Cryopreservation and long-term in vitro maintenance of second-stage larvae of Toxocara canis

Second stage larvae of Toxocara canis were isolated from developed eggs, frozen in Eagle's Minimal Essential Medium with 5% dimethyl sulfoxide or 10% glycerol as cryoprotectants according to two cooling schedules and maintained in liquid nitrogen for 1 week. After thawing, the previously frozen larvae (FL) and unfrozen controls (CL) were maintained in a chemically defined medium in vitro for 35 weeks. While CL had motility rates around 95% to 97% throughout the experiment, previously frozen larvae (FL) exhibited rates of 48%–58% at the beginning and of 19%–39% at the end of the 35 week in vitro maintenance period. The surviving FL and CL larvae proved to be infective for mice. Excretory/secretory (ES) antigens isolated from several batches of culture medium in which FL and CL had been maintained reacted in the ELISA with human sera containing antibodies against Toxocara. Antigens from FL and CL separated by SDS-PAGE and silver-stained showed some differences in polypeptide patterns. Western-blot analysis revealed that these differences were not related to antigenic polypeptides but were most likely caused by substances without antigenic properties originating from dead and/or degenerating larvae. It can be concluded that ES antigens produced by previously frozen larvae are essentially the same as those derived from unfrozen controls.The value of cryopreservation of T. canis larvae for routine production of ES antigens will be further evaluated.     

Bi-allelic loss of function variants in SLC30A5 as cause of perinatal lethal cardiomyopathy

Perinatal mortality is a heavy burden for both affected parents and physicians. However, the underlying genetic causes have not been sufficiently investigated and most cases remain without diagnosis. This impedes appropriate counseling or therapy. We describe four affected children of two unrelated families with cardiomyopathy, hydrops fetalis, or cystic hygroma that all deceased perinatally. In the four patients, we found the following homozygous loss of function (LoF) variants in SLC30A5 NM_022902.4:c.832_836del p.(Ile278Phefs*33) and NM_022902.4:c.1981_1982del p.(His661Tyrfs*10). Knockout of SLC30A5 has previously been shown a cardiac phenotype in mouse models and no homozygous LoF variants in SLC30A5 are currently described in gnomAD. Taken together, we present SLC30A5 as a new gene for a severe and perinatally lethal form of cardiomyopathy.Subject terms: Cardiovascular diseases, Development, Medical genetics, Medical genomics     

Micronuclei containing whole chromosomes harbouring the selectable gene do not lead to mutagenesis

Loss of heterozygosity is one genetic change observed in manytumours. We do not know whether the loss of chromosomal materialthrough micronucleus formation is a viable mechanism associatedwith, and possibly leading to, genetic disease. Previously,we treated L5178Y mouse lymphoma cells with four aneugens. Althoughthese aneugens induced micronuclei containing predominantlywhole chromosomes, they did not induce mutations at Tk1, theselectable gene, under the same non-toxic conditions in whichthey induced micronuclei. This suggested that the inductionof micronuclei containing whole chromosomes was not an earlyevent leading to phenotypically expressed mutations in thesecells under the conditions used. However, it is possible thatchromosome 11, on which Tk1 resides, may be under-representedin the micronucleus population. To find out the frequency ofinduction of micronuclei containing chromosome 11, we appliedfluorescence in situ hybridization using a chromosome 11 paintto micronuclei induced by colcemid and vinblastine. We foundthat the numbers of micronuclei containing chromosome 11 aremore than sufficient to be detectable as mutations if thesemicronuclei lead to viable mutants. We conclude that the formationof micronuclei containing whole chromosomes does not lead toviable, dividing mutants in this system. ⁵To whom correspondence should be addressed     

The methionine allele of the COMT polymorphism impairs prefrontal cognition in children and adolescents with ADHD

ADHD is a highly heritable psychiatric disorder of childhood. A functional polymorphism (Val158Met) of the catechol-O-methyltransferase (COMT) gene has attracted interest as a candidate gene for ADHD. The high-activity valine variant of this polymorphism degrades prefrontal dopamine three to four times more quickly than the low-activity methionine variant and could therefore contribute to the proposed hypodopaminergic state in ADHD. Here we tested for association of this polymorphism with ADHD and examined its influence on prefrontal cognition in ADHD. We have previously reported no association of the Val158Met COMT gene polymorphism in 94 Irish ADHD families (Hawi et al. (2000) Am J Med Genet 96:282–284). Here we re-examined this finding with an extended sample of 179 ADHD cases using a family control design. We also examined the performance of children and adolescents with ADHD (n=61) on a standardised test of sustained attention. Analysis confirmed the absence of an association between the Val158Met COMT gene polymorphism and the clinical phenotype of ADHD. COMT genotype, however, affected prefrontal cognition in ADHD: ADHD children who were homozygous for the valine variant had significantly better sustained attention than those ADHD children possessing at least one copy of the methionine variant. Children possessing the methionine variant performed significantly below age-related norms on tests of sustained attention. Contrary to expectations, the methionine variant of the Val158Met COMT gene polymorphism impaired prefrontally-mediated cognition in ADHD. This effect may be understood by positing a hyper-functioning of prefrontal dopaminergic systems. Against this background, the slower clearance of dopamine associated with the methionine variant of the COMT gene polymorphism may be disadvantageous to cognition in ADHD.Mark Bellgrove and Katharina Domschke contributed equally to this work and should therefore both be considered first authors. The work reported herein was supported by a grant from the Irish Health Research Board.     

Humoral and cellular immune reactions 'in vitro' against allogeneic and autologous human melanoma cells.

Based on 51Cr release from an allogeneic human melanoma cell (M1) cell-mediated (CMC) and antibody-dependent cellular cytotoxicity (ADCC) were determined in twenty-eight melanoma patients, thirty-one healthy controls, ten patients with other tumours and eleven chronic lymphocytic leukaemia (CLL) patients. The results were related to simultaneously performed microcytotoxicity (MC) tests, HL-A typing, plasma membrane fluorescence and B:T cell ratios in the peripheral blood. Furthermore, lymphocytes from six melanoma patients were tested in CMC and ADCC assays against autologous tumour cells. The following results were obtained. (1) A large number of healthy controls possessed lymphocytes which readily lysed M1 target cells in CMC and MC assays. (2) CMC activity of lymphocytes from melanoma patients was generally lower than that of control lymphocytes and decreased further with progression of the disease. (3) CLL lymphocytes were virtually non-toxic for M1 cells, even at high aggressor:target cell ratios. (4) ADCC assays with a heterologous rabbit-anti-M1 serum showed generally higher isotope release than CMC assays; this was particularly pronounced in the melanoma group and in the group of patients with other tumours. (5) No tumour-specific blocking factor could be detected in melanoma sera, as judged by the capacity of the sera to block CMC activity. (6) No obvious correlation was found between the results obtained in short-term CMC and long-term MC assays. (7) T lymphocytes, as determined by E-rosette formation, were significantly diminished in melanoma patients. (8) The HL-A type of lymphocytes from normal donors and melanoma patients did not appear to be related to high or low activity in CMC and MC assays. (9) Preliminary results of 51Cr release tests with autologous melanoma cells were encouraging with respect to the correlation of the results to the clinical course of the disease.     

Differences in maintenance of CD8+ and CD4+ bacteria-specific effector-memory T cell populations

Our knowledge about the kinetics and dynamics of complex pathogen-specific CD8(+) T cell responses and the in vivo development of CD8(+) memory T cells has increased substantially over the past years; in comparison, relatively little is known about the CD4(+) T cell compartment. We monitored and directly compared the phenotypical changes of pathogen (Listeria monocytogenes)-specific CD8(+) and CD4(+) T cell responses under conditions leading to effective and long-lasting protective immunity. We found that the general kinetics of bacteria-specific CD8(+) and CD4(+) T cells during the effector and post-effector phases are synchronized. However, later during the memory phase, CD8(+) and CD4(+) T cell populations differ substantially. Whereas CD8(+) memory T cell populations with immediate effector function are readily detectable in lymphoid and non-lymphoid tissues and remain remarkably stable in size, antigen-specific CD4(+) effector-memory T cells decline continuously in frequency over time. These findings have important implications for the better understanding of the in vivo development of protective immunity towards intracellular pathogens.     

Evaluation of cellular substrates for antinuclear antibody determinations.

The immunofluorescent technique was employed to evaluate the sensitivity of 10 human and animal cell monolayers and tissue sections as substrates for titering the antinuclear antibody content of serum samples. The highest mean ranks of sensitivity, the relative ability of each substrate to maintain its sensitivity rank when 21 selected positive sera were tested, were achieved by two fibroblast cell lines, baby hamster kidney (BHK 21/C13) and human lung (WI-38), respectively. The least sensitive substrates were commercial rat kidney and liver tissue sections.     

Evaluation of a medical interviewing course. Use of the helping relationship inventory

Changes in beginning medical students' preferred interview responses appear attributable to a course that emphasizes communication techniques for developing patient rapport. For each of five successive classes, pre/postcourse preferences were obtained for alternative response modes (categorized as understanding, probing, interpretive, supportive, and evaluative. Analysis indicated significant increases in students' preferences for understanding responses and decreases in preferences for evaluative responses (p less than .001). Changes are in the desired direction with respect to course goals, since rapport is generally enhanced by conveying understanding and refraining from premature evaluation. Effects on response preferences of some instructor characteristics are analyzed. Implications for health professions education and research are discussed.