Myoblasts fail to stimulate T cells but induce tolerance

Recent interest in myoblast transfer and in the use of myoblastsas vehicles in gene therapy has made it important to understandthe potential immunogenicity of allogeneic or neoantlgen-expresslngmyoblasts. Given the problems of producing a pure populationof myoblasts, In this study we used a tumour-derived musclecell line (TE671), with phenotyplc features of myoblasts, whichwe transfected to express HLA-DR1. However, this cell line wasunable to stimulate either established HLA-DR1-specific alloreactlveT cell clones or a primary alloresponse. Nor could it presenthaemagglutlnln peptide HA 306–324 to DR1-restricted, HA306–324-speciflc T cell clones or lines. Indeed, prelncubatlonwith DR1-expressing TE671 and HA 306–324 rendered suchT cells tolerant as Judged by their subsequent inability toproliferate in response to a DR1⁺ B cell line plus peptide HA306–324. These results imply that myoblasts do not providecostlmulatory signals, and are therefore unlikely to stimulateallospeclfic T cells following myoblasts transplantation orto initiate neoantlgen-speclfic immune responses following Invivo transfection.     

Four-color flow cytometric analysis of peripheral blood donor cell chimerism

Passenger leukocytes have been demonstrated to play significant roles in initiating and also regulating immune reactions after organ transplantation. Reliable techniques to detect donor leukocytes in recipients after organ transplantation are essential to analyze the role, function, and behavior of these leukocytes. In this report we describe a simple, reliable method to detect donor cells with low frequencies using peripheral blood samples. Detection of small numbers of major histocompatibility complex (MHC) mismatched cells was first studied using four-color flow cytometry in artificially created cell mixtures. By selecting the CD45(+) population and simultaneous staining with several leukocyte lineage markers (CD3, CD4, CD8, CD56, and CD19), MHC-mismatched leukocytes were consistently detected in cell suspensions prepared from directly stained whole blood samples with a threshold sensitivity as low as 0.1%-0.2%. When the fresh peripheral blood mononuclear cells were separated by conventional Ficoll gradient purification, similar, but slightly lower levels of donor cells were detected. Blood samples obtained 1-5 months after liver, kidney, and intestine transplants revealed that the kind of organ allograft influenced levels and lineage pattern of the circulating donor cells. This procedure provided a simple and reliable method in determining early chimerism in transplant recipients. However, the detection of MHC-mismatched leukocytes of all lineages was much lower when frozen peripheral blood mononuclear cells were used.     

Evaluation of the cause of nasal and ocular symptoms associated with lawn mowing

A number of individuals with perennial or seasonal rhinoconjunctivitis state that their symptoms may suddenly worsen on exposure to lawn mowing. Many allergists have believed that this was related to the agitation of molds deposited on the grass. We studied 50 consecutive new patients with rhinitis using history, skin testing, total and specific IgE assays, and nasal smears. Twenty-five patients gave histories of minimal or no change in their nasal symptoms with exposure to lawn mowing, whereas 11 patients had definite, but mild, symptoms, and 14 patients had severe symptoms. Positive skin tests to grasses, trees, and weed pollens were more frequent in those patients with symptoms exacerbated by lawn mowing (p less than 0.03). Symptomatic patients also had higher serum concentrations of total IgE (p less than 0.008) and grass-pollen specific IgE (p = 0.0004). The frequencies of positive skin tests to ragweed pollens, house dust, molds, and grass leaves, as well as the percentage of nasal eosinophils, were not different in the symptomatic and asymptomatic groups. No significant association was found between symptoms and IgE antibodies to molds or grass-leaf extract. We believe that the increased nasal and ocular symptoms coincident with lawn mowing are allergic phenomena significantly associated with skin test sensitivity and specific IgE antibodies to grass pollens but not with sensitivity or specific IgE to molds or grass-leaf extract.     

Absence of coding mutations in the X-linked genes neuroligin 3 and neuroligin 4 in individuals with autism from the IMGSAC collection.

Neuroligin abnormalities have been recently implicated in the aetiology of autism spectrum disorders (ASD), given the finding of point mutations in the two X-linked genes NLGN3 and NLGN4X and the important role of neuroligins in synaptogenesis. To enquire on the relevance and frequency of neuroligin mutations in ASD, we performed a mutation screening of NLGN3 and NLGN4X in a sample of 124 autism probands from the International Molecular Genetic Study of Autism Consortium (IMGSAC). We identified a new non-synonymous variant in NLGN3 (Thr632Ala), which is likely to be a rare polymorphism. Our data indicate that coding mutations in these genes are very rarely associated to ASD.     

Rapid molecular cytogenetic analysis of X-chromosomal microdeletions: Fluorescence in situ hybridization (FISH) for complex glycerol kinase deficiency

Diagnosis of X-chromosomal microdeletions has relied upon the traditional methods of Southern blotting and DNA amplification, with carrier identification requiring timeconsuming and unreliable dosage calculations. In this report, we describe rapid molecular cytogenetic identification of deleted DNA in affected males with the Xp21 contiguous gene syndrome (complex glycerol kinase deficiency, CGKD) and female carriers for this disorder. CGKD deletions involve the genes for glycerol kinase, Duchenne muscular dystrophy, and/or adrenal hypoplasia congenita. We report an improved method for diagnosis of deletions in individuals with CGKD and for identification of female carriers within their families, using fluorescence in situ hybridization (FISH) with a cosmid marker (cosmid 35) within the glycerol kinase gene. When used in combination with an Xq control probe, affected males demonstrate a single signal from the control probe, while female carriers demonstrate a normal chromosome with two signals, as well as a deleted chromosome with a single signal from the control probe. FISH analysis for CGKD provides the advantages of speed and accuracy for evaluation of submicroscopic X-chromosomal deletions, particularly in identification of female carriers. In addition to improving carrier evaluation, FISH will make prenatal diagnosis of CGKD more readily available. © 1995 Wiley-Liss, Inc.     

Down-modulation of CD4+ T helper type 2 and type 0 cells by T helper type 1 cells via Fas/Fasligand interaction

Fas was recently demonstrated to be the major target molecule engaged by CD4⁺ cytolytic T lymphocytes (CTL). We examined Fas expression on various cloned T cell subpopulations and their susceptibility to lysis by CD4⁺ or CD8⁺ CTL. A reciprocal relationship in Fas and Fas-ligand expression was observed in CD4⁺ T helper (Th)1- and Th2-type clones, and Fas mRNA was predominantly detected in Th2 clones, whereas Fas-ligand mRNA was principally found in Th1 clones. The two Th0 clones tested expressed both Fas and Fas-ligand, but only one exhibited cytolytic activity, whereas both were sensitive to CD4-mediated lysis. A functional consequence of the inverse Fas-Fas-ligand expression pattern was that Th2 and Th0 cells were sensitive to lysis by both Th1 CD4⁺ CTL and a CD8⁺ CTL clone in a Fas-dependent manner. These results suggest that cytolytic CD4⁺ Th1 cells may play an immunomodulatory role, regulating a Th2/Th0 response by Fas-mediated lysis.     

Optimization of selective hyperthermia

Selective hyperthermia can be a feasible treatment modality for deep tissue abnormalities. It is accomplished by using a laser or ultrasound noninvasively to transfer energy to a desired target causing tissue damage. This process has two potential benefits to medical professionals: simplicity of procedure and safety to patient. However, optimizing these selective interactions is difficult due to the number of variables. We propose an optimization coefficient relating the dynamic and geometric parameters of selective hyperthermia, and proceed to measure it in an experimental setup consisting of a near-infrared laser and laser-absorbing dye. To simulate tissue, gelatin phantoms are created using a combination of water, intralipid, and gelatin. Our experiments use a 1.00-cm-diam spherical phantom that is homogeneously enhanced with an indocyanine green (ICG) solution and placed inside a nontarget phantom and irradiated by an 805-nm diode laser. Temperature measurements taken at different locations are analyzed so optimization coefficients can be calculated for different parameters. This optimization coefficient compares the difference in temperatures from inside and outside the target. Analysis of the values after thermal equilibrium provides information about the best parameter selection. Our findings indicate that the optimal ICG concentration and power combination for our tested parameters are 0.083% and 0.97 W, respectively. Based on our analysis, optimization can be obtained by using this coefficient to compare the selectivity of several parameter combinations.     

The effects of indoramin on histamine and antigen-induced changes in respiration in guinea-pigs

Indoramin, a drug which blocks alpha-adrenergie, histamine and serotonin receptors, was tested as a protective agent during challenge with bronchoconstrictor agents in guinea-pigs. In conscious guinea-pigs, the time of onset of respiratory distress during continuous administration of aerosolized solutions of histamine, serotonin or ovalbumin (with animals pre-sensitized to this antigen) was measured using a force-displacement transducer applied to the animal's back. This time interval for each guinea-pig was compared with and without indoramin pre-treatment. Indoramin was administered by intraperitoneal injection or by aerosol treatment. In anaesthetized animals under artificial respiration, respiratory distress was induced by intravenous injection of histamine and measured by the Konzett-Rössler technique. Indoramin treatment significantly protected guinea-pigs in both types of experiment from the effects of each challenging agent.