Publications

BACKGROUND

Conditional knockout mice are a useful tool to study the function of gene products in a tissue-specific or inducible manner. Classical approaches to generate targeting vectors for conditional alleles are often limited by the availability of suitable restriction sites. Furthermore, plasmid-based targeting vectors can only cover a few kB of DNA which precludes the generation of targeting vectors where the two loxP sites are placed far apart. These limitations have been overcome in the recent past by using homologous recombination of bacterial artificial chromosomes (BACs) in Escherichia coli to produce large targeting vector containing two different loxP-flanked selection cassettes so that a single targeting event is sufficient to introduce loxP-sites a great distances into the mouse genome. However, the final targeted allele should be free of selection cassettes and screening for correct removal of selection cassettes can be a laborious task. Therefore, we developed a new strategy to rapidly identify ES cells containing the desired allele.

RESULTS

Using BAC recombineering we generated a single targeting vector which contained two different selection cassettes that were flanked by loxP-loxP sites or by FRT-FRT/loxP sites so that they could be deleted sequentially by Cre- and FLPe-recombinases, respectively. Transfected ES cells were first selected in the presence of both antibiotics in vitro before correctly targeted clones were identified by Southern blot. After transfection of a Cre recombinase expression plasmid ES cell clones were selected on replicate plates to identify those clones which maintained the FRT-FRT/loxP flanked cassette and lost the loxP-loxP flanked cassette. Using this strategy facilitated the identification of ES cell clones containing the desired allele before blastocyst injection.

CONCLUSION

The strategy of ES cell cultures in replicate plates proved to be very efficient in identifying ES cells that had undergone the correct recombination event. This approach facilitates the generation of conditional knock-out mice when large parts of the genome are intended to be flanked by loxP sites.

The division of labor among two types of T helper (Th) subsets, first described over 20 yr ago, has been buffeted by the discovery of new subsets and new cytokines that can be coaxed out of T cells with increasing disregard for the subset of origin. Although Th17 cells and regulatory T (T reg) cells are widely accepted subsets, and others are being proposed, their plasticity is difficult to reconcile with the definitions of Th subsets as put forth in the initial description of Th1 and Th2 cells. A deeper molecular context will be required to reconcile the ever-increasing complexity of effector T cells.

CONTEXT

Asthma self-management education is critical for high-quality asthma care for children. A number of studies have assessed the effectiveness of providing asthma education in schools to augment education provided by primary care providers.

OBJECTIVE

To conduct a systematic review of the literature on school-based asthma education programs.

METHODS

As our data sources, we used 3 databases that index peer-reviewed literature: MEDLINE, the Cochrane Central Register of Controlled Trials, and the Cumulative Index to Nursing and Allied Health Literature. Inclusion criteria included publication in English and enrollment of children aged 4 to 17 years with a clinical diagnosis of asthma or symptoms consistent with asthma.

RESULTS

Twenty-five articles met the inclusion criteria. Synthesizing findings across studies was difficult because the characteristics of interventions and target populations varied widely, as did the outcomes assessed. In addition, some studies had major methodologic weaknesses. Most studies that compared asthma education to usual care found that school-based asthma education improved knowledge of asthma (7 of 10 studies), self-efficacy (6 of 8 studies), and self-management behaviors (7 of 8 studies). Fewer studies reported favorable effects on quality of life (4 of 8 studies), days of symptoms (5 of 11 studies), nights with symptoms (2 of 4 studies), and school absences (5 of 17 studies).

CONCLUSIONS

Although findings regarding effects of school-based asthma education programs on quality of life, school absences, and days and nights with symptoms were not consistent, our analyses suggest that school-based asthma education improves knowledge of asthma, self-efficacy, and self-management behaviors.

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) threatens public health worldwide, and epidemiologic data suggest that the Panton-Valentine Leukocidin (PVL) expressed by most CA-MRSA strains could contribute to severe human infections, particularly in young and immunocompetent hosts. PVL is proposed to induce cytolysis or apoptosis of phagocytes. However, recent comparisons of isogenic CA-MRSA strains with or without PVL have revealed no differences in human PMN cytolytic activity. Furthermore, many of the mouse studies performed to date have failed to demonstrate a virulence role for PVL, thereby provoking the question: does PVL have a mechanistic role in human infection? In this report, we evaluated the contribution of PVL to severe skin and soft tissue infection. We generated PVL mutants in CA-MRSA strains isolated from patients with necrotizing fasciitis and used these tools to evaluate the pathogenic role of PVL in vivo. In a model of necrotizing soft tissue infection, we found PVL caused significant damage of muscle but not the skin. Muscle injury was linked to induction of pro-inflammatory chemokines KC, MIP-2, and RANTES, and recruitment of neutrophils. Tissue damage was most prominent in young mice and in those strains of mice that more effectively cleared S. aureus, and was not significant in older mice and mouse strains that had a more limited immune response to the pathogen. PVL mediated injury could be blocked by pretreatment with anti-PVL antibodies. Our data provide new insights into CA-MRSA pathogenesis, epidemiology and therapeutics. PVL could contribute to the increased incidence of myositis in CA-MRSA infection, and the toxin could mediate tissue injury by mechanisms other than direct killing of phagocytes.

Population-level genetic diversity in the obligate symbiosis between the bivalve Solemya velum and its thioautotrophic bacterial endosymbiont was examined. Distinct populations along the New England coast shared a single mitochondrial genotype but were fixed for unique symbiont genotypes, indicating high levels of symbiont genetic structuring and potential symbiont-host decoupling.