mobilize an immunosuppressive myeloid CD11b+ Ly-6G− population in the NOD mouse spleen in addition to a well-identified neutrophil CD11b+ Ly-6G+ population. CD11b+ Ly-6G− cells, in contrast with CD11b+ Ly-6G+ cells, were able to inhibit in vitro mitogen-induced syngeneic T cell proliferation. CD11b+ Ly-6G− cells represented a heterogeneous population mainly made of CD31hi cells and Ly-6C+ monocytes. Only these last ones supported the immunosuppressive in vitro activity and resembled circulating inflammatory monocytes according to flow cytometry, cytology and RT-PCR data. Although CD11b+ Ly-6G− Ly-6C+ cells exhibited immunosuppressive function in vitro, they were not able to control the autoimmune response following CTX injection. Our data show that these CTX-induced immunosuppressive myeloid cells actually behaved as very plastic cells in vitro. Likewise, in the model of prediabetic NOD/SCID mice, CD11b+ Ly-6G− Ly-6C+ were able to differentiate into CD11c+ cells after i.v. injection. Herein, we described a new mechanism by which CTX might induce diabetes acceleration in the NOD mouse. In summary, recruited immunosuppressive cells might participate in the immunopotentiating effect of CTX on the autoimmune response by their further differentiation into immunostimulatory cells. [Copyright &y& Elsevier]

Background: Pancreatic duodenal homeobox-1 (Pdx-1) or Pdx-1-VP16 gene transfer has been shown to induce in vitro rat liver-stem WB cell conversion into pancreatic endocrine precursor cells. High glucose conditions were necessary for further differentiation into functional insulin-producing cells. Pdx-1 has the ability to permeate different cell types due to an inherent protein transduction domain (PTD). In this study, we evaluated liver-to-pancreas conversion of WB cells following Pdx-1 or Pdx-1-VP16 protein transduction. Findings: WB cells were grown in high glucose medium containing Pdx-1 or Pdx-1-VP16 recombinant proteins for two weeks. β-like cell commitment was analysed by RT-PCR of pancreatic endocrine genes. We found that WB cells in high glucose culture spontaneously express pancreatic endocrine genes (Pdx-1, Ngn3, Nkx2.2, Kir6.2). Their further differentiation into β-like cells expressing genes related to endocrine pancreas development (Ngn3, NeuroD, Pax4, Nkx2.2, Nkx6.1, Pdx-1) and β-cell function (Glut-2, Kir6.2, insulin) was achieved only in the presence of Pdx-1(-VP16) protein. Conclusion: These results demonstrate that Pdx-1(-VP16) protein transduction is instrumental for in vitro liver-to-pancreas conversion and is an alternative to gene therapy for β-cell engineering for diabetes cell therapy. [ABSTRACT FROM AUTHOR]

OBJECTIVE--Islet-reactive CD8+ T-cells play a key role in the pathogenesis of type 1 diabetes in the NOD mouse. The predominant T-cell specificities change over time, but whether similar shifts also occur after clinical diagnosis and insulin treatment in type 1 diabetic patients is unknown. RESEARCH DESIGN AND METHODS--We took advantage of a recently validated islet-specific CD8+ T-cell γ-interferon enzyme-linked immunospot (ISL8Spot) assay to follow responses against preproinsulin (PPI), GAD, insulinoma-associated protein 2 (IA-2), and islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) epitopes in 15 HLA-A2+ adult type 1 diabetic patients close to diagnosis and at a second time point 7-16 months later. RESULTS--CD8+ T-cell reactivities were less frequent at follow-up, as 28.6% of responses tested positive at type 1 diabetes diagnosis vs. 13.2% after a median of 11 months (P = 0.003). While GAD and IA-2 autoantibody (aAb) titers were unchanged in 75% of cases, the fraction of patients responding to PPI and/or GAD epitopes by ISL8Spot decreased from 60-67 to 20% (P < 0.02). The previously subdominant IA-2206-214 and IGRP265273 peptides were newly targeted, thus becoming the immunodominant epitopes. CONCLUSIONS--Shifts both in frequency and in immunodominance of CD8+ T-cell responses occur more rapidly than do changes in aAb titers. These different kinetics may suggest complementary clinical applications for T-cell and aAb measurements. Diabetes 57:1312-1320, 2008 [ABSTRACT FROM AUTHOR]

Abstract: During the past decade, glutamic acid decarboxylase (GAD) has been considered a crucial β-cell autoantigen involved in type 1 diabetes in the NOD mouse and human. Recently, the etiological role of GAD has remained controversy. In the NOD mouse, some previous studies argued in favor of a regulatory role for GAD-specific CD4+ T cells, and no diabetogenic CD8+ T cells specific for GAD have been identified so far, discrediting the importance of GAD in β-cell injury. Here, we identified, in the NOD model, a relevant GAD CD8+ T cell epitope (GAD90–98) using immunization with a plasmid encoding GAD, a protocol relying on in vivo processing of peptides from the autoantigenic protein. In pancreatic lymph nodes of naïve female NOD mice, CD8+ T lymphocytes recognizing GAD90–98 peptide were detected during the initial phase of invasive insulitis (between 4 and 8 weeks of age), suggesting an important role for these cells in the first stage of the disease. GAD90–98 specific CD8+ lymphocytes lysed efficiently islet cells in vitro and transferred diabetes into NODSCID mice (100%). Finally, diabetes was accelerated greatly in 3-week-old female NOD mice injected i.p. with GAD90–98, strengthening the role of GAD-specific CTLs in diabetes pathogenesis. [Copyright &y& Elsevier]

Despite the understanding that type 1 diabetes pathogenesis is mediated by T-cells, detection of these rare lymphocytes remains largely elusive. Suitable T-cell assays are highly needed, since they could offer preclinical diagnoses and immune surrogate end points for clinical trials. Although CD4+ T-cell assays have met with limited success, CD8+ T-cells are increasingly recognized as key actors in the diabetes of the NOD mouse. CD8+ T-cells are likely to play a role also in humans and may provide new markers of β-cell autoimmunity. Taking advantage of a panel of HLA-A2-restricted β-cell epitopes derived from preproinsulin, GAD, and islet glucose-6-phosphatase catalytic subunit-related protein (IGRP), we have implemented an islet-specific CD8+ T-cell interferon-γ enzyme-linked immunospot (ISL8Spot) assay. The ISL8Spot assay is capable of detecting and quantifying β-cell-reactive CD8+ T-cells directly ex vivo, without any preliminary expansion, using either fresh or frozen samples. Positive ISL8Spot responses separate new-onset diabetic and healthy samples with high accuracy (86% sensitivity, 91% specificity), using as few as five immunodominant epitopes. Moreover, sensitivity reaches 100% when the ISL8Spot assay is complemented by antibody determinations. Combination of CD8+ T-cell measurements with immune intervention strategies may open new avenues toward type 1 diabetes prediction and prevention. Diabetes 56:613-621, 2007 [ABSTRACT FROM AUTHOR]

Type 1 diabetes mellitus (T1DM) results from the destruction of β cells by autoantigen-specific T cells. In the non-obese diabetic (NOD) mouse model, CD8+ T cells play an essential role in both the initial triggering of insulitis and its destructive phase, and proinsulin (PI) is one of the dominant target antigens (Ags). However, little is known about the beta cell epitopes presented by HLA class I molecules and recognized by human CD8+ T cells. We and other groups recently applied reverse immunology approaches to identify HLA class I-restricted PI epitopes. To establish an inventory of potential naturally processed epitopes, whole human PI or the transitional region between the B-chain and C-peptide were digested with purified proteasome complexes. By combining proteasome digestion data with epitope prediction algorithms, candidate epitopes restricted by HLA-A2.1 and other HLA class I molecules were identified. We validated immunogenicity and natural processing of the identified PI epitopes in HLA-A2.1-transgenic mice, while others demonstrated recognition of multiple PI epitopes by CD8+ T cells from T1DM and healthy subjects in the context of different HLA class I molecules. These results demonstrate the power of reverse immunology strategies for epitope discovery. DNA vaccination of HLA-transgenic mice may be another rapid and efficient reverse immunology approach to map additional epitopes derived from other T1DM Ags, such as IA-2 and glutamic acid decarboxylase 65 (GAD 65). Transfer of this information to Elispot- and MHC tetramer-based assay formats should allow to reliably detect and characterize autoreactive CD8+ T cell responses in T1DM, and may open new avenues for early T1DM diagnosis and immune intervention. [ABSTRACT FROM AUTHOR]

Two well‐characterized carbohydrate epitopes are absent in humans but present in other mammals. These are galactose‐α1,3‐galactose (αGal) and N‐glycolylneuraminic acid (Neu5Gc) which are introduced by the activities of two enzymes including α(1,3) galactosyltransferase (encoded by the GGTA1 gene) and CMP‐Neu5Gc hydroxylase (encoded by the CMAH gene) that are inactive in humans but present in cattle. Hence, bovine‐derived products are antigenic in humans who receive bioprosthetic heart valves (BHVs) or those that suffer from red meat syndrome. Using programmable nucleases, we disrupted (knockout, KO) GGTA1 and CMAH genes encoding for the enzymes that catalyse the synthesis of αGal and Neu5Gc, respectively, in both male and female bovine fibroblasts. The KO in clonally selected fibroblasts was detected by polymerase chain reaction (PCR) and confirmed by Sanger sequencing. Selected fibroblasts colonies were used for somatic cell nuclear transfer (SCNT) to produce cloned embryos that were implanted in surrogate recipient heifers. Fifty‐three embryos were implanted in 33 recipients heifers; 3 pregnancies were carried to term and delivered 3 live calves. Primary cell cultures were established from the 3 calves and following molecular analyses confirmed the genetic deletions. FACS analysis showed the double‐KO phenotype for both antigens confirming the mutated genotypes. Availability of such cattle double‐KO model lacking both αGal and Neu5Gc offers a unique opportunity to study the functionality of BHV manufactured with tissues of potentially lower immunogenicity, as well as a possible new clinical approaches to help patients with red meat allergy syndrome due to the presence of these xenoantigens in the diet. [ABSTRACT FROM AUTHOR]

Antibodies of non‐human mammals are glycosylated with carbohydrate antigens, such as galactose‐α‐1‐3‐galactose (α‐Gal) and N‐glycolylneuraminic acid (Neu5Gc). These non‐human carbohydrate antigens are highly immunogenic in humans due to loss‐of‐function mutations of the key genes involved in their synthesis. Such immunogenic carbohydrates are expressed on therapeutic polyclonal rabbit anti‐human T‐cell IgGs (anti‐thymocyte globulin; ATG), the most popular induction treatment in allograft recipients. To decipher the quantitative and qualitative response against these antigens in immunosuppressed patients, particularly against Neu5Gc, which may induce endothelial inflammation in both the graft and the host. We report a prospective study of the antibody response against α‐Gal and Neu5Gc‐containing glycans following rabbit ATG induction compared to controls. We show a drop in the overall levels of anti‐Neu5Gc antibodies at 6 and 12 months post‐graft compared to the pre‐existing levels due to the major early immunosuppression. However, in contrast, in a cross‐sectional study there was a highly significant increase in anti‐Neu5Gc IgGs levels at 6 months post‐graft in the ATG‐treated compared to non‐treated patients(P = 0.007), with a clear hierarchy favouring anti‐Neu5Gc over anti‐Gal response. A sialoglycan microarray analysis revealed that the increased anti‐Neu5Gc IgG response was still highly diverse against multiple different Neu5Gc‐containing glycans. Furthermore, some of the ATG‐treated patients developed a shift in their anti‐Neu5Gc IgG repertoire compared with the baseline, recognizing different patterns of Neu5Gc‐glycans. In contrast to Gal, Neu5Gc epitopes remain antigenic in severely immunosuppressed patients, who also develop an anti‐Neu5Gc repertoire shift. The clinical implications of these observations are discussed. [ABSTRACT FROM AUTHOR]

Background: The sustainability of farming and animal welfare requires the reconsideration of current selection schemes. In particular, implementation of new selection criteria related to animal health and welfare should help to produce more robust animals and to reduce anti-microbial use. The hypothalamo-pituitary-adrenocortical (HPA) axis plays a major role in metabolic regulation and adaptation processes and its activity is strongly influenced by genetic factors. A positive association between HPA axis activity and robustness was recently described. To explore whether selecting pigs upon HPA axis activity could increase their robustness, a divergent selection experiment was carried out in the Large White pig breed. This allowed the generation of low (HPAlo) and high (HPAhi) responders to adrenocorticotropic hormone administration. Results: In this study, we compared 23 hematologic and immune parameters of 6-week-old, HPAlo and HPAhi piglets and analysed their response to a low dose of lipopolysaccharide (LPS) two weeks later. At six weeks of age, HPAhi piglets displayed greater red blood cell and leucocyte number including CD8α+ γδ cells, cytotoxic T lymphocytes, naive T helper (Th) cells and B lymphocytes as compared to HPAlo individuals. The ability of blood cells to secrete TNFα in response to LPS ex vivo was higher for HPAhi pigs. At week eight, the inflammatory response to the LPS in vivo challenge was poorly affected by the HPA axis activity. Conclusions: Divergent selection upon HPA axis activity modulated hematologic and immune parameters in 6-week-old pigs, which may confer an advantage to HPAhi pigs at weaning. However, HPAlo and HPAhi piglets did not exhibit major differences in the parameters analysed two weeks later, i. e. in 8-week-old pigs. In conclusion, chronic exposure to high cortisol levels in HPAhi pigs does not negatively impact immunity. [ABSTRACT FROM AUTHOR]

The last decade has seen a sharp increase in the number of scientific publications describing physiological and pathological functions of extracellular vesicles (EVs), a collective term covering various subtypes of cell-released, membranous structures, called exosomes, microvesicles, microparticles, ectosomes, oncosomes, apoptotic bodies, and many other names. However, specific issues arise when working with these entities, whose size and amount often make them difficult to obtain as relatively pure preparations, and to characterize properly. The International Society for Extracellular Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles ("MISEV") guidelines for the field in 2014. We now update these "MISEV2014" guidelines based on evolution of the collective knowledge in the last four years. An important point to consider is that ascribing a specific function to EVs in general, or to subtypes of EVs, requires reporting of specific information beyond mere description of function in a crude, potentially contaminated, and heterogeneous preparation. For example, claims that exosomes are endowed with exquisite and specific activities remain difficult to support experimentally, given our still limited knowledge of their specific molecular machineries of biogenesis and release, as compared with other biophysically similar EVs. The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV-associated functional activities. Finally, a checklist is provided with summaries of key points. [ABSTRACT FROM AUTHOR]

The last decade has seen a sharp increase in the number of scientific publications describing physiological and pathological functions of extracellular vesicles (EVs), a collective term covering various subtypes of cell-released, membranous structures, called exosomes, microvesicles, microparticles, ectosomes, oncosomes, apoptotic bodies, and many other names. However, specific issues arise when working with these entities, whose size and amount often make them difficult to obtain as relatively pure preparations, and to characterize properly. The International Society for Extracellular Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles ("MISEV") guidelines for the field in 2014. We now update these "MISEV2014" guidelines based on evolution of the collective knowledge in the last four years. An important point to consider is that ascribing a specific function to EVs in general, or to subtypes of EVs, requires reporting of specific information beyond mere description of function in a crude, potentially contaminated, and heterogeneous preparation. For example, claims that exosomes are endowed with exquisite and specific activities remain difficult to support experimentally, given our still limited knowledge of their specific molecular machineries of biogenesis and release, as compared with other biophysically similar EVs. The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV-associated functional activities. Finally, a checklist is provided with summaries of key points. [ABSTRACT FROM AUTHOR]