Pneumocystis carinii f. sp. hominis is not infectious for SCID mice

The infectious power of Pneumocystis carinii f. sp. hominis was explored by inoculating SCID mice intranasally with either P. carinii f. sp. hominis or P. carinii f. sp. muris isolates. Only mice inoculated with mouse parasites developed Pneumocystis pneumonia, as assessed by microscopy and PCR. These results suggest that humans do not contract pneumocystosis from animals.     

Lipoteichoic acids from Lactobacillus johnsonii strain La1 and Lactobacillus acidophilus strain La10 antagonize the responsiveness of human intestinal epithelial HT29 cells to lipopolysaccharide and gram-negative bacteria

Intestinal epithelial cells (IECs) respond to lipopolysaccharide (LPS) from gram-negative bacteria in the presence of the soluble form of CD14 (sCD14), a major endotoxin receptor. Since sCD14 is also known to interact with gram-positive bacteria and their components, we looked at whether sCD14 could mediate their effects on human IECs. To this end, we examined the production of proinflammatory cytokines following exposure of the IECs to specific gram-positive bacteria or their lipoteichoic acids (LTAs) in the absence and presence of human milk as a source of sCD14. In contrast to LPS from Escherichia coli or Salmonella enteritidis, neither the gram-positive bacteria Lactobacillus johnsonii strain La1 and Lactobacillus acidophilus strain La10 nor their LTAs stimulated IECs, even in the presence of sCD14. However, both LTAs inhibited the sCD14-mediated LPS responsiveness of IECs. We have previously hypothesized that sCD14 in human milk is a means by which the neonate gauges the bacterial load in the intestinal lumen and liberates protective proinflammatory cytokines from IECs. The present observations suggest that gram-positive organisms, via their LTAs, temper this response and prevent an exaggerated inflammatory response.     

Effects of long-term lamivudine therapy in renal-transplant patients.

BACKGROUND: Following renal transplantation (RT), chronic immunosuppression is associated in hepatitis B virus (HBV) (+) patients with a flare-up of the disease, which might be harmful in the long term. OBJECTIVES: We report on the effect of long-term lamivudine therapy given at an initial daily dose of 100mg in 18 HBV (+) RT patients. RESULTS: When lamivudine therapy was commenced, 14 patients (77%) had an increase in their aspartate (AST) and alanine (ALT) aminotransferase levels. During a mean follow-up, under treatment, of 36.5 +/- 3.5 months (up to 66 months), 10 patients (55%) had a sustained partial (HBV DNA < 4 x 10(5)copies/ml) (n = 4) or complete (HBV DNA < 400 copies/ml) (n = 6) virological response. Overall, 12 virological breakthroughs were observed. Of those who were HBe Ag(+) prior to lamivudine therapy (n = 4), one seroconverted to HBe Ab during therapy. At the last follow-up, AST and ALT levels were normal in 13 patients. When liver biopsy was repeated during treatment (n = 15), the virological responders showed a significant decrease in total Knodell score from 10 +/- 0.6 to 7 +/- 1 (P = 0.04), but no significant change in the stage of fibrosis. Conversely, in those patients with high HBV DNA titers, there were no significant changes in the total Knodell score or in the grade of fibrosis. CONCLUSION: In conclusion, lamivudine therapy is safe in HBV(+)ve renal-transplant patients. However, even if the full and partial virological response rates are still high (55%) in the long term, relapse or primary non-responses occur. The implementation of alternative efficient strategies is warranted.     

Multiplex PCR targeting tpi (triose phosphate isomerase), tcdA (Toxin A), and tcdB (Toxin B) genes for toxigenic culture of Clostridium difficile

A multiplex PCR toxigenic culture approach was designed for simultaneous identification and toxigenic type characterization of Clostridium difficile isolates. Three pairs of primers were designed for the amplification of (i) a species-specific internal fragment of the tpi (triose phosphate isomerase) gene, (ii) an internal fragment of the tcdB (toxin B) gene, and (iii) an internal fragment of the tcdA (toxin A) gene allowing distinction between toxin A-positive, toxin B-positive (A+B+) strains and toxin A-negative, toxin B-positive (A−B+) variant strains. The reliability of the multiplex PCR was established by using a panel of 72 C. difficile strains including A+B+, A−B−, and A−B+ toxigenic types and 11 other Clostridium species type strains. The multiplex PCR assay was then included in a toxigenic culture approach for the detection, identification, and toxigenic type characterization of C. difficile in 1,343 consecutive human and animal stool samples. Overall, 111 (15.4%) of 721 human samples were positive for C. difficile; 67 (60.4%) of these samples contained A+B+ toxigenic isolates, and none of them contained A−B+ variant strains. Fifty (8%) of 622 animal samples contained C. difficile strains, which were toxigenic in 27 (54%) cases, including 1 A−B+ variant isolate. Eighty of the 721 human stool samples (37 positive and 43 negative for C. difficile culture) were comparatively tested by Premier Toxins A&B (Meridian Bioscience) and Triage C. difficile Panel (Biosite) immunoassays, the results of which were found concordant with toxigenic culture for 82.5 and 92.5% of the samples, respectively. The multiplex PCR toxigenic culture scheme described here allows combined diagnosis and toxigenic type characterization for human and animal C. difficile intestinal infections.     

Widespread lipoplex-mediated gene transfer to vascular endothelial cells and hemangioblasts in the vertebrate embryo.

We report here a method that allows fast, efficient, and low-cost screening for gene function in the vascular system of the vertebrate embryo. Through intracardiac delivery of nucleic acids optimally compacted by a specific cationic lipid, we are able to induce in vivo endothelial cell-specific gain-of-function during development of the vascular network in the chick embryo. When the nucleic acids are delivered during the period of intraembryonic hematopoiesis, aortic hemangioblasts, the forerunners of the hematopoietic stem cells known to derive from the aortic endothelium, are also labeled. Similarly, we show that siRNA could be used to induce loss-of-function in vascular endothelial cells. This gene transfer technique was also applied to the mouse embryo with a high efficiency. The present method allows large-scale analysis and may represent a new and versatile tool for functional genomics.     

Dysferlin mutations in LGMD2B, Miyoshi myopathy, and atypical dysferlinopathies

DYSF encoding dysferlin is mutated in Miyoshi myopathy and Limb-Girdle Muscular Dystrophy type 2B, the two main phenotypes recognized in dysferlinopathies. Dysferlin deficiency in muscle is the most relevant feature for the diagnosis of dysferlinopathy and prompts the search for mutations in DYSF. DYSF, located on chromosome 2p13, contains 55 coding exons and spans 150 kb of genomic DNA. We performed a genomic analysis of the DYSF coding sequence in 34 unrelated patients from various ethnic origins. All patients showed an absence or drastic decrease of dysferlin expression in muscle. A primary screening of DYSF using SSCP or dHPLC of PCR products of each of 55 exons of the gene was followed by sequencing whenever a sequence variation was detected. All together, 54 sequence variations were identified in DYSF, 50 of which predicting either a truncated protein or one amino-acid substitution and most of them (34 out of 54) being novel. In 23 patients, we identified two pathogenic mutations, while only one was identified in 11 patients. These mutations were widely spread in the coding sequence of the gene without any mutational "hotspot."     

Emergence of extended-spectrum-beta-lactamase (CTX-M-9)-producing multiresistant strains of Salmonella enterica serotype Virchow in poultry and humans in France

During 2002 to 2003, eight Salmonella enterica serotype Virchow poultry and poultry product isolates from various sources (chicken farms, poultry slaughterhouse, or retail store) and one S. enterica rough strain isolated from human feces were found to produce extended-spectrum beta-lactamase CTX-M-9. Poultry and poultry product isolates were recovered from different locations in the southwest of France. The human rough isolate had sequences of flagellin genes (fliC and fljB) typical of serotype Virchow and ribotyping and pulsed-field gel electrophoresis (PFGE) patterns closely similar to those of serotype Virchow strains. PFGE confirmed the clonal relationship between the poultry isolates, while the human isolate displayed a pattern with 94% homology. The bla(CTX-M-9) gene was located on a conjugative plasmid and was shown to be linked to orf513. Plasmid profiling found a very similar EcoRI restriction pattern in six transconjugants studied, including transconjugants obtained from the human isolate. A single hatchery, supplying chicks to the six farms, was identified. Emergence of extended-spectrum beta-lactamase-producing S. enterica strains in food animals is a major concern, as such strains could disseminate on a large scale and lead to antibiotic therapy difficulties.     

Basis for the age-related decline in intestinal mucosal immunity

The elderly are characterized by mucosal immunosenescence and high rates of morbidity and mortality associated with infectious diseases of the intestinal tract. Little is known about how the differentiation of immunoglobulin A (IgA) plasma cells in Peyer's patches (PPs) and their subsequent homing to the small intestinal lamina propria (LP) is affected by aging. Quantitative immunohistochemical analyses demonstrated a 2-fold increase in the number of IgA+ cells in the PPs, coupled with significant declines in the numbers of IgA+ and antibody-positive cells in the intestinal LP of senescent rats compared to young adult animals. These data suggest that aging diminishes the emigration of IgA immunoblasts from these lymphoid aggregates, as well as their migration to the intestinal LP. Flow cytometry and lymphocyte adoptive transfer studies showed 3- to 4-fold age-related declines in the homing of antibody-containing cells and mesenteric lymph node lymphocytes to the small intestines of rhesus macaques and rats, respectively. The number of peripheral blood IgA immunoblasts expressing the homing molecule alpha4beta7 declined 30% in senescent rats. This was accompanied by a > 17% decrease in the areal density of LP blood vessels staining positive for the cell adhesion molecule MAdCAM-1. Cumulatively, declines in expression of these homing molecules constitute a substantial age-related diminution of IgA immunoblast homing potential. In vitro antibody secretion by LP plasma cells, i.e. antibody secreted per antibody-positive cell, remains unchanged as a function of donor age. Intestinal mucosal immunosenescence is a consequence of reduced homing of IgA plasma cells to the intestinal LP as a result of declines in homing molecule expression.