Isolation of neonatal porcine islet tissue and transplantation into diabetic mice. A methodological evaluation

Diabetes mellitus type I can be a disabling disease with a high risk of complications (i.e. neuropathy, atherosclerosis and nephropathy). Furthermore, the treatment with insulin can be complicated with severe episodes of hypoglycaemia.As diabetes type 1 is caused by an autoimmune destruction of the islets of Langerhans, replacement of these islets could be the treatment of choice for this disease provided the immunological process is halted. Centres world wide perform islet allotransplantation (i.e. transplantation within species) as an experimental treatment of diabetes (Ricordi et al. 1992, Secchi et al. 1997, Warnock et al. 1992, Birkeland et al. 1995). However the results are poor with only 8 % of cases having more than one year of insulin independence after transplantation (international islet transplant registry, 1999).Islet transplantation faces two major obstacles : rejection and shortage of human donors. Rejection is avoided by immunosuppression and in experimental models by immunoprotection of the grafts with micro-encapsulation or larger encapsulating devices (Lanza et al. 1995, Lacy et al. 1991, Monaco et al. 1991). Shortage of human donors has necessitated the search for alternative donors, and in this context the pig has drawn most attention, because the physiology of the pig is comparable to that of humans. Indeed, pig insulin has been successfully used for decades in the treatment of human diabetics. Pigs breed fast, have large litters and can be bred under standardised conditions, which is important, as factors such as strain and age influence the outcome of porcine islet isolations (Socci et al. 1990, Heiser et al. 1994).By small modifications of the automated method developed for human islet isolation, it is possible to isolate large numbers of adult porcine islets (Ricordi et al. 1990). These islets are well functioning in vivo after transplantation to rodents. However, unlike human islets, the adult porcine islets have a very thin peri-insular capsule and are therefore very fragile disintegrating easily in overnight culture (van Deijnen et al. 1992, Warnock G.L. et al. 1995). Islet tissue from foetuses possess a potential for growth, and has been extensively examined in the rat (Yderstraede et al. 1995), pig (Korsgren et al. 1988) and humans (Tuch et al. 1991). Foetal porcine islet tissue has even been transplanted to eight diabetic humans with histological signs of graft survival after three weeks, and porcine C-peptide production detectable up to eight months after transplantation, but without any detectable metabolic improvement in the patients (Groth et al. 1993).Recently, a method has been developed for the isolation of islet tissue from neonatal pigs (Korbutt et al. 1996). The neonatal islet-like cell clusters (NICC’s) can be isolated in large numbers, and are able to differentiate and maybe even proliferate in vitro and in vivo.The purpose of this study is to describe the methods for isolation, transplantation and in vivo evaluation of porcine neonatal islet-like cell clusters. Focus is on the transplantation procedure, but aspects of isolation and functional outcome in vitro and in vivo are also included

Dimethyl fumarate is an allosteric covalent inhibitor of the p90 ribosomal S6 kinases

Dimethyl fumarate (DMF) has been applied for decades in the treatment of psoriasis and now also multiple sclerosis. However, the mechanism of action has remained obscure and involves high dose over long time of this small, reactive compound implicating many potential targets. Based on a 1.9 Å resolution crystal structure of the C-terminal kinase domain of the mouse p90 Ribosomal S6 Kinase 2 (RSK2) inhibited by DMF we describe a central binding site in RSKs and the closely related Mitogen and Stress-activated Kinases (MSKs). DMF reacts covalently as a Michael acceptor to a conserved cysteine residue in the αF-helix of RSK/MSKs. Binding of DMF prevents the activation loop of the kinase from engaging substrate, and stabilizes an auto-inhibitory αL-helix, thus pointing to an effective, allosteric mechanism of kinase inhibition. The biochemical and cell biological characteristics of DMF inhibition of RSK/MSKs are consistent with the clinical protocols of DMF treatment.</p

Evaluation of Multi-Frequency Bioelectrical Impedance Analysis against Dual-Energy X-ray Absorptiometry for Estimation of Low Muscle Mass in Older Hospitalized Patients

The accuracy of multi-frequency (MF) bioelectrical impedance analysis (BIA) to estimate low muscle mass in older hospitalized patients remains unclear. This study aimed to describe the ability of MF-BIA to identify low muscle mass as proposed by The Global Leadership Initiative on Malnutrition (GLIM) and The European Working Group on Sarcopenia in Older People (EWGSOP-2) and examine the association between muscle mass, dehydration, malnutrition, and poor appetite in older hospitalized patients. In this prospective exploratory cohort study, low muscle mass was estimated with MF-BIA against dual-energy X-ray absorptiometry (DXA) in 42 older hospitalized adults (≥65 years). The primary variable for muscle mass was appendicular skeletal muscle mass (ASM), and secondary variables were appendicular skeletal muscle mass index (ASMI) and fat-free mass index (FFMI). Cut-off values for low muscle mass were based on recommendations by GLIM and EWGSOP-2. MF-BIA was evaluated against DXA on the ability to estimate absolute values of muscle mass by mean bias, limits of agreement (LOA), and accuracy (5% and 10% levels). Agreement between MF-BIA and DXA to identify low muscle mass was evaluated with sensitivity, specificity, negative predictive value (NPV), and positive predictive value (PPV). The association between muscle mass, dehydration, malnutrition, and poor appetite was visually examined with boxplots. MF-BIA overestimated absolute values of ASM with a mean bias of 0.63 kg (CI: -0.20:1.46, LOA: -4.61:5.87). Agreement between MF-BIA and DXA measures of ASM showed a sensitivity of 86%, specificity of 94%, PPV of 75% and NPV of 97%. Boxplots indicate that ASM is lower in patients with malnutrition. This was not observed in patients with poor appetite. We observed a tendency toward higher ASM in patients with dehydration. Estimation of absolute ASM values with MF-BIA should be interpreted with caution, but MF-BIA might identify low muscle mass in older hospitalized patients.</p