Sherilyn Smith

Deletion of RD1 from Mycobacterium tuberculosis Mimics Bacille Calmette-Guérin Attenuation

The tuberculosis (TB) vaccine bacille Calmette-Guérin (BCG) is a live attenuated organism, but the mutation responsible for its attenuation has never been defined. Recent genetic studies identified a single DNA region of difference, RD1, which is absent in all BCG strains and present in all Mycobacterium tuberculosis (MTB) strains. The 9 open-reading frames predicted within this 9.5-kb region are of unknown function, although they include the TB-specific immunodominant antigens ESAT-6 and CFP-10. In this study, RD1 was deleted from MTB strain H37Rv, and virulence of H37Rv:DeltaRD1 was assessed after infections of the human macrophage-like cell line THP-1, human peripheral blood monocyte-derived macrophages, and C57BL/6 mice. In each of these systems, the H37Rv:DeltaRD1 strain was strikingly less virulent than MTB and was very similar to BCG controls. Therefore, it was concluded that genes within or controlled by RD1 are essential for MTB virulence and that loss of RD1 was important in BCG attenuation.

Individual RD1-region genes are required for export of ESAT-6/CFP-10 and for virulence of Mycobacterium tuberculosis

The RD1 genomic region is present in virulent strains of Mycobacterium tuberculosis (MTB), missing from the vaccine strain M. bovis BCG, and its importance to virulence has been established experimentally. Based on in silico analysis, it has been suggested that RD1 may encode a novel secretion system, but the mechanism by which this region affects virulence is unknown. Here we examined mutants disrupted in five individual RD1 genes. Both in vitro and in vivo, each mutant displayed an attenuated phenotype very similar to a mutant missing the entire RD1 region. Genetic complementation of individual genes restored virulence. Attenuated mutants could multiply within THP‐1 cells, but they were unable to spread to uninfected macrophages. We also examined export of two immunodominant RD1 proteins, CFP‐10 and ESAT‐6. Export of these proteins was greatly reduced or abolished in each attenuated mutant. Again, genetic complementation restored a wild‐type phenotype. Our results indicate that RD1 genes work together to form a single virulence determinant, and argue that RD1 encodes a novel specialized secretion system that is required for pathogenesis of MTB.

T Cell Expression Cloning of a Mycobacterium tuberculosis Gene Encoding a Protective Antigen Associated with the Early Control of Infection

Infection of C57BL/6 mice with Mycobacterium tuberculosis results in the development of a progressive disease during the first 2 wk after challenge. Thereafter, the disease is controlled by the emergence of protective T cells. We have used this infection model in conjunction with direct T cell expression cloning to identify Ags involved with the early control of the disease. A protective M. tuberculosis-specific CD4 T cell line derived from mice at 3 wk postchallenge was used to directly screen an M. tuberculosis genomic expression library. This screen resulted in the identification of a genomic clone comprising two putative adjacent genes with predicted open reading frames of 10 and 41 kDa, MTB10 and MTB41, respectively (the products of Rv0916c and Rv0915c, respectively, in the TubercuList H37Rv database). MTB10 and MTB41 belong to the PE and PPE family of proteins recently identified to comprise 10% of the M. tuberculosis genome. Evaluation of the recombinant proteins revealed that MTB41, but not MTB10, is the Ag recognized by the cell line and by M. tuberculosis-sensitized human PBMC. Moreover, C57BL/6 mice immunized with MTB41 DNA developed both CD4- (predominantly Th1) and CD8-specific T cell responses to rMTB41 protein. More importantly, immunization of C57BL/6 mice with MTB41 DNA induced protection against infection with M. tuberculosis comparable to that induced by bacillus Calmette-Guérin. Thus, the use of a proven protective T cell line in conjunction with the T cell expression cloning approach resulted in the identification of a candidate Ag for a subunit vaccine against tuberculosis.

Multi-institutional development and utilization of a computer-assisted learning program for the pediatrics clerkship: The CLIPP project

Computer-assisted instruction (CAI) holds significant promise for meeting the current challenges of medical education by providing consistent and quality teaching materials regardless of training site. The Computer-assisted Learning in Pediatrics Project (CLIPP) was created over three years (2000–2003) to meet this potential through multi-institutional development of interactive Internet-based patient simulations that comprehensively teach the North American core pediatrics clerkship curriculum. Project development adhered to four objectives: (1) comprehensive coverage of the core curriculum; (2) uniform approach to CAI pedagogy; (3) multi-institutional development by educators; and (4) extensive evaluation by users. Pediatrics clerkship directors from 30 institutions worked in teams to develop a series of 31 patient case simulations. An iterative process of case content and pedagogy development, case authoring, peer review, and pilot-testing ensured that the needs of clerkship directors and medical students were met. Fifty medical schools in the United States and Canada are presently using CLIPP. More than 8,000 students have completed over 98,000 case sessions, with an average of 2,000 case sessions completed per week at this time. Each CLIPP case has been completed by more than 3,000 students. The current cost of CLIPP development is approximately

During Trypanosoma cruzi Infection CD1d-Restricted NK T Cells Limit Parasitemia and Augment the Antibody Response to a Glycophosphoinositol-Modified Surface Protein

70 per student user, or

Promoting fundamental clinical skills: a competency-based college approach at the University of Washington.

6 per case session. The project’s success demonstrates that multi-institutional development and implementation of a peer-reviewed comprehensive CAI learning program by medical educators is feasible and provides a useful model for other organizations to develop similar programs. Although CAI development is both time-consuming and costly, the initial investment decreases significantly with broad use over time.

Teaching Patient Communication Skills to Medical Students A Review of Randomized Controlled Trials

ABSTRACT Trypanosoma cruzi is a protozoan parasite that chronically infects many mammalian species and in humans causes Chagas’ disease, a chronic inflammatory disease. The parasite expresses glycophosphoinositol (GPI), which potently stimulates interleukin 12 (IL-12) production. During T. cruzi infection IL-12, and possibly GPI, might stimulate NK T cells to affect the protective and chronic inflammatory responses. Here we report that during T. cruzi infection CD1d-restricted NK T cells are stimulated as NK T-cell-deficient mice have greater parasitemia. Furthermore, during T. cruzi infection the percentages of NK T cells in the liver and spleen become decreased for prolonged periods of time, and in vitro stimulation of NK T cells derived from livers of chronically infected mice, compared to uninfected mice, results in increased gamma interferon and IL-4 secretion. Moreover, in NK T-cell-deficient mice the chronic-phase antibody response to a GPI-modified surface protein is decreased. These results indicate that, during the acute infection, NK T cells limit parasitemia and that, during the chronic phase, NK T cells augment the antibody response. Thus, during T. cruzi infection the quality of an individual’s NK T-cell response can affect the level of parasitemia and parasite tissue burden, the intensity of the chronic inflammatory responses, and possibly the outcome of Chagas’ disease.

Finding Effective Strategies for Teaching Ethics: A Comparison Trial of Two Interventions

The focus on fundamental clinical skills in undergraduate medical education has declined over the last several decades. Dramatic growth in the number of faculty involved in teaching and increasing clinical and research commitments have contributed to depersonalization and declining individual attention to students. In contrast to the close teaching and mentoring relationship between faculty and students 50 years ago, today’s medical students may interact with hundreds of faculty members without the benefit of a focused program of teaching and evaluating clinical skills to form the core of their four-year curriculum. Bedside teaching has also declined, which may negatively affect clinical skills development. In response to these and other concerns, the University of Washington School of Medicine has created an integrated developmental curriculum that emphasizes bedside teaching and role modeling, focuses on enhancing fundamental clinical skills and professionalism, and implements these goals via a new administrative structure, the College system, which consists of a core of clinical teachers who spend substantial time teaching and mentoring medical students. Each medical student is assigned a faculty mentor within a College for the duration of his or her medical school career. Mentors continuously teach and reflect with students on clinical skills development and professionalism and, during the second year, work intensively with them at the bedside. They also provide an ongoing personal faculty contact. Competency domains and benchmarks define skill areas in which deepening, progressive attention is focused throughout medical school. This educational model places primary focus on the student.

Local Role for Tumor Necrosis Factor Alpha in the Pulmonary Inflammatory Response to Mycobacterium tuberculosis Infection

Tools to examine the effects of teaching interventions across a variety of studies are needed. The authors perform a meta-analysis of 24 randomized controlled trials evaluating the effects of teaching on medical students’ patient communication skills. Study quality is rated using a modified Jadad score, and standardized mean difference effect size (d) measures are calculated. Fifteen of 24 studies have sufficient data for analysis. Students’ ability to establish rapport improves after teaching. The effects are large when the teaching intervention was small group discussion (n = 5) or giving structured feedback on a student-patient interview (n = 6). A similar effect of teaching is seen on student data gathering skills (n = 5). Teaching medical students patient communication skills using small group discussion or providing feedback on a student-patient interview results in improvement in student performance.

Integration strategies for using virtual patients in clinical clerkships.

Purpose To compare the effects of two teaching methods (written case analyses and written case analyses with group discussion) on students’ recognition and assessment of common ethical dilemmas. Method In 1999–2000, all third-year students at the University of Washington School of Medicine on a pediatrics clinical rotation participated in the study. Eighty students were based in Seattle and 66 were in community sites in a five-state area. All students received three scenarios with written instructions for ethical analysis, submitted written answers, and received written feedback from a single evaluator. The Seattle students also participated in an hour-long, one-time discussion group about the cases. All students submitted a final case analysis. Four components of the case analyses were evaluated: ability to identify ethical issues, see multiple viewpoints, formulate an action plan, and justify their actions. One investigator evaluated a masked subset of the case analyses from both groups to assess whether teaching method affected the students’ ability to recognize and assess ethical problems. Results Forty-eight of 146 available case analysis sets (each set included three initial analyses plus one final analysis) were masked and coded. Performances on the initial analyses were similar in both groups (p > .2–.8). The discussion group had a higher absolute increase in total score (p = .017) and in ability to formulate a plan (p = .013) on the final case analysis. Performances otherwise remained largely similar. Conclusions Students’ recognition and assessment of ethical issues in pediatrics improves following a case-based exercise with structured feedback. Group discussion may optimize the learning experience and increase students’ satisfaction.