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Xenocell therapy from neonate or adult pig pancreatic islets is one of the most promising alternatives to allograft in type 1 diabetes for addressing organ shortage. In humans, however, natural and elicited antibodies specific for pig xenoantigens, α-(1,3)-galactose (GAL) and N -glycolylneuraminic acid (Neu5Gc), are likely to significantly contribute to xenoislet rejection. We obtained double-knockout (DKO) pigs lacking GAL and Neu5Gc. Because Neu5Gc -/- mice exhibit glycemic dysregulations and pancreatic β-cell dysfunctions, we evaluated islet function and glucose metabolism regulation in DKO pigs. Isolation of islets from neonate piglets yielded identical islet equivalent quantities to quantities obtained from control wild-type pigs. In contrast to wild-type islets, DKO islets did not induce anti-Neu5Gc antibody when grafted in cytidine monophosphate- N -acetylneuraminic acid hydroxylase KO mice and exhibited in vitro normal insulin secretion stimulated by glucose and theophylline. Adult DKO pancreata showed no histological abnormalities, and immunostaining of insulin and glucagon was similar to that from wild-type pancreata. Blood glucose, insulin, C-peptide, the insulin-to-glucagon ratio, and HOMA-insulin resistance in fasted adult DKO pigs and blood glucose and C-peptide changes after intravenous glucose or insulin administration were similar to wild-type pigs. This first evaluation of glucose homeostasis in DKO pigs for two major xenoantigens paves the way to their use in (pre)clinical studies., (© 2017 by the American Diabetes Association.)

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.

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 CD31(hi) 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 2010 Elsevier B.V. All rights reserved.)

Four cases of operated odontoid process fractures associated with a fracture of the posterior arch of the atlas are presented. Three types of surgery were performed: atlas-axis fusion, occipitocervical fusion, and odontoid process screw fixation. Based on a literature review and our experience, the therapeutic management is discussed according to the type of odontoid fracture and the presence of neurological involvement, with a reminder that wiring is not indicated when C1 posterior arch continuity is compromised.

Aims/hypothesis: Pig islets could transmit porcine endogenous retroviruses (PERV) to diabetic patients. Our previous work showed that pig islets expressed low levels of PERV mRNA and were not likely to transmit PERV to human cells in vitro. The real risk of infection during pig tissue xenografts can only be evaluated by in vivo experiments., Methods: Nude mice bearing tumours containing human 293 cells were grafted with specific pathogen-free pig islets or PERV-producing pig PK15 cells to determine whether pig cells could transmit PERV to mouse and human cells in vivo. Infection was monitored by PCR, long PCR, RT-PCR and long RT-PCR. As detection of PERV sequences could be due to the presence of residual pig cells, we looked for pig mitochondrial (mt) DNA. Quantitative PCR for PERV and pig mt DNA was done to compare the PERV-to-pig mt (P-to-M) ratio of each sample with the reference ratio for grafted pig cells., Results: Among 78 mouse tissues from PK15-grafted mice, 54 and 72 were positive for gag and pig mt DNA, respectively. Human tumours developed in these mice were positive for PERV (78%) and pig mt (89%). The P-to-M ratios for mouse tissues and PERV-positive human tumours from PK15-grafted mice were higher than the ratio in PK15 cells. Among 41 tissues from pig islet cell-grafted mice, 7 were positive for PERV (3 lymph nodes, 1 kidney, 2 salivary glands, 1 ovary), and 14 were positive for pig mt DNA. Three of these samples (1 lymph node, 1 kidney and 1 salivary gland) were positive for gag DNA, but negative for pig mt DNA. One human tumour in these mice was positive for PERV DNA. P-to-M reference ratio in grafted islet cells was 0.05+/-0.03. The three PERV-positive lymph nodes contained 78 gag/3 mt copies (P-to-M: 26), 101 gag/3 mt copies (P-to-M: 34), and 4 gag/0 mt copies. The two PERV-positive salivary glands contained 14 gag/1 mt copies, and 28 gag/0 mt copies. The ovary and the kidney contained 46 gag/3 mt and 69 gag/0 mt copies, respectively. The PERV-positive human tumour contained 47 gag/3 mt copies., Conclusions/interpretation: Microchimerism and PERV transmission were frequently observed in both mouse and human tissues during grafting of pig PK15 cells into nude mice bearing human tumours, and sometimes during pig islet xenograft in this model. This strengthens the notion that there is a risk of transmitting PERV during pig islet xenograft.

Purpose of the Study: We report our experience with the medial and medioposterior approaches to the humeral diaphysis for plate fixation of the proximal humerus., Material and Methods: Fifteen patients were treated for shaft fracture (n=13) or nonunion (n=2) of the distal third of the humerus without radial nerve involvement. The medial approach was performed in the supine position and the medioposterior approach in the prone position. Bone healing was achieved in all cases. Three patients experienced sensorial irritation in the territory of the median nerve after the medial approach; one case did not regress. There were no complications with the medioposterior approach., Discussion: Dissection of the radial nerve can be avoided with these two approaches which also have a cosmetic advantage. The medioposterior approach appears to be preferable because of the comfortable operative position and the absence of risk for the median nerve. Both approaches are however contraindicated if there is preoperative involvement of the radial nerve.

Aims/hypothesis: Islets from specific pathogen-free (SPF) pigs could prevent the transmission of conventional zoonosis, but not endogenous retroviruses (PERV), from pigs to diabetic patients. We previously reported that the pancreas showed the lowest expression of PERV mRNA among pig tissues intended for grafting. This study aimed to determine whether PERV from pig islets infect human cells during co-incubation., Methods: Human cells (including highly PERV-sensitive 293 cells) were incubated with SPF pig islet cells under conditions designed to increase contact (a high islet to human cell ratio, extended period of co-culture, and repeated contacts). PK15 and G2 retrovirus-producing pig cells were used in place of islet cells as "positive infection controls". Infection of human cells was monitored on cellular extracts and supernatants by PCR or long PCR, and RT-PCR or long RT-PCR, to detect PERV DNA and mRNA, respectively. Reverse-transcriptase activity was monitored by PERT., Results: Despite the presence of all PERV sequences in pig islet cells, including full-length inserts, no DNA or RNA for gag, pol, and the 3 env sub-types were detected in any human cell line or blood mononuclear cells incubated with pig islet cells, during an 18-week follow-up period. No PERV sequences or RT activity were detected in supernatants. PERV signals were negative even when the pig islet to human cell ratio was increased to 100:1, the time of co-culture was extended to 5 days and two sequential co-incubations were done. By contrast, all PERV DNA and mRNA were detected in all human cells co-incubated with PK15 or G2 cells. Depending on human cell types, productive or non-productive infections were obtained: full-length PERV RNA and RT activity in supernatants were detected or not; and PERV sequences to previously unexposed human cells by PERV-infected human cells were transmitted or not. Some human cells were not productively infected by PK15 cells but became productively infected after co-incubation with PERV-infected 293 cells., Conclusion/interpretation: SPF pig islet cells, even with PERV inserts and transcripts, have very little probability of transmitting PERV to human cells during co-incubation. The sensitivity of human cells to stable and productive infection by PERV depends on the cell type. Human adaptation of PERV was observed.