Evaluation of the MicroScan Urinary Combo Panel and API 20E system for identification of glucose-nonfermenting gram-negative bacilli isolated from clinical veterinary materials

Many isolates of glucose-nonfermenting gram-negative bacilli (NFB) cultured from clinical veterinary specimens are not identified because of the large number of identification tests required. We evaluated two commercial identification systems to determine if they could accurately identify NFB isolated from animals. Of 182 strains of NFB, the MicroScan Urinary Combo Panel (MicroScan, Inc., Campbell, Calif.) correctly identified 72%, and the API 20E system (Analytab Products, Plainview, N.Y.) correctly identified 74%. Of the 118 strains of the three most common species of NFB isolated from animals, the MicroScan Urinary Combo Panel identified 86% correctly, and the API 20E system identified 92% correctly. The use of either of these systems could improve the accuracy of identification of NFB from clinical veterinary materials.     

Two novel presenilin-1 mutations (Y256S and Q222H) are associated with early-onset Alzheimer's disease

Mutations in the gene encoding presenilin 1 (PS-1) account for 50% of early-onset familial Alzheimer's disease (EOFAD) cases. In this study, we identified two missense mutations in the coding sequence of the presenilin (PS-1) gene in two EOFAD pedigrees. AD was confirmed in one pedigree by autopsy. Mutation analysis of PCR products amplified from genomic DNA templates showed two novel PS-1 mutations resulting in Gln222His and Tyr256Ser. The two novel mutations are located within predicted transmembrane domains five (TM-5) and six (TM-6), respectively, and are associated with very early ages of onset. The Tyr256Ser is associated with one of the youngest age of AD onset, 25 years, which is consistent with a drastic change in function of the altered PS-1 protein. A morphometric analysis of the cortical degenerative changes of the Tyr256Ser case, showed severe involvement of the primary motor cortex, which correlated well with the pyramidal changes, including tetraspasticity. Immunoblot analysis showed the Tyr256Ser case had the greatest expression of Abeta(1-40) and Abeta(1-42), which was confirmed by ELISA, compared to other PS-1 mutant FAD cases and age-matched controls and, thus, contributes to the severity of the disease pathology.     

Experience with prenatal fragile X detection

We have attempted the prenatal detection of the fra(X) 9 times. Three fra(X) positive fetuses have been diagnosed: 2 males and one female. The diagnosis on the 2 males has been confirmed. The testes of the 2 fra(X) positive fetuses appeared large for gestational age. However, results of anthropometric, bone age, anatomical and neurohistological studies were normal. Normal outcome was confirmed after birth in 2 males and one female on the basis of whole blood fra(X) studies. A presumptively positive female and a presumptively negative female await confirmation. Two presumptively negative males remain unborn. Further experience is needed to establish the reliability of the prenatal detection of fra(X) (q27).     

In vitro genotoxicity of dyes present in colored smoke munitions

Genetic toxicology studies were conducted on organic dyes and mixtures used in colored smoke munitions. The dyes studied included Solvent Red 1; two different batches (Lot 1 and Lot 2) of Disperse Red 11; terephthalic acid; and a mixture of 25 parts Solvent Red 1, 5 parts Disperse Red 11, and 16 parts terephthalic acid. The dyes were evaluated for their ability to produce mutations in Salmonella bacterial strains and in Chinese hamster ovary (CHO) cells. The dyes were also tested in CHO cells to determine cytotoxicity and the induction of sister chromatid exchanges and chromosome aberration. None of the dyes were genotoxic in the standard Ames assay using bacterial strain TA1535 or TA100 with or without the addition of S-9 or in TA98 and TA1538 without S-9. With S-9, Disperse Red 11 (Lot 2) showed significant mutagenic activity in TA98 and TA1538 which increased as a function of S-9 concentration. However, the maximum level of mutagenic activity detected was low (3.8 revertants/micrograms). The azo dye Solvent Red 1 was also negative in a pre-incubation assay designed to reduce azo compounds to free amines. Solvent Red 1 was cytotoxic to mammalian cells, caused a significant increase in SCE, but was not mutagenic or clastogenic. Disperse Red 11 (Lot 1 and Lot 2) were not cytotoxic or clastogenic but produced an increase in cell cycle time and SCE frequency. Only Disperse Red 11 (Lot 2) increased mutations in the CHO/hypoxanthine-guanine phosphoribosyltransferase (HGPRT) assay. The mutagenic activity of the dye mixture was not significant, suggesting no synergistic interaction between the dyes. These studies demonstrated that none of the dyes was clastogenic and that a contaminant in Disperse Red 11 (Lot 2) may be responsible for the weak mutagenic activity in both mammalian and bacterial cell systems.     

Hypothalamic-immune interactions: neuromodulation of natural killer activity by lesioning of the anterior hypothalamus

Placement of bilateral electrolytic lesions in the preoptic-anterior hypothalamic area (AHT) of Fischer 344 rats results in decreased splenic NK activity as compared to control and normal animals. Animals with AHT lesions have a decrease in NK activity 4 and 7 days after lesion placement, with a return to normal activity by day 14. Fractionation of spleen cells on glass bead columns results in nonadherent and adherent cell fractions with NK activity. AHT lesions affect NK activity only in the adherent cell fraction. The removal of macrophages from this cell fraction did not restore NK activity. Moreover, this NK activity is not the result of cytotoxic macrophages. Hypophysectomy decreases NK activity in lesioned and non-lesioned animals, suggesting the influence of pituitary factors. These data indicate that the anterior hypothalamus is capable of modulating the cytotoxic activity of NK cells. Thus, neuroimmunomodulation may be a potential factor in susceptibility to some disease states such as viral infections and neoplasia.     

Gentamicin-mediated suppression of Hurler syndrome stop mutations restores a low level of alpha-L-iduronidase activity and reduces lysosomal glycosaminoglycan accumulation

Hurler syndrome is the most severe form of a lysosomal storage disease caused by loss of the enzyme alpha-L-iduronidase (encoded by the IDUA gene), which participates in the degradation of glycosaminoglycans (GAGs) within the lysosome. In some populations, premature stop mutations represent roughly two-thirds of the mutations that cause Hurler syndrome. In this study we investigated whether the aminoglycoside gentamicin can suppress stop mutations within the IDUA gene. We found that a Hurler syndrome fibroblast cell line heterozygous for the IDUA stop mutations Q70X and W402X showed a significant increase in alpha-L-iduronidase activity when cultured in the presence of gentamicin, resulting in the restoration of 2.8% of normal alpha-L-iduronidase activity. Determination of alpha-L-iduronidase protein levels by an immunoquantification assay indicated that gentamicin treatment produced a similar increase in alpha-L-iduronidase protein in Hurler cells. Both the alpha-L-iduronidase activity and protein level resulting from this treatment have previously been correlated with mild Hurler phenotypes. Although Hurler fibroblasts contain a much higher level of GAGs than normal, we found that gentamicin treatment reduced GAG accumulation in Hurler cells to a normal level. We also found that a reduced GAG level could be sustained for at least 2 days after gentamicin treatment was discontinued. The reduction in the GAG level was also reflected in a marked reduction in lysosomal vacuolation. Taken together, these results suggest that the suppression of premature stop mutations may provide an effective treatment for Hurler syndrome patients with premature stop mutations in the IDUA gene.     

Generation of monoclonal antibodies to cryptic collagen sites by using subtractive immunization

The extracellular matrix (ECM) plays a fundamental role in the regulation of normal and pathological processes. The most abundantly expressed component found in the ECM is collagen. Triple helical collagen is known to be highly resistant to proteolytic cleavage except by members of the matrix metalloproteinase (MMP) family of enzymes. To date little is known concerning the biochemical consequences of collagen metabolism on human diseases. This is due in part to the lack of specific reagents that can distinguish between proteolyzed and triple helical forms of collagen. Here we used the technique of Subtractive Immunization (SI) to generate two unique monoclonal antibodies (MAbs HUIV26 and HUI77) that react with denatured and proteolyzed forms of collagen, but show little if any reaction with triple helical collagen. Importantly, HUIV26 and HUI77 react with cryptic sites within the ECM of human melanoma tumors, demonstrating their utility for immunohistochemical analysis in vivo. Thus, the generation of these novel MAbs not only identify specific cryptic epitopes within triple helical collagen, but also provide important new reagents for studying the roles of collagen remodeling in normal as well as pathological processes.     

Detection of either rapidly cytolytic macrophages or NK cells in "activated" peritoneal exudates depends on the method of analysis and the target cell type

The nature of the cytotoxic cells present in the peritoneal cavity of rats treated with Bacille Calmette-Guérin (BCG) or Corynebacterium parvum was investigated using a 6 hr chromium release assay and a quantitative method of analysis based on consideration of target-cell killing as an enzyme-substrate reaction. When the results of cell-fractionation experiments were evaluated in terms of recovery of total lytic units and when appropriate target cells (such as sarcoma Mc7) were used, the simultaneous presence of both cytotoxic macrophages and NK cells in peritoneal exudates could be readily demonstrated. With certain other target cells different results were obtained. Thus, with normal thymocytes, normal hepatocytes, or myeloma P3NSI as targets, NK cells were preferentially detected, whereas with leukaemias L5178Y, P815, and EL4 as targets, cytotoxic macrophages were preferentially detected. These findings resolve the previously conflicting reports concerning the nature of cytotoxic cells in activated peritoneal exudates.