Interleukin-1 Receptor Antagonist Modulates the Early Phase of Liver Regeneration after Partial Hepatectomy in Mice

Cytokine administration is a potential therapy for acute liver failure by reducing inflammatory responses and favour hepatocyte regeneration. The aim of this study was to evaluate the role of interleukin-1 receptor antagonist (IL-1ra) during liver regeneration and to study the effect of a recombinant human IL-1ra on liver regeneration

Synthesis strategies to extend the variety of alginate-based hybrid hydrogels for cell microencapsulation

The production of hydrogel microspheres (MS) for cell immobilization, maintaining the favorable properties of alginate gels but presenting enhanced performance in terms of in vivo durability and physical properties, is desirable to extend the therapeutic potential of cell transplantation. A novel type of hydrogel MS was produced by straightforward functionalization of sodium alginate (Na-alg) with heterotelechelic poly(ethylene glycol) (PEG) derivatives equipped with either end thiol or 1,2-dithiolane moieties. Activation of the hydroxyl moieties of the alginate backbone in the form of imidazolide intermediate allowed for fast conjugation to PEG oligomers through a covalent carbamate linkage. Evaluation of the modified alginates for the preparation of MS combining fast ionic gelation ability of the alginate carboxylate groups and slow covalentcross-linking provided by the PEG-end functionalities highlighted the influence of the chemical composition of the PEG-grafting units on the physical characteristics of the MS. The mechanical properties of the MS (resistance and shape recovery) and durability of PEG-grafted alginates in physiological environment can be adjusted by varying the nature of the end functionalities and the length of the PEG chains. In vitro cell microencapsulation studies and preliminary in vivo assessment suggested the potential of these hydrogels for cell transplantation applications

Multipotent mesenchymal stromal cells enhance insulin secretion from human islets via N-Cadherin interaction and prolong function of transplanted encapsulated islets in mice

Background: Multipotent mesenchymal stromal cells (MSC) enhance viability and function of islets of Langerhans. We aimed to examine the interactions between human MSC and human islets of Langerhans that influence the function of islets. Methods: Human MSC and human islets (or pseudoislets, obtained after digestion and reaggregation of islet cells) were cocultured with or without cellular contact and glucose-stimulated insulin secretion assays were performed to assess cell function. The expression of several adhesion molecules, notably ICAM-1 and N-cadherin on islets and MSC, was investigated by qPCR. The role of N-cadherin was analyzed by adding an anti-N-cadherin antibody in islets cultured with or without MSC for 24 h followed by insulin measurements in static incubation assays. Islets and MSC were coencapsulated in new hydrogel microspheres composed of calcium alginate and covalently crosslinked polyethylene glycol. Encapsulated cells were transplanted intraperitoneally in streptozotocin-induced diabetic mice and glycemia was monitored. Islet function was evaluated by the intraperitoneal glucose tolerance test. Results: In vitro, free islets and pseudoislets cocultured in contact with MSC showed a significantly increased insulin secretion when compared to islets or pseudoislets cultured alone or cocultured without cell-to-cell contact with MSC (p < 0.05). The expression of ICAM-1 and N-cadherin was present on islets and MSC. Blocking N-cadherin prevented the enhanced insulin secretion by islets cultured in contact with MSC whereas it did not affect insulin secretion by islets cultured alone. Upon transplantation in diabetic mice, islets microencapsulated together with MSC showed significantly prolonged normoglycemia when compared with islets alone (median 69 and 39 days,respectively, p < 0.01). The intraperitoneal glucose tolerance test revealed an improved glycemic response in mice treated with islets microencapsulated together with MSC compared to mice transplanted with islets alone (p < 0.001). Conclusions: MSC improve survival and function of islets of Lan gerhans by cell-to-cell contact mediated by the adhesion molecule N-cadherin

Anti-CD154 mAb and Rapamycin Induce T Regulatory Cell Mediated Tolerance in Rat-to-Mouse Islet Transplantation

Anti-CD154 (MR1) monoclonal antibody (mAb) and rapamycin (RAPA) treatment both improve survival of rat-to-mouse islet xenograft. The present study investigated the effect of combined RAPA/MR1 treatment on rat-to-mouse islet xenograft survival and analyzed the role of CD4(+)CD25(+)Foxp3(+) T regulatory cells (Treg) in the induction and maintenance of the ensuing tolerance. C57BL/6 mice were treated with MR1/RAPA and received additional monoclonal anti-IL2 mAb or anti CD25 mAb either early (0-28 d) or late (100-128 d) post-transplantation. Treg were characterised in the blood, spleen, draining lymph nodes and within the graft of tolerant and rejecting mice by flow cytometry and immunohistochemistry. Fourteen days of RAPA/MR1 combination therapy allowed indefinite islet graft survival in &gt;80% of the mice. Additional administration of anti-IL-2 mAb or depleting anti-CD25 mAb at the time of transplantation resulted in rejection (100% and 89% respectively), whereas administration at 100 days post transplantation lead to lower rejection rates (25% and 40% respectively). Tolerant mice showed an increase of Treg within the graft and in draining lymph nodes early post transplantation, whereas 100 days post transplantation no significant increase of Treg was observed. Rejecting mice showed a transient increase of Treg in the xenograft and secondary lymphoid organs, which disappeared within 7 days after rejection. These results suggest a critical role for Treg in the induction phase of tolerance early after islet xenotransplantation. These encouraging data support the need of developing further Treg therapy for overcoming the species barrier in xenotransplantation

Characterization of SNAP-25 in insulin secreting cells

GRENOBLE1-BU Sciences (384212103) / SudocSudocFranceF

Methods for Isolation and Purification of Murine Liver Sinusoidal Endothelial Cells: A Systematic Review

To study the biological functions of liver sinusoidal endothelial cells (LSEC) and to identify their interplay with blood or liver cells, techniques allowing for the isolation and purification of LSEC have been developed over the last decades. The objective of the present review is to summarize and to compare the efficiency of existing methods for isolating murine LSEC. Toward this end, the MEDLINE database was searched for all original articles describing LSEC isolation from rat and mouse livers. Out of the 489 publications identified, 23 reported the main steps and outcomes of the procedure and were included in our review. Here, we report and analyse the technical details of the essential steps of the techniques used for LSEC isolation. The correlations between the prevalence of some steps and the efficiency of LSEC isolation were also identified. We found that centrifugal elutriation, selective adherence and, more recently, magnetic-activated cell sorting were used for LSEC purification. Centrifugal elutriation procured high yields of pure LSEC (for rats 30-141.9 million cells for 85-98% purities; for mice 9-9.25 million cells for >95% purities), but the use of this method remained limited due to its high technical requirements. Selective adherence showed inconsistent results in terms of cell yields and purities in rats (5-100 million cells for 73.7-95% purities). In contrast, magnetic-activated cell sorting allowed for the isolation of highly pure LSEC, but overall lower cell yields were reported (for rats 10.7 million cells with 97.6% purity; for mice 0.5-9 million cells with 90-98% purities). Notably, the controversies regarding the accuracy of several phenotypic markers for LSEC should be considered and their use for both magnetic sorting and characterization remain doubtful. It appears that more effort is needed to refine and standardize the procedure for LSEC isolation, with a focus on the identification of specific antigens. Such a procedure is required to identify the molecular mechanisms regulating the function of LSEC and to improve our understanding of their role in complex cellular processes in the liver

Phosphorylation of SNAP-25 on serine-187 is induced by secretagogues in insulin-secreting cells, but is not correlated with insulin secretion.

The tSNARE (the target-membrane soluble NSF-attachment protein receptor, where NSF is N -ethylmaleimide-sensitive fusion protein) synaptosomal-associated protein of 25 kDa (SNAP-25) is implicated in regulated insulin secretion. In pheochromocytoma PC12 cells, SNAP-25 is phosphorylated at Ser(187), which lies in a region that is important for its function. The aims of the present study were to determine whether SNAP-25 is phosphorylated at Ser(187) in insulin-secreting cells and, if so, whether this is important for regulated insulin secretion. The major findings are: (i) SNAP-25 is rapidly and reversibly phosphorylated on Ser(187) in both rat insulinoma INS-1 cells and rat islets in response to the phorbol ester, PMA; (ii) less than 35% of SNAP-25 in INS-1 cells is phosphorylated in response to PMA, and phosphorylation is limited to plasma-membrane-associated SNAP-25; (iii) both SNAP-25 isoforms (a and b) are phosphorylated, with 1.8-fold greater phosphorylation for SNAP-25b in response to PMA; (iv) in rat islets, Ser(187) phosphorylation is stimulated by glucose or carbachol, albeit to a lesser extent than by PMA, but not by cAMP; (v) insulin secretion from botulinum neurotoxin E-treated hamster insulinoma tumour (HIT) cells, transfected with toxin-resistant Ser(187)-->Ala or Ser(187)-->Asp mutant SNAP-25, was similar to that of wild-type HIT cells. Furthermore, in rat islets no correlation was found between the extent of SNAP-25 phosphorylation at Ser(187) in response to secretagogues and stimulation of insulin release; (vi) use of protein kinase C (PKC) inhibitors suggests that glucose stimulates SNAP-25 phosphorylation via conventional and non-conventional PKC isoforms. In summary, although SNAP-25 phosphorylation at Ser(187) occurs in insulin-secreting cells and is mediated by PKC, it does not appear to play a major role in regulated insulin secretion

Transplantation of mesenchymal stem cells for the treatment of liver diseases, is there enough evidence?

Mesenchymal stem cells or multipotent mesenchymal stromal cells (MSCs) have been extensively investigated in small animal models to treat both acute and chronic liver injuries. Mechanisms of action are not clearly elucidated but may include their ability to differentiate into hepatocyte-like cells, to reduce inflammation, and to enhance tissue repair at the site of injury. This approach is controversial and evidence in large animals is missing. Side effects of MSC infusion such as the contribution to a fibrotic process have been reported in experimental settings. Nevertheless, MSCs moved quickly from bench to bedside and over 280 clinical trials are registered, of which 28 focus on the treatment of liver diseases. If no severe side-effects were observed so far, long-term benefits remain uncertain. More preclinical data regarding mechanisms of action, long term safety and efficacy are warranted before initiating large scale clinical application. The proposal of this review is to visit the current state of knowledge regarding mechanisms behind the therapeutic effects of MSCs in the treatment of experimental liver diseases, to address questions about efficacy and risk, and to discuss recent clinical advances involving MSC-based therapies