Optimisation of the CT h4S bioassay for detection of human interleukin-4 secreted by mononuclear cells stimulated by phytohaemaglutinin or by human leukocyte antigen mismatched mixed lymphocyte culture

Limiting dilution analysis has been used in the context of allogeneic bone marrow transplantation to determine anti-recipient interleukin-2 (IL-2) producing helper T lymphocyte precursor (HTLp) frequencies, which in several studies have been predictive of graft-versus-host disease (GVHD). Recently high anti-recipient IL-4 producing HTLp frequencies have been reported and associated with a decreased risk of GVHD. The aim of the present study was to define the optimal conditions for combined determination of IL-2 and IL-4 producing anti-recipient HTLp frequencies. We have optimised the CT.h4S bioassay with regards to specificity, sensitivity, detection limit, and reproducibility. We have found the optimal assay conditions to be 1 x 10 (4) CT.h4S cells/well deprived of IL-4 for 24 h and preincubated for 7 h followed by 18 h of incubation with tritiated methyl-thymidine. In this setting the CT.h4S bioassay detects 5 pg/ml of human recombinant IL-4 with no detection of IL-2 in concentrations below 500 pg/ml. We have found 72 h of culture optimal for detection of IL-2 and IL-4 produced by human mononuclear cells (MNC) in response to stimulation with phytohaemaglutinin and for detection of IL-2 in human leukocyte antigen (HLA)-mismatched mixed leukocyte culture (MLC). An interindividual variation in cytokine accumulation was demonstrated for IL-4 but not for IL-2. With the use of 5x10(4) responder cells/well no IL-4 could be detected in HLA-mismatched MLC between days 1 and 16. The lack of IL-4 detection was not due to high amounts of soluble IL-4 receptor. With the use of 1x10(6) responder cells/well in HLA-mismatched MLC, we found limited IL-4 accumulation still increasing at day 12. We conclude that the CT.h4S bioassay is a reliable and specific method for quantification of IL-4 accumulation in cultures of human MNC. The difference in optimal timing for IL-2 (day 3) and IL-4 (>/=day 12) detection and evidence of very low IL-4 producing HTLp frequencies makes the relevance of a combined IL-2/IL-4 HTLp assay questionable.     

Recirculating and germinal center B cells differentiate into cells responsive to polysaccharide antigens

Antibodies against bacterial capsular polysaccharides play a critical protective role. Responses to these antigens can occur without the help or control of T cells and are associated with marginal zone (MZ) B cells. Capsular antigens are diverse and some cross-react with self-carbohydrate epitopes. This diversity may explain the recruitment of non-autoreactive recirculating B cells and memory B cells to the MZ in addition to other B cells, some of which are weakly autoreactive cells, that are recruited to the MZ without entering the recirculating pool. To test whether memory B cells respond to polysaccharide-based antigens, mice with hapten-specific memory B cells were challenged with hapten-polysaccharide. Hapten-specific plasma cells producing high affinity antibody with Ig V-region mutations were induced. To test whether naive recirculating B cells can form MZ cells that respond to polysaccharide, recirculating B cells from lymph nodes were transferred into Rag-1-deficient mice. MZ cells differentiated from the donor cells without proliferation or T cell help and responded to polysaccharide-based antigen. The differentiation of B cells both from germinal centers and the recirculating pool to the MZ phenotype is likely to make an important contribution to the repertoire of B cells that respond to polysaccharide antigens.     

Pentobarbital—Ein Abbauprodukt von Thiopental

Journal of Molecular Medicine -     

ADULT-syndrome: An autosomal-dominant disorder with pigment anomalies,ectrodactyly, nail dysplasia,and hypodontia

We describe a family with at least seven living persons who are affected by an hitherto undescribed autosomal-dominant syndrome with variable expression, bearing close resemblance to the EEC syndrome and related disorders. The main manifestations are hypodontia and/or early loss of permanent teeth, ectrodactyly, obstruction of lacrimal ducts, onychodysplasia, and excessive freckling. We propose the acronym ADULT (acro-der-mato-ungual-lacrimal-tooth)-syndrome for this condition. © 1993 Wiley-Liss, Inc.     

The effect of sodium ions on the light-induced86Rb release from the isolated crayfish retina

The effect of low external Na⁺ concentrations on the light-induced K⁺ release from crayfish photoreceptor cells was tested by labelling intracellular K⁺ with the isotope⁸⁶Rb. The amount of isotope released per light stimulus is roughly proportional to the external Na⁺ concentration if the osmolarity is kept constant by replacing Na⁺ with Tris, choline or sucrose. When sucrose is used to replace the depleted Na⁺ the light-induced K⁺ release is a linear function of the external Na⁺ concentration and is reduced by approx. 95% at an external Na⁺ concentration of 5 mmol/l. For choline and Tris substitutions the relationships are less clear but at Na⁺ concentrations 56 mmol/l it seems that in comparison with sucrose the light-induced K⁺ release is smaller in a Tris solution and larger in a choline solution. It is suggested that the light-induced K⁺ release is due mainly to an activation of voltage sensitive K⁺ channels.     

Effects of cyclosporin A on the immune system of the mouse. I. Evidence for a direct selective effect of cyclosporin A on B cells responding to anti-immunoglobulin antibodies

Previous in vivo studies have shown that the immunsuppressive peptide cyclosporin A (CsA) selectively suppresses antibody responses to nonmitogenic T-independent (TI-2) antigens, but not those to mitogenic (TI-1) antigens. This report demonstrates that in vitro CsA suppresses the polyclonal proliferative response of B cells to anti-Ig (anti-mu) antibodies at 300-400 fold lower doses than are required to inhibit B cell proliferation induced by lipopolysaccharide. Since the proliferative response to anti-mu requires neither T cells nor macrophages, it is concluded that CsA has a direct inhibitory effect on the B cells responding to this mitogen. The data support the concept that the B cells responding to TI-2 antigens are contained within the population which is stimulated polyclonally by anti-mu, and that lipopolysaccharide stimulates a distinct B cell subpopulation. They do not, however, exclude the possibility that anti-mu and lipopolysaccharide stimulate the same B cell population via two biochemically distinct triggering mechanisms, one of which is CsA-sensitive and the other CsA-resistant.     

Cardiomyopathy associated with Leigh's disease

Summary Clinical and postmortem findings in a female infant, suffering from Leigh's disease and cardiomegaly are described. The cardiac enlargement was due to symmetrical thickening of both ventricular walls and the septum. On light microscopy a widespread fibre disarray with a slight predilection for the ventricular septum was observed. Ultrastructural changes included an extreme reduction in the number of myofibrils and an excess of mitochondria. Abnormalities of the mitochondrial structure with tubular and myelinic transformation of the cristae suggested that a mitochondriopathy is responsible for the cardiomegaly in Leigh's disease.Dedicated to Prof. Dr. Waldemar Hort on the occasion to his 60th birthday