Aims/hypothesis: Efficient islet isolation is an important prerequisite for successful clinical islet transplantation. Although progressively improved, islet yield and quality are, however, unpredictable and variable and require standardisation. Methods: Since 1989 we have processed 437 pancreases using the automated method. The donor characteristics, pancreas procurement, and digestion and purification procedures including a wide enzyme characterisation of these pancreases were analysed and correlated with islet yield and transplant outcome. Results: By univariate analysis, islet yield was significantly associated with donor age (r=0.16; p=0.0009), BMI (r=0.19; p= 0.0004), good pancreas condition (p=0.0031) and weight (r=0.15; p=0.0056), total collagenase activity (r=0.22; p= 0.0001), adjusted collagenase activity/mg (r=0.18; p=0.0002), collagenase activity/solution volume (r=0.18; p=0.0002) and neutral protease activity/solution volume (r=0.14; p= 0.0029). A statistically significant contribution to the variability of islet yield in a multivariate analysis performed on donor variables was found for donor BMI (p=0.0008). In a multivariate analysis performed on pancreas variables a contribution was found for pancreas weight (p=0.0064), and for a multivariate analysis performed on digestion variables we found a contribution for digestion time (p= 0.0048) and total collagenase activity (p=0.0001). Twenty-four patients with type 1 diabetes received single islet preparations from single donors. In these patients, multivariate analyses showed that the reduction in insulin requirement was significantly associated with morphological aspects of islets (p= 0.0010) and that 1-month C-peptide values were associated with islet purity (p=0.0071). Conclusions/interpretation: These data provide baseline donor, digestion and purification selection criteria for islet isolation using the automated method and indicate that the morphological aspect may be a clinically relevant measure of islets on which the decision for transplant can be based.

Collectively, our findings suggest that humoral and cellular components of the natural immune response against heterologous species independently evolved recognition patterns directed against overlapping carbohydrate determinants.

No abstract

In clinical transplantation, the occurrence of cyclosporin A (CsA)‐resistant production of IL‐2 in vitro correlates with graft rejection in vivo. In this study we investigated the role of the costimulatory molecules CD28 and LFA‐1 in this process in the setting of TCR‐induced proliferation of primary T lymphocytes in vitro. Co‐stimulation with ICAM‐1 and B7.2 led to strong and CsA‐resistant proliferation, which was found to be largely IL‐2 dependent. All of the known calcineurin‐dependent events, such as induction of NF‐AT and NF‐κB or stress‐activated protein kinase activation, were markedly modulated by CsA independently of costimulation. In contrast, both ICAM‐1 and B7.2 enhanced the half‐life of the inducible IL‐2 transcript in a CsA‐resistant manner. LFA‐1‐ but not CD28‐induced IL‐2 mRNA stabilization required the integrity of the actin‐based cytoskeleton, suggesting that the two costimulatory molecules impact on qualitatively different signaling pathways. This is further suggested by the demonstration that LFA‐1 and CD28 acted synergistically to confer CsA resistance in a model of co‐stimulation using superantigen‐pulsed dendritic cells. We propose that IL‐2 transcript accumulation and subsequent T cell proliferation at the low transcriptional rate imposed by CsA are the result of co‐stimulation‐dependent stabilization of IL‐2 mRNA.

Th1 and Th2 cells are functionally distinct subsets of CD4+ T lymphocytes whose tissue-specific homing to sites of inflammation is regulated in part by the differential expression of P- and E-selectin ligands and selected chemokine receptors. Here we investigated the expression and function of β1 integrins in Th1 and Th2 cells polarized in vitro. Th1 lymphocytes adhere transiently to the extracellular matrix ligands laminin 1 and fibronectin in response to chemokines such as RANTES and stromal cell-derived factor-1, and this process is paralleled by the activation of the Rac1 GTPase and by a rapid burst of actin polymerization. Selective inhibitors of phosphoinositide-3 kinase prevent efficiently all of the above processes, whereas the protein kinase C inhibitor bisindolylmaleimide prevents chemokine-induced adhesion without affecting Rac1 activation and actin polymerization. Notably, chemokine-induced adhesion to β1 integrin ligands is markedly reduced in Th2 cells. Such a defect cannot be explained by a reduced sensitivity to chemokine stimulation in this T cell subset, nor by a defective activation of the signaling cascade involving phosphoinositide-3 kinase, Rac1, and actin turnover, as all these processes are activated at comparable levels by chemokines in the two subsets. We propose that reduced β1 integrin-mediated adhesion in Th2 cells may restrain their ability to invade and/or reside in sites of chronic inflammation, which are characterized by thickening of basement membranes and extensive fibrosis, requiring efficient interaction with organized extracellular matrices.