Fluorescent Cell Barcoding as a Tool to Assess the Age-Related Development of Intracellular Cytokine Production in Small Amounts of Blood from Infants

Fluorescent Cell Barcoding (FCB) is a flow cytometric technique which has been used for assessing signaling proteins. This FCB technique has the potential to be applied in other multiparameter analyses. Since data on antigen (Ag)-specific T-cell immune responses, like intracellular cytokine production, are still lacking in infants because limited blood volumes can be obtained for analysis, the FCB technique could be very useful for this purpose. The objectives of this study were to modify the FCB method to be able to measure multiple Ag-specific cytokine reponses in T-cells upon simultaneous stimulation by various antigens and mitogens in small amounts of blood and to investigate the cytokine pattern of T-cell subsets in healthy infants aged six and twelve months. Blood samples, collected from 20 healthy infants aged six and twelve months, were stimulated in vitro with the antigens: phorbol-myristate-acetate (PMA), purified-protein-derivative (PPD), Tetanus-toxoid (TT), Staphylococcal-enterotoxin-B (SEB), and phytohemagglutinin (PHA). Each stimulus was barcoded by labelling with different intensities of fluorescent cell barcoding (FCB) markers. Intracellular production of interleukin-2, interferon-gamma, and tumor necrosis factor-alpha was measured simultaneously in just one blood sample of 600 µl whole blood. Significant age-related differences in cytokine production were shown for PMA, PHA, and TT in CD4+ T-cells, and for PMA, PHA, SEB, and TT in CD8+ T-cells. The intracellular cytokine production by CD4+ and CD8+ T-cells was higher at twelve months compared to six months of age for all antigens, except for PMA, which was lower at the age of twelve months. Based on the consistency in both T-cell subsets, we conclude that the new FCB method is a promising tool to investigate the age-related development of intracellular cytokine production in infants

Stress Hormones Receptors in the Amygdala Mediate the Effects of Stress on the Consolidation, but Not the Retrieval, of a Non Aversive Spatial Task

This study examined the effects of the arousal level of the rat and exposure to a behavioral stressor on acquisition, consolidation and retrieval of a non-aversive hippocampal-dependent learning paradigm, the object location task. Learning was tested under two arousal conditions: no previous habituation to the experimental context (high novelty stress/arousal level) or extensive prior habituation (reduced novelty stress/arousal level). Results indicated that in the habituated rats, exposure to an out-of-context stressor (i.e, elevated platform stress) impaired consolidation and retrieval, but not acquisition, of the task. Non-habituated animals under both stressed and control conditions did not show retention of the task. In habituated rats, RU-486 (10 ng/side), a glucocorticoid receptor (GR) antagonist, or propranolol (0.75 µg/side), a beta-adrenergic antagonist, injected into the basolateral amygdala (BLA), prevented the impairing effects of the stressor on consolidation, but not on retrieval. The CB1/CB2 receptor agonist WIN55,212-2 (WIN, 5 µg/side) microinjected into the BLA did not prevent the effects of stress on either consolidation or retrieval. Taken together the results suggest that: (i) GR and β-adrenergic receptors in the BLA mediate the impairing effects of stress on the consolidation, but not the retrieval, of a neutral, non-aversive hippocampal-dependent task, (ii) the impairing effects of stress on hippocampal consolidation and retrieval are mediated by different neural mechanisms (i.e., different neurotransmitters or different brain areas), and (iii) the effects of stress on memory depend on the interaction between several main factors such as the stage of memory processing under investigation, the animal's level of arousal and the nature of the task (neutral or aversive)

Testing relationships: ethical arguments for screening with HbA1C

Since the 1990s, glycated haemoglobin (HbA1C) has been the gold standard for monitoring glycaemic control in people diagnosed as having either type 1 diabetes mellitus (T1DM) or type 2 diabetes mellitus (T2DM). Discussions are underway about diagnosing diabetes mellitus on the basis of HbA1C titres and using HbA1C tests to screen for T2DM. These discussions have focused on the relative benefits for individual patients, with some attention directed towards reduced costs to healthcare systems and benefits to society. We argue that there are strong ethical reasons for adopting HbA1C-based diagnosis and T2DM screening that have not yet been articulated. The rationale includes the differential impact of HbA1C-based diabetic testing on disadvantaged groups, and what we are beginning to learn about HbA1C vis-à-vis population health. Although it is arguable that screening must primarily benefit the individual, using HbA1C to diagnose and screen for T2DM may promote a more just distribution of health resources and lead to advances in investigating, monitoring and tackling the social determinants of health

POU5F1 (OCT3/4) identifies cells with pluripotent potential in human germ cell tumors

Human germ cell tumors (GCTs) may have variable histology and clinical behavior, depending on factors such as sex of the patient, age at clinical diagnosis, and anatomical site of the tumor. Some types of GCT, i.e., the seminomas/germinomas/dysgerminomas and embryonal carcinomas (the stem cell component of nonseminomas), have pluripotent potential, which is demonstrated by their capacity to differentiate into somatic and/or extraembryonic elements. Although embryonal carcinoma cells are intrinsically pluripotent, seminoma/germinoma/dysgerminoma cells, as well as their precursor carcinoma in situ/gonadoblastoma cells, have the phenotype of early germ cells that can be activated to pluripotency. The other types of GCT (teratomas and yolk sac tumors of infants and newborn, dermoid cyst of the ovary, and spermatocytic seminoma of elderly) are composed of (fully) differentiated tissues and lack the appearance of undifferentiated and pluripotent stem cells. OCT3/4, a transcription factor also known as OTF3 and POU5F1, is involved in regulation of pluripotency during normal development and is detectable in embryonic stem and germ cells. We analyzed the presence of POU5F1 in GCT and other tumor types using immunohistochemistry. The protein was consistently detected in carcinoma in situ/gonadoblasto

PIRCHE-II Is Related to Graft Failure after Kidney Transplantation

Individual HLA mismatches may differentially impact graft survival after kidney transplantation. Therefore, there is a need for a reliable tool to define permissible HLA mismatches in kidney transplantation. We previously demonstrated that donor-derived Predicted Indirectly ReCognizable HLA Epitopes presented by recipient HLA class II (PIRCHE-II) play a role in de novo donor-specific HLA antibodies formation after kidney transplantation. In the present Dutch multi-center study, we evaluated the possible association between PIRCHE-II and kidney graft failure in 2,918 donor–recipient couples that were transplanted between 1995 and 2005. For these donors–recipients couples, PIRCHE-II numbers were related to graft survival in univariate and multivariable analyses. Adjusted for confounders, the natural logarithm of PIRCHE-II was associated with a higher risk for graft failure [hazard ratio (HR): 1.13, 95% CI: 1.04–1.23, p = 0.003]. When analyzing a subgroup of patients who had their first transplantation, the HR of graft failure for ln(PIRCHE-II) was higher compared with the overall cohort (HR: 1.22, 95% CI: 1.10–1.34, p < 0.001). PIRCHE-II demonstrated both early and late effects on graft failure in this subgroup. These data suggest that the PIRCHE-II may impact graft survival after kidney transplantation. Inclusion of PIRCHE-II in donor-selection criteria may eventually lead to an improved kidney graft survival