Genetic analysis of susceptibility to dextran sulfate sodium-induced colitis in mice

The genetic basis for differential sensitivity of inbred mice to inflammatory bowel disease induced by dextran sulfate sodium (DSS) is unknown. Susceptible C3H/HeJ were outcrossed to partially resistant C57BL/6J mice. F2 and N2 progeny were phenotyped by evaluating histopathologic lesions in large intestine detected 16 days after a 5-day period of feeding 3.5% DSS. Screening for DSS colitis (Dssc) loci revealed quantitative trait loci (QTL) on Chr 5 (Dssc1) and Chr 2 (Dssc2). These traits contributed additively, explaining 17.5% of the variation in total colonic lesions. Additional QTL on Chr 18 and 1 that collectively explained 11% of the variation in total colon lesions were indicated. In the cecum, only a putative QTL on Chr 11 was associated with pathology (lesion severity) in the cecum. Reduced DSS susceptibility was observed in congenic stocks in which the highly susceptible NOD/Lt strain carried putative resistance alleles from either B6 on Chr 2 or from the highly resistant NON/Lt strain on Chr 9. We conclude that multiple genes control susceptibility to DSS colitis in mice. Possible Dssc candidate genes are discussed in terms of current knowledge of inflammatory bowel disease susceptibility loci in humans.     

Substrate recognition by the Pvu II endonuclease: binding and cleavage of CAG5mCTG sites

The Pvu II restriction endonuclease (R. Pvu II) cleaves CAG downward arrowCTG sequences as indicated, leaving blunt ends. Its cognate methyltransferase (M. Pvu II) generates N4-methylcytosine, yielding CAGN4mCTG, though the mechanism by which this prevents cleavage by R. Pvu II is unknown. The heterologous 5-methylcytosinemethylation CAG5mCTG has also been reported to prevent cleavage by R. Pvu II and this has been used in some cloning methods. Since this heterologousmethylation occurs at the native methylated base, it can provide insights into the detection of DNAmethylation by R. Pvu II. We found that the cloned gene for R. Pvu II could not stably transform cells protected only by M. Alu I (AG5mCT) and then determined that R. Pvu II cleaves CAG5mCTG in vitro, even when both strands are methylated. DNase I footprint analysis and competition experiments reveal that R. Pvu II binds to CAG5mCTG specifically, though with reduced affinity relative to the unmethylated sequence. These results provide biochemical support for the publishedstructures of R. Pvu II complexed with DNA containing CAGCTG and CAG5-iodoCTG and support a model for how methylation interferes with DNA cleavage by this enzyme.     

Organization of an echinoderm Hox gene cluster

The Strongylocentrotus purpuratus genome contains a single ten-gene Hox complex >0.5 megabase in length. This complex was isolated on overlapping bacterial artificial chromosome and P1 artificial chromosome genomic recombinants by using probes for individual genes and by genomic walking. Echinoderm Hox genes of Paralog Groups (PG) 1 and 2 are reported. The cluster includes genes representing all paralog groups of vertebrate Hox clusters, except that there is a single gene of the PG4-5 types and only three genes of the PG9-12 types. The echinoderm Hox gene cluster is essentially similar to those of the bilaterally organized chordates, despite the radically altered pentameral body plans of these animals.     

Environmentally superior technologies for swine waste management.

The high nitrogen content of animal waste provides opportunities for processing to marketable byproducts and challenges for proper management to avoid harmful impacts. Technologies are being developed to conserve and utilize nitrogen as well as other valuable constituents in animal waste. Advanced treatment technologies are also being developed for housing/waste management systems that address public concerns and protect soil, water and air quality. Smithfield Foods, Premium Standard Farms and Frontline Farmers have entered into an agreement with North Carolina to develop environmentally superior technologies that meet these goals. The 18 candidate technologies are identified and three with the longest operating period, and thus most data to date are discussed. Methods for distributing this information for implementation of cost-effective technologies through the Curriculum Project and the National Center for Manure and Animal Waste Management will be presented. This work supports priority goals to conserve and utilize valuable animal waste constituents while also protecting against negative impacts.     

Consistent, persistent expression from modified retroviral vectors in murine hematopoietic stem cells

Retroviral vectors based on the Moloney murine leukemia virus (MoMuLV) have shown inconsistent levels and duration of expression as well as a propensity for the acquisition of de novo methylation in vivo. MoMuLV-based vectors are known to contain sequences that are capable of suppressing or preventing expression from the long terminal repeat. Previously, we constructed a series of modified retroviral vectors and showed that they function significantly better than MoMuLV-based vectors in vitro. To test the efficacy of the modified vectors in hematopoietic stem cells in vivo, we examined gene expression and proviral methylation in differentiated hematopoietic colonies formed in the spleens of mice after serial transplantation with transduced bone marrow (2 degreesCFU-S). We found a significant increase in the frequency of expression with our modified vectors (>90% expression in vector DNA containing 2 degreesCFU-S) over the frequency observed with the standard MoMuLV-based vector (28% expression in vector containing 2 degreesCFU-S). Expression from the modified vectors was highly consistent, with expression in >50% of the vector-containing 2 degreesCFU-S from all 20 transplant recipients analyzed, whereas expression from the standard MoMuLV-based vector was inconsistent, with expression in 0-10% of the vector containing 2 degreesCFU-S from 8 recipients and expression in >50% of the vector-containing 2 degreesCFU-S from 4 other recipients. In addition, we established that the modified vectors had a lower level of DNA methylation than the control vector. These findings represent significant advances in the development and evaluation of effective retroviral vectors for application in vivo.     

Faecal egg count reduction percentage calculations to detect anthelmintic resistance in Oesophagostomum spp. in pigs

The results of four alternative methods of mean faecal egg count reduction percentage (FECR%) calculations were evaluated and compared using data obtained for Oesophagostomum spp. from ten sow herds. The estimates of FECR% and 95% confidence limits obtained using the four methods were different. However, there were few discrepancies in the final decision as to whether a given herd carried drug resistant isolates or not. The methods that used geometric means were more appropriate than those that used the arithmetic mean as the measure of central tendency for eggs per gram of faeces (EPG) values. The use of geometric mean EPG values in calculations has been criticized from several viewpoints, one of which is that its use reduces the comparability of reports between laboratories. If the geometric mean is to be used as we suggest in FECR% calculations, the appropriate references, number of animals in each group, minimum and maximum EPG values and the factor added to zero EPG counts should be reported in order to improve the comparability. The difficulty in obtaining groups with similar pre-treatment EPG values in field situations suggested the inclusion of pre-treatment EPG values in the calculations as an adjustment procedure. The importance of including a non-treated control group in calculations was demonstrated during this study. Therefore, we suggest the use of geometric mean EPG values, to include pre-treatment EPG values and to include the egg counts from the control group in FECR% calculations. The interpretation of the resulting FECR estimate may be different according to the purpose for which the testing procedure is carried out, e.g. survey in detecting anthelmintic resistance, control field tests, etc.     

Association between incidence of Aspergillus antigenemia and exposure to construction works at a hospital site

During the course of extensive building activity in the vicinity of bone marrow transplantation wards, the patients were routinely screened for the occurrence of Aspergillus galactomannan antigen in serum. In 19 (6.7%) out of 285 patients, an antigenemia was detected. Eleven (58%) of the 19 antigenemic patients suffered from autopsy-proven or clinically suspected invasive aspergillosis. The yearly incidence of antigenemic patients differed significantly, ranging from 0% in the year without building activities to 20.9% in the year with major activities, particularly interior completion works and landscaping. It is concluded that Aspergillus antigen monitoring of bone marrow transplant recipients has a limited value for the diagnosis of manifest invasive aspergillosis. However, it it epidemiologically useful to assess the extent of intensive contact with aspergilli and to control the effectivity of preventive measures.