Simple in vitro migration assay for neural crest cells and the opposite effects of all‐trans‐retinoic acid on cephalic‐ and trunk‐derived cells

Here, we describe a simple in vitro neural crest cell (NCC) migration assay and the effects of all‐trans‐retinoic acid (RA) on NCCs. Neural tubes excised from the rhombencephalic or trunk region of day 10.5 rat embryos were cultured for 48 h to allow emigration and migration of NCCs. Migration of NCCs was measured as the change in the radius (radius ratio) calculated from the circular spread of NCCs between 24 and 48 h of culture. RA was added to the culture medium after 24 h at embryotoxic concentrations determined by rat whole embryo culture. RA (10 μM) reduced the migration of cephalic NCCs, whereas it enhanced the migration of trunk NCCs, indicating that RA has opposite effects on these two types of NCCs.     

CD154‐expressing CMV‐specific T cells associate with freedom from DNAemia and may be protective in seronegative recipients after liver or intestine transplantation

Cell‐mediated immunity to CMV, if known, could improve antiviral drug therapy in at‐risk children and young adults with LT and IT. Host immunity has been measured with CMV‐specific T cells, which express IFNγ, but not those which express CD154, a possible substitute for IFNγ. CMV‐specific CD154+ T cells and their subsets were measured with flow cytometry after stimulating PBL from recipient blood samples with an overlapping peptide mix of CMV‐pp65 antigen for up to 6 hours. CMV‐specific CD154+ T cells co‐expressed IFNγ in PBL from three healthy adults and averaged 3.8% (95% CI 3.2%‐4.4%) in 40 healthy adults. CMV‐specific T cells were significantly lower in 19 CMV DNAemic LT or IT recipients, compared with 126 non‐DNAemic recipients, 1.3% (95% CI 0.8‐1.7) vs 4.1 (95% CI 3.6‐4.6, P < .001). All T‐cell subsets demonstrated similar between‐group differences. In logistic regression analysis of 46 training set samples, 12 with DNAemia, all obtained between days 0 and 60 from transplant, CMV‐specific T‐cell frequencies ≥1.7% predicted freedom from DNAemia with NPV of 93%. Sensitivity, specificity, and PPV were 83%, 74%, and 53%, respectively. Test performance was replicated in 99 validation samples. In 32 of 46 training set samples, all from seronegative recipients, one of 19 recipients with CMV‐specific T‐cell frequencies ≥1.7% experienced DNAemia, compared with 8 of 13 recipients with frequencies <1.7% (P = .001). CMV‐specific CD154+ T cells are associated with freedom from DNAemia after LT and IT. Among seronegative recipients, CMV‐specific T cells may protect against the development of CMV DNAemia.     

Impaired β‐cell sensitivity to glucose and maximal insulin secretory capacity in adolescents with type 2 diabetes

Elder DA, Woo JG, D’Alessio DA. Impaired β‐cell sensitivity to glucose and maximal insulin secretory capacity in adolescents with type 2 diabetes. Background: Adults with type 2 diabetes mellitus (T2DM) have broad impairments in β‐cell function, including severe attenuation of the first‐phase insulin response to glucose, and reduced β‐cell mass. In adolescents with T2DM, there is some evidence that β‐cell dysfunction may be less severe. Our objective was to determine β‐cell sensitivity to glucose and maximal insulin secretory capacity (AIR_max) in teenagers with T2DM. Methods: Fifteen adolescents with T2DM [11 F/4 M, age 18.4 ± 0.3 yr, body mass index (BMI) 39.8 ± 2.2 kg/m²] and 10 non‐diabetic control subjects (7 F/3 M, age 17.4 ± 0.5 yr, BMI 41.5 ± 2.2 kg/m²) were studied. T2DM subjects had a mean duration of diabetes of 48.8 ± 6.4 months, were treated with conventional therapies, and had good metabolic control [hemoglobin A1c (HbA1c) 6.7 ± 1.2%]. Insulin and C‐peptide were determined before and after a graded glucose infusion and after intravenous arginine at a whole blood glucose level of ≥22 mM. Results: The insulin response to increasing plasma glucose concentrations was blunted in the diabetic compared with control subjects (34.8 ± 11.9 vs. 280.5 ± 57.8 pmol/mmol; p < 0.0001), and AIR_max was also significantly reduced in the diabetic group (1868 ± 330 vs. 4445 ± 606; p = 0.0005). Conclusion: Even adolescents with well‐controlled T2DM have severe impairments of insulin secretion. These data support β‐cell dysfunction as central in the pathogenesis of T2DM in young people, and indicate that these abnormalities can develop over a period of just several years.     

Controlling the blood glucose of type 1 diabetes mice by co‐culturing MIN‐6 β cells on 3D scaffold

T1D is an autoimmune disease, which may be caused by lack of insulin‐secreting β cells due to damage of autoimmune system. Living with T1D is a challenge for the child and the family; cell transplantation is a treatment option for diabetes in children. To establish a microenvironment suitable for cell growth and proliferation as well as for sustained cellular function, we used MIN‐6 β cells as seed cells and SF‐IV collagen as a 3D composite scaffold to construct artificial pancreas in this experiment. The cell viabilities were determined by MTT assay, and the response of cells to different glucose concentrations was observed by glucose stimulation test. Artificial pancreas was transplanted into the abdominal cavity of T1D mice, and the changes of blood glucose were monitored. After 10 days, insulin expression was detected by immunohistochemical method, and the claybank stained area showed effectiveness of insulin secretion. A series of experiments showed that implantation of 3D cell scaffold into the abdominal cavity can effectively control the blood glucose level of T1D mice. It also had longer‐lasting hypoglycemic effects than simple cell transplantation, which was expected to become a new method for the treatment of T1D.