Search

The bioartificial pancreas encapsulating pancreatic islets in immunoprotective hydrogel is a promising therapy for Type 1 diabetes. As pancreatic islets are highly metabolically active and exquisitely sensitive to hypoxia, maintaining O2 supply after transplantation remains a major challenge. In this study, we address the O2 limitation by combining silicone‐encapsulated CaO2 (silicone‐CaO2) to generate O2 with an extracellular hemoglobin O2‐carrier coencapsulated with islets. We showed that the hemoglobin improved by 37% the O2‐diffusivity through an alginate hydrogel and displayed antioxidant properties neutralizing deleterious reactive O2 species produced by silicone‐CaO2. While the hemoglobin alone failed to maintain alginate macroencapsulated neonate pig islets under hypoxia, silicone‐CaO2 alone or combined to the hemoglobin restored islet viability and insulin secretion and prevented proinflammatory metabolism (PTGS2 expression). Interestingly, the combination took the advantages of the two individual strategies, improved neonate pig islet viability and insulin secretion in normoxia, and VEGF secretion and PDK1 normalization in hypoxia. Moreover, we confirmed the specific benefits of the combination compared to silicone‐CaO2 alone on murine pseudo‐islet viability in normoxia and hypoxia. For the first time, our results show the interest of combining an O2 provider with hemoglobin as an effective strategy to overcome O2 limitations in tissue engineering. Mouré et al. adress the O2 limitation in a bioartificial pancreas, composed of neonate pig islets encapsulated in an alginate hydrogel, by combining an O2 generator (silicone‐CaO2) with an extracellular hemoglobin O2‐carrier. By increasing the O2 diffusivity in the alginate hydrogel and neutralising reactive O2 species produced by the silicone‐CaO2, the hemoglobin aims to improve the integration of the O2 generator into the device and the adaptation of the bioartificial pancreas to the varying O2 tensions encountered after transplantation. [ABSTRACT FROM AUTHOR]

Heme oxygenase-1 ( HO-1) inhibits immune responses and inflammatory reactions via the catabolism of heme into carbon monoxide ( CO), Fe2+, and biliverdin. We have previously shown that either induction of HO-1 or treatment with exogenous CO inhibits LPS-induced maturation of dendritic cells ( DCs) and protects in vivo and in vitro antigen-specific inflammation. Here, we evaluated the capacity of HO-1 and CO to regulate antigen presentation on MHC class I and MHC class II molecules by LPS-treated DCs. We observed that HO-1 and CO treatment significantly inhibited the capacity of DCs to present soluble antigens to T cells. Inhibition was restricted to soluble OVA protein, as no inhibition was observed for antigenic OVA-derived peptides, bead-bound OVA protein, or OVA as an endogenous antigen. Inhibition of soluble antigen presentation was not due to reduced antigen uptake by DCs, as endocytosis remained functional after HO-1 induction and CO treatment. On the contrary, CO significantly reduced the efficiency of fusion between late endosomes and lysosomes and not by phagosomes and lysosomes. These data suggest that HO-1 and CO can inhibit the ability of LPS-treated DCs to present exogenous soluble antigens to naïve T cells by blocking antigen trafficking at the level of late endosome-lysosome fusion. [ABSTRACT FROM AUTHOR]

Carbon monoxide (CO) treatment improves pathogenic outcome of autoimmune diseases by promoting tolerance. However, the mechanism behind this protective tolerance is not yet defined. Here, we show in a transgenic mouse model for autoimmune diabetes that ex vivo gaseous CO (gCO)-treated DCs loaded with pancreatic β-cell peptides protect mice from disease. This protection is peptide-restricted, independent of IL-10 secretion by DCs and of CD4+ T cells. Although no differences were observed in autoreactive CD8+ T-cell function from gCO-treated versus untreated DC-immunized groups, gCO-treated DCs strongly inhibited accumulation of autoreactive CD8+ T cells in the pancreas. Interestingly, induction of β1-integrin was curtailed when CD8+ T cells were primed with gCO-treated DCs, and the capacity of these CD8+ T cells to lyse isolated islet was dramatically impaired. Thus, immunotherapy using CO-treated DCs appears to be an original strategy to control autoimmune disease. [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]

Plasmid-DNA gene-gun immunization may be an efficient approach for investigating the role of skin dendritic cells (DCs) in type 1 diabetes (T1D) pathogenesis and the significance of the presentation of peptides that mimic autoantigenic epitopes in aggravating or modulating the autoimmune reaction. Gene-gun immunization has been described as producing longlasting immune responses elicited by skin DCs, especially Langerhans cells (LCs). Therefore, we tested the immune response and diabetes modulation in nonobese diabetic (NOD) mice and in control BALB/c mice, by gene-gun administration of plasmid-DNA encoding (1) human 65 kDa glutamic acid decarboxylase (hGAD65) mimicking the crucial mouse autoantigen GAD65 (similarity of 95.7%) or (2) β-galactosidase (βGAL) as a negative control. Expression of GAD and βGAL in skin of pc-GAD- and pc-LacZ-injected mice, respectively, was confirmed. It was surprising that both pc-LacZ-injected BALB/c and NOD mice exhibited a βGAL-specific Th1 immune response: spleen cells of pc-LacZ mice proliferated specifically to βGAL (P < 10-4) and secreted significant amounts of IFNγ (P < 10-4). pc-LacZ mice also developed a βGAL-specific Th1-related (IgG2a/2c) and Th2-related (IgG1) humoral response. Although pc-GAD BALB/c mice showed Th2-related GAD-specific IgG1 production and a significant secretion of IL4 (P < .03), pc-GAD NOD mice did not generate either an antibody response or a T cell response specific to GAD. Moreover, gene-gun immunization encoding hGAD65 did not clearly modulate diabetes onset in NOD mice. This absence of detectable GAD-specific response may implicate skin DC deficiencies in NOD mice. The gene-gun technique could thus provide an interesting model for studying skin DC abnormalities in NOD mice and their potential implication of presenting mimetic peptides that modulate the autoimmune response in T1D. [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]

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]