Importance of the collagen adhesin ace in pathogenesis and protection against Enterococcus faecalis experimental endocarditis.

Ace is an adhesin to collagen from Enterococcus faecalis expressed conditionally after growth in serum or in the presence of collagen. Here, we generated an ace deletion mutant and showed that it was significantly attenuated versus wild-type OG1RF in a mixed infection rat endocarditis model (P<0.0001), while no differences were observed in a peritonitis model. Complemented OG1RFDeltaace (pAT392::ace) enhanced early (4 h) heart valve colonization versus OG1RFDeltaace (pAT392) (P = 0.0418), suggesting that Ace expression is important for early attachment. By flow cytometry using specific anti-recombinant Ace (rAce) immunoglobulins (Igs), we showed in vivo expression of Ace by OG1RF cells obtained directly from infected vegetations, consistent with our previous finding of anti-Ace antibodies in E. faecalis endocarditis patient sera. Finally, rats actively immunized against rAce were less susceptible to infection by OG1RF than non-immunized (P = 0.0004) or sham-immunized (P = 0.0475) by CFU counts. Similarly, animals given specific anti-rAce Igs were less likely to develop E. faecalis endocarditis (P = 0.0001) and showed fewer CFU in vegetations (P = 0.0146). In conclusion, we have shown for the first time that Ace is involved in pathogenesis of, and is useful for protection against, E. faecalis experimental endocarditis.

Importance of the ebp (endocarditis- and biofilm-associated pilus) locus in the pathogenesis of Enterococcus faecalis ascending urinary tract infection.

BACKGROUND: We recently demonstrated that the ubiquitous Enterococcus faecalis ebp (endocarditis- and biofilm-associated pilus) operon is important for biofilm formation and experimental endocarditis. Here, we assess its role in murine urinary tract infection (UTI) by use of wild-type E. faecalis OG1RF and its nonpiliated, ebpA allelic replacement mutant (TX5475). METHODS: OG1RF and TX5475 were administered transurethrally either at an ~1 : 1 ratio (competition assay) or individually (monoinfection). Kidney pairs and urinary bladders were cultured 48 h after infection. These strains were also tested in a peritonitis model. RESULTS: No differences were observed in the peritonitis model. In mixed UTIs, OG1RF significantly outnumbered TX5475 in kidneys (P=.0033) and bladders (P< or =.0001). More OG1RF colony-forming units were also recovered from the kidneys of monoinfected mice at the 4 inocula tested (P=.015 to P=.049), and 50% infective doses of OG1RF for kidneys and bladder (9.1x10(1) and 3.5x10(3) cfu, respectively) were 2-3 log(10) lower than those of TX5475. Increased tropism for the kidney relative to the bladder was observed for both OG1RF and TX5475. CONCLUSION: The ebp locus, part of the core genome of E. faecalis, contributes to infection in an ascending UTI model and is the first such enterococcal locus shown to be important in this site.

Regulation of sex hormone dependent growth by a serum borne inhibitor

This thesis project focused on understanding the basic process controlling cell proliferation in sex-steroid hormone dependent cancers. The availability of inculture models using cloned cell lines offers the greatest advantage for studying the control of this event. Incubation of cloned sex-hormone sensitive cells in medium containing increasing concentrations of sex-hormone stripped serum, results in a dose dependent growth inhibition; this inhibition is reversed by the addition of physiological concentrations of steroid hormones. The mechanisms explaining this phenomenon are not yet fully understood, but different theories propose the existence in serum of a sex hormone binding protein with growth inhibitory properties. We were able to identify a protein that specifically binds sex hormones in rat and horse serum with affinities 10-fold lower to the ones observed with the classic sex-hormone binding globulin (SHBG) in humans. Purification of this protein on a large scale Lowed a more detailed analysis. The putative sex-hormone binding protein has an apparent molecular weight of 386 KDa. SDS-PAGE with commassie staining of the purified product, displayed a pattern non-characteristic of SMG, but all bands cross-reacted with a commercial anti-SMG antibody in western analysis. Titrations of the purified product on cell proliferation assays using sex-hormone dependent lines, resulted in a dose dependent growth inhibition. This inhibition was reversed by the addition of sex hormones. Our results indicate that we have identified and purified a sex-hormone binding protein in serum with characteristics similar to SHBG and with cell growth inhibitory properties. ^

Importance of sperm transition proteins demonstrated in mice carrying Tnp1- and Tnp2-null alleles

Chromatin condensation within the nucleus of developing spermatids involves replacement of histones by transition proteins, which are in turn replaced by protamines. The importance of transition proteins in the complex process of spermiogenesis has, to date, been only speculative. This study sought to investigate the extent to which transition proteins are essential or have redundant functions by characterizing sperm produced in mice expressing all combinations of Tnp-null alleles. Results from breeding trials of 8 weeks duration revealed that, on average, wildtype males produced about 14 offspring whereas TP2 and TP1 single-knockout males produced about 8 and 1 offspring, respectively, demonstrating their subfertility. Genotypes with less than two Tnp wildtype alleles, as well as double-knockout mutants, were completely infertile. Sperm from males with impaired fertility had poor progressive motility, heterogeneous chromatin condensation, incompletely processed protamine 2 and head and tail abnormalities. Generally, as the number of Tnp-null alleles increased so did the severity of abnormalities. However, specific morphological abnormalities were associated with the absence of an individual TP. Studies which sought to identify possible root causes for abnormalities in thiol-rich sperm structures revealed no differences in thiol content or sulfhydryl oxidation status within the nucleus but nuclei and tails from single-knockout mutants were severely disrupted following thiol reduction. Binding of fluorescent dyes to DNA was normal in sperm recovered from caput but abnormal in cauda epididymal sperm from TP1 knockouts and infertile double mutants. Injection of cauda epididymal sperm from double knockouts into oocytes produced very few offspring; however, after injection with testicular sperm, the efficiency was no different from wildtype. These results suggest DNA structural alterations or degradation during epididymal transport of sperm resulting in a diminished capacity of the paternal DNA of these sperm to produce offspring. The overall importance of transition proteins for normal chromatin condensation and production of fertile sperm has been demonstrated. Furthermore, identification of specific morphological abnormalities associated with the absence of an individual transition protein provides new evidence that the proteins are not completely redundant and each fulfills some unique function. ^

A conditional Mdm2 allele shows the importance of Mdm2 in heart development

Over 50% of sporadic tumors in humans have a p53 mutation highlighting its importance as a tumor suppressor. Considering additional mutations in other genes involved in p53 pathways, every tumor probably has mutant p53 or impaired p53-mediated functions. In response to a variety of cellular and genotoxic stresses, p53, mainly through its transcriptional activity, induces pathways involved in apoptosis and growth arrest. In these circumstances and under normal situations, p53 must be tightly regulated. Mdm2 is an important regulator of p53. Mdm2 inhibits p53 function by binding and blocking its transactivation domain. In addition, Mdm2 helps target p53 for degradation through its E3 ligase activity. Mdm2 null mice are embryonic lethal due to apoptosis in the blastocysts. However, a p53 null background rescues this lethality demonstrating the importance of the p53-Mdm2 interaction, particularly during development. The lethality of the Mdm2 null mouse prior to implantation limits the ability to investigate the role of Mdm2 in regulating p53 in a temporal and tissue specific manner. Does p53 need to be regulated in all tissues throughout the life of a mouse? Does Mdm2 always have to regulate it? To address these questions, we created a conditional Mdm2 allele. The conditional allele, Mdm2FM, in the presence of Cre recombinase results in the deletion of exons 5 and 6 of Mdm2 (most of the p53 binding domain) and represents a null allele. ^ The Mdm2FM allele was crossed with a heart muscle specific Cre expressing mouse (α-myosin heavy chain promoter driven Cre) to ask whether Mdm2 acts as a negative regulator of p53 in the heart. The heart is the most prominent organ early in embryogenesis and is shaped by cell death and proliferation. p53 does not appear to be active in the heart in response to some types of stress, so it remained to be determined if it has to be regulated in normal heart development. Loss of Mdm2 in the heart results in heart defects as early as E9.5. Loss of Mdm2 results in stabilized p53 and apoptosis. This apoptosis leads to a thinning of the myocardial wall particularly in the ventricles and abnormal ventricular structure. Eventually the abnormal heart fails resulting in lethality by E13.5. The embryonic lethality is rescued in a p53 null background. Thus, Mdm2 is important in regulating p53 in the development of the heart. ^

Irritable bowel syndrome (IBS) in U.S. adult travelers': The importance of travel to the development of IBS

Background. Irritable bowel syndrome is a gastrointestinal disorder that is potentially linked to international travel at an undetermined frequency.^ Methods. A self-administered questionnaire was distributed through mail to five hundred and ninety-one patients that were twice diagnosed with irritable bowel syndrome at Kelsey Seybold Clinic in Houston, TX. Responses to survey questions were used to assess patient travel history, IBS symptomology, and disease classification.^ Results. Of the five hundred and ninety-one patients that were mailed a questionnaire, two hundred and twenty one patients returned questionnaires and two hundred and one met inclusion criteria. Of the participants reporting international travel within six months of developing their chronic intestinal disorder, 60% were classified as having PI-IBS, while 25% had IBS, 10% had PI-UFBD, and 5% had UFBD. A majority of the subjects who traveled six months before onset of their functional bowel disease had a post-infectious form of IBS and reported a start and worsening of symptoms with an acute bout of diarrhea. It was common for those traveling six months before travel and labeled PI-IBS to have enteric symptoms that led to lifestyle adjustments. ^ Conclusion. International travel had a significant effect on the classification of IBS among patients which relates to the differences in IBS symptoms and perhaps pathogenesis among travelers versus non-travelers. ^

Importance of antibiotic class to the development of Clostridium difficile infection (CDI) in adults: A systematic review

C. difficile causes gastrointestinal infections in humans, including severe diarrhea. It is implicated in 20%-30% of cases of antibiotic-associated diarrhea, in 50%-70% of cases of antibiotic-associated colitis, and in >90% of cases of antibiotic-associated pseudomembranous colitis. Exposure to antimicrobial agent, hospitalization and age are some of the risk factors that predispose to CDI. Virtually all hospitalized patients with nosocomially-acquired CDI have a history of treatment with antimicrobials or neoplastic agent within the previous 2 months. The development of CDI usually occurs during treatment with antibiotics or some weeks after completing the course of the antibiotics. ^ After exposure to the organism (often in a hospital), the median incubation period is less than 1 week, with a median time of onset of 2days. The difference in the time between the use of antibiotic and the development of the disease relate to the timing of exogenous acquisition of C. difficile. ^ This paper reviewed the literature for studies on different classes of antibiotics in association with the rates of primary CDI and RCDI from the year 1984 to 2012. The databases searched in this systematic review were: PubMed (National Library of Medicine) and Medline (R) (Ovid). RefWorks was used to store bibliographic data. ^ The search strategy yielded 733 studies, 692 articles from Ovid Medline (R) and 41 articles from PubMed after removing all duplicates. Only 11 studies were included as high quality studies. Out of the 11 studies reviewed, 6 studies described the development of CDI in non-CDI patients taking antibiotics for other purposes and 5 studies identified the risk factors associated with the development of recurrent CDI after exposure to antibiotics. ^ The risk of developing CDI in non-CDI patients receiving beta lactam antibiotics was 2.35%, while fluoroquinolones, clindamycin/macrolides and other antibiotics were associated with 2.64%, 2.54% and 2.35% respectively. Of those who received beta lactam antibiotic, 26.7% developed RCDI, while 36.8% of those who received any fluoroquinolone developed RCDI, 26.5% of those who received either clindamycin or macrolides developed RCDI and 29.1% of those who received other antibiotics developed RCDI. Continued use of non-C. difficile antibiotics especially fluoroquinolones was identified as an important risk factor for primary CDI and recurrent CDI. ^

Highlighting the Importance of Researching Family Homelessness

This invited commentary responds to and builds upon Tobin and Murphy's article, “Addressing the Challenges of Child and Family Homelessness.” In affirming the ideas emerging from this article, Hallett and Tierney provide three points of extension: (1) more research needs to be conducted with doubled-up families; (2) the role of shame needs further exploration; and, (3) additional work needs to be done to increase access to postsecondary institutions.