Molecular analysis of a hospital cafeteria-associated salmonellosis outbreak using modified repetitive element PCR fingerprinting

A hospital cafeteria-associated outbreak of gastroenteritis due to Salmonella enterica serotype Infantis was retrospectively evaluated using modified repetitive element PCR (rep-PCR) fingerprinting with the ERIC2 and BOXA1R primers and computer-assisted gel analysis and dendrogram construction. Rep-PCR yielded objective between-cycler, same-strain similarity values of from 92% (composite fingerprints) to 96% (ERIC2 fingerprints). The 70 Salmonella isolates (which included 19 serotype Infantis isolates from the hospital outbreak, 10 other serotype Infantis isolates, and 41 isolates representing 14 other serotypes) were resolved well to the serotype level with each of the three fingerprint types (ERIC2, BOXA1R, and composite). Rep-PCR typing uncovered several historical serotyping errors and provided presumptive serotype assignments for other isolates with incomplete or undetermined serotypes. Analysis of replicate fingerprints for each isolate, as generated on two different thermal cyclers, indicated that most of the seeming subserotype discrimination noted in single-cycler dendrograms actually represented assay variability, since it was not reproducible in combined-cycler dendrograms. Rep-PCR typing, which would have been able to identify the presence of the hospital-associated serotype Infantis outbreak after the second outbreak isolate, could be used as a simple surrogate for serotyping by clinical microbiology laboratories that are equipped for diagnostic PCR.     

Predominance of null mutations in ataxia-telangiectasia

Ataxia-telangiectasia (A-T) is an autosomal recessive disorder involving cerebellar degeneration, immunodeficiency, chromosomal instability, radiosensitivity and cancer predisposition. The responsible gene, ATM, was recently identified by positional cloning and found to encode a putative 350 kDa protein with a Pl 3-kinase-like domain, presumably involved in mediating cell cycle arrest in response to radiation-induced DNA damage. The nature and location of A-T mutations should provide insight into the function of the ATM protein and the molecular basis of this pleiotropic disease. Of 44 A-T mutations identified by us to date, 39 (89%) are expected to inactivate the ATM protein by truncating it, by abolishing correct initiation or termination of translation, or by deleting large segments. Additional mutations are four smaller in-frame deletions and insertions, and one substitution of a highly conserved amino acid at the Pl 3-kinase domain. The emerging profile of mutations causing A-T is thus dominated by those expected to completely inactivate the ATM protein. ATM mutations with milder effects may result in phenotypes related, but not identical, to A-T.      

Difference in cell binding patterns of two monoclonal antibodies recognizing distinct epitopes on a human melanoma-associated oncofetal antigen

Two monoclonal antibodies (MAbs), 140.240 and 96.5, generated independently in different laboratories, have been shown to detect the target structures of 87,000 (gp87) and 97,000 (p97) glycoproteins, respectively, both strongly expressed by melanoma cells and fetal small intestine. To determine whether MAb 140.240 and MAb 696.5 recognized a same target structure, they were tested in immunoprecipitation/SDS-PAGE using NP-40 lysates of melanoma cells labelled with [35S]methionine for 18 hr. Both antibodies precipitated a single band with Mr = 87,000. Reciprocal immunodepletion studies showed that neither of the two antibodies detected the 87,000 band in the lysate immuno depleted by either antibody, suggesting that these two antibodies recognize the same or extremely similar molecules. Two-dimensional tryptic peptide mapping analysis showed that the two identified molecules shared the same finger-printing pattern. A 40,000 fragment of the 87,000 molecule produced by protease digestion was precipitated by MAb 96.5 but not MAb 140.240, indicating that the epitopes recognized by the two antibodies are localized at discrete sites on the molecule. Serological studies on these two antibodies revealed slightly different binding patterns in the MAb 140.240 exhibited a more melanoma-restricted specificity, while MAb 96.5 had a specificity to melanoma and to some other cell types. The observed difference in epitope specificity may be important in the clinical applications of these antibodies.     

Further refinement of Pendred syndrome locus by homozygosity analysis to a 0.8 cM interval flanked by D7S496 and D7S2425.

Pendred syndrome is an autosomal recessive disease characterised by congenital sensorineural deafness and goitre. The gene responsible for Pendred syndrome has been mapped to chromosome 7q31 in a 5.5 centimorgan (cM) interval flanked by D7S501 and D7S523. This interval was recently refined a to 1.7 cM interval located between D7S501 and D7S692. In the present study, we report linkage analysis data on a large consanguineous family genotyped with eight microsatellite markers located between D7S501 and D7S523. Complete cosegregation with the disease locus was observed with the loci analysed, which further supports locus homogeneity for Pendred syndrome and close linkage to this region. Haplotype analysis placed the Pendred syndrome gene between D7S496 and D7S2425 in a 0.8 cM interval. This additional refinement of the Pendred syndrome region will facilitate the construction of a physical map of the region and will help the identification of candidate genes.     

Immunohistochemistry of hepatocellular carcinoma associated with cirrhosis

Tumor cell marker antibodies were used to analyze ten cases of hepatocellular carcinoma associated with cirrhosis. Clinically, eight of these cases gave a history of chronic alcoholism and the other two of hepatitis B virus infection. Formalin-fixed, paraffin-embedded sections from these cases were screened with antibodies against alpha fetoprotein (AFP), hepatitis B surface antigen (HBsAg) and carcinoembryonic antigen (CEA) using the peroxidase antiperoxidase and avidin-biotin immunoperoxidase procedures. Three cases were positive for AFP, four for HBsAg, and three for CEA; two cases had both HBsAg and CEA. Alpha fetoprotein was present only in the cytoplasm of tumor cells in three cases. Hepatitis B surface antigen, on the other hand, was present in the cytoplasm of hepatocytes in cirrhotic areas and, in one out of the four cases, was also present in hepatocellular carcinoma cells. Carcinoembryonic antigen was seen in three cases; it was present on the surface and in the cytoplasm of proliferating ducts within the cirrhotic areas and between cell surfaces of individual tumor cells in two cases. The presence of different markers was not related to the microscopic appearance of the tumors. In one case, positivity for AFP was of diagnostic help in a tissue sample obtained by needle biopsy. The avidin-biotin immunoperoxidase procedure was more sensitive than the peroxidase antiperoxidase (PAP technique in the pathological assessment of autopsy specimens. Our findings are in agreement with those of other reports and indicate that AFP and HBsAg are the most commonly found markers in hepatoma associated with cirrhosis, and that CEA staining is variable and hepatoma associated with cirrhosis, and that CEA staining is variable and probably non-contributory.