Establishment and characterization of an antigen-specific T-cell line from liver granulomas of Schistosoma mansoni-infected mice.

Granulomatous inflammations in schistosomiasis mansoni are the result of T-cell-mediated reactions to soluble egg antigens (SEA) secreted by parasite ova. To study TDH effector cell function, a granuloma T-cell line was established from collagenase-digested liver granulomas of acutely infected CBA/J mice. Dispersed nonadherent granuloma cells were cultured with feeder layer cells and SEA or with feeder layer cells alone in alternate cycles for 32 weeks. The granuloma T-cell line was L3T4+ Lyt-1+. In vitro, the SEA-stimulated T cells showed proliferation and interleukin 2 production. One million T cells adoptively transferred SEA-specific footpad swelling, and 7.5 X 10(6) T cells adoptively transferred granulomatous hypersensitivity to injected ova or SEA-coated beads. Anti-L3T4 monoclonal antibody blocked the SEA-specific cell proliferation. Depletion of L3T4+ cells abrogated, while that of Lyt-1+ cells diminished the adoptive transfer of SEA-specific footpad swelling. These experiments demonstrate that the granuloma T-lymphocyte population contains TDH-type effector cells. Establishment of an SEA-specific granuloma T-cell line will allow the study of the effector functions of the hitherto uncharacterized intralesional granuloma T lymphocyte.     

A convenient multiplex PCR system for the detection of dystrophin gene deletions: a comparative analysis with cDNA hybridisation shows mistypings by both methods.

Existing reactions for the multiplex PCR amplification of exons in the dystrophin gene have been modified to produce two multiplex reactions which separately cover the 5' and 3' major deletion 'hotspots' in the gene, and together detect approximately 98% of all deletions detectable by Southern cDNA hybridisation. A comparative study of 148 patients showed mistypings in both the cDNA hybridisation data (4%) and the PCR analysis (1.2%). We suggest means of circumventing the underlying problems in order to avoid mistyping and subsequent misdiagnosis, and conclude that, with appropriate precautions, multiplex PCR amplification can be the method of choice for detecting deletions in the dystrophin gene.     

Are three sputum acid-fast bacillus smears necessary for discontinuing tuberculosis isolation?

To evaluate the efficacy of three sputum acid-fast bacillus (AFB) smears to rule out pulmonary tuberculosis, sputum AFB smear and culture results were analyzed at two university-affiliated teaching hospitals. The negative predictive value of the smear increased by only 0.2% on days 2 and 3 each, indicating that in low-prevalence populations, there is limited value in requiring three negative sputum AFB smears before discontinuing tuberculosis isolation.     

Prenatal prediction of spinal muscular atrophy.

Spinal muscular atrophy (SMA) is a common cause of inherited morbidity and mortality in childhood. The wide range of phenotypes in SMA, uncertainty regarding its mode of inheritance, and the suggestion of linkage heterogeneity have complicated the genetic counselling of parents of affected children. The locus responsible for autosomal recessive SMA has been mapped to 5q11.2-q13.3. The most likely order of loci is cen-D5S6-(SMA,D5S125)-(JK53CA1/2,D5S112)-D5S3 9-qter, with highly polymorphic loci being identified at JK53CA1/2 and D5S39. We describe linkage studies with another highly polymorphic locus, D5S127, that is closely linked to D5S39. This genetic map can be used as the basis for genetic counselling in families with autosomal recessive SMA. Appropriate allowance can be made for sporadic cases owing to non-inherited causes and for linkage heterogeneity or misdiagnoses.     

Association of DLG5 R30Q variant with inflammatory bowel disease

Crohn's disease (CD) and ulcerative colitis (UC) are chronic inflammatory diseases of the gastrointestinal system known as the inflammatory bowel diseases (IBD). Recently, Stoll and colleagues reported a novel finding of genetic variation in the DLG5 gene that is associated with IBD (CD and UC combined). We present here a study of the genetic variation described in that report in two well-powered, independent case-control cohorts and one family-based collection, and confirm the proposed association between IBD and the R30Q variant of DLG5 in two of the three studies. We are, however, unable to replicate the other proposed association to the common haplotype described in Stoll et al and suggest that this other finding could conceivably have been partially a statistical fluctuation and partially a result of LD with the replicated R30Q association. This study provides support for the hypothesis that DLG5 constitutes a true IBD risk factor of modest effect.     

Spectrum of mutations in the Fanconi anaemia group G gene, FANCG/XRCC9

FANCG was the third Faconi anaemia gene identified and proved to be identical to the previously cloned XRCC9 gene. We present the pathogenic mutations and sequence variants we have so far identified in a panel of FA-G patients. Mutation screening was performed by PCR, single strand conformational polymorphism analysis and protein truncation tests. Altogether 18 mutations have been determined in 20 families - 97% of all expected mutant alleles. All mutation types have been found, with the exception of large deletions, the large majority is predicted to lead to shortened proteins. One stop codon mutation, E105X, has been found in several German patients and this founder mutation accounts for 44% of the mutant FANCG alleles in German FA-G patients. Comparison of clinical phenotypes shows that patients homozygous for this mutation have an earlier onset of the haematological disorder than most other FA-G patients. The mouse Fancg sequence was established in order to evaluate missense mutations. A putative missense mutation, L71P, in a possible leucine zipper motif may affect FANCG binding of FANCA and seems to be associated with a milder clinical phenotype.     

Synectin/syndecan-4 regulate coronary arteriolar growth during development.

Syndecan-4 and its cytoplasmic binding partner, synectin, are known to play a role in FGF-2 signaling and vascular growth. To determine their roles in coronary artery/arteriolar formation and growth, we compared syndecan-4 and synectin null mice with their wild-type counterparts. Image analysis of arterioles visualized by smooth muscle alpha-actin immunostaining revealed that synectin (-/-) mice had lower arteriolar length and volume densities than wild-type mice. As shown by electron microscopic analysis, arterioles from the two did not differ in morphology, including their endothelial cell junctions, and the organization and distribution of smooth muscle. Using micro-computer tomography, we found that the size and branching patterns of coronary arteries (diameters > 50 microm) were similar for the two groups, a finding that indicates that the growth of arteries is not influenced by a loss of synectin. Syndecan-4 null male mice also had lower arteriolar length densities than their gender wild-type controls. However, female syndecan-4 null mice were characterized by higher arteriolar length and volume densities than their gender-matched wild-type controls. Thus, we conclude that both synectin and syndecan-4 play a role in arteriolar development, a finding that is consistent with previous evidence that FGF-2 plays a role in coronary arterial growth. Moreover, our data reveal that gender influences the arteriolar growth response to syndecan-4 but not to synectin.     

Tissue zinc levels in precancerous tissue in the gastrointestinal tract of azoxymethane (AOM)-treated rats

Alterations in tissue zinc levels have been documented in patients with gastrointestinal tract malignancies and more frequently, in those with colonic cancer. However, the precise role of tissue zinc in carcinogenesis is not well elucidated. This study, using a well-established colon cancer model in rats, was designed to investigate the relationship of tissue zinc to the carcinogenic process. The aim was to examine tissue zinc levels in the preneoplastic tissues and to study the changes that occur during transition of mucosa from normal to preneoplastic state. Six-week old rats were given a single dose subcutaneous injection of azoxymethane (AOM) (30mg/kg body weight) and sacrificed after 1, 2, 5, and 9 months of the treatment. Plasma zinc levels showed a significant decrease (p<0.05) at 9 months compared with controls. Tissue zinc levels showed a significant decrease in the large intestine at 1 and 2 months (p<0.05) and at 5 and 9 months (p<0.01), in the small intestine at 2, 5, and 9 months (p<0.05), and in the stomach at 5 and 9 months (p<0.05). The maximum percent decrease (45%) in tissue zinc was observed in the large intestine at 9 months. Tissue copper zinc super oxide dismutase (CuZnSOD) activity was assessed in the body of the stomach, small intestine, and large intestine and compared with the control group. There was a significant fall in CuZnSOD levels in the small intestine at 9 months (p<0.05) and in the large intestine at 5 and 9 months (p<0.01). Two of these six rats showed histological evidence of precancerous lesions in the mucosa of the colon. This study suggests that the decrease in plasma zinc, tissue zinc and activity of CuZnSOD is associated with development of preneoplastic lesions in the colonic mucosa.