Targeted disruption of the mouse Csrp2 gene encoding the cysteine- and glycine-rich LIM domain protein CRP2 result in subtle alteration of cardiac ultrastructure.

BACKGROUND: The cysteine and glycine rich protein 2 (CRP2) encoded by the Csrp2 gene is a LIM domain protein expressed in the vascular system, particularly in smooth muscle cells. It exhibits a bimodal subcellular distribution, accumulating at actin-based filaments in the cytosol and in the nucleus. In order to analyze the function of CRP2 in vivo, we disrupted the Csrp2 gene in mice and analysed the resulting phenotype. RESULTS: A approximately 17.3 kbp fragment of the murine Csrp2 gene containing exon 3 through 6 was isolated. Using this construct we confirmed the recently determined chromosomal localization (Chromosome 10, best fit location between markers D10Mit203 proximal and D10Mit150 central). A gene disruption cassette was cloned into exon 4 and a mouse strain lacking functional Csrp2 was generated. Mice lacking CRP2 are viable and fertile and have no obvious deficits in reproduction and survival. However, detailed histological and electron microscopic studies reveal that CRP2-deficient mice have subtle alterations in their cardiac ultrastructure. In these mice, the cardiomyocytes display a slight increase in their thickness, indicating moderate hypertrophy at the cellular level. Although the expression of several intercalated disc-associated proteins such as beta-catenin, N-RAP and connexin-43 were not affected in these mice, the distribution of respective proteins was changed within heart tissue. CONCLUSION: We conclude that the lack of CRP2 is associated with alterations in cardiomyocyte thickness and hypertrophy.

Urology residency and research: round table discussion and plea for innovation.

OBJECTIVES: To evaluate the current and future states of resident research experience in urology residencies in the United States. METHODS: Round table discussion with leading educators and Urology faculty from a university urology residency. RESULTS: Research exposure has rapidly diminished in urology residencies for a variety of reasons. There are multiple barriers to resident research and only a small number of residencies will be able to provide protected time. Nevertheless, an understanding of research methodology and biostatistics is required to be a successful clinician. CONCLUSIONS: Some barriers to resident research can be addressed by better integration of residency and fellowships. Flexibility in the format of resident education may allow introduction of new methods to encourage resident research scholarship. An education program with a research curriculum is needed for all residencies.

Report of the national survey of Urology Program Directors: attitudes and actions regarding the accreditation council for graduate medical education regulations.

PURPOSE: Residency programs in the United States are held accountable for achieving outcomes based on the new Accreditation Council for Graduate Medical Education (ACGME) regulations for improving resident fatigue and education. A national survey of Urology Program Directors was conducted to determine the attitudes toward the new regulations and the actions taken to implement them. MATERIALS AND METHODS: A national survey was developed by an ad hoc committee and distributed electronically. A total of 24 multiple-choice customized Likert scale questions were used to obtain opinions from Urology Program Directors across the country. The data were evaluated using statistical analysis. RESULTS: There was an 88% response rate to the survey. Of the respondents 53% demonstrated a statistically significant association between the size of the program and the degree to which the program was involved in addressing the duty hours standard (p = 0.015). A majority of program directors reported involvement in educating residents and faculty about the ACGME competencies. According to program directors, faculty members were negative about training residents in an 80-hour workweek and residents seemed to be apathetic. CONCLUSIONS: The national survey of Urology Program Directors has demonstrated that most programs have started to attend to the cultural change occurring in GME. Although there are some pockets of resistance to the ACGME regulations, the duty hours have been folded into most programs with little apprehension. Barriers to implementation of the Core Competencies discovered in this survey included inadequate validated instrumentation, inadequate staffing, and insufficient financial support.

Brief communication of the Residency Review Committee-Surgery (RRC-S) on residents' surgical volume in general surgery.

BACKGROUND: The Residency Review Committee-Surgery (RRC--S), 1 of 10 surgical specialties of the Accreditation Council for Graduate Medical Education (ACGME) has monitored the surgical volume of all general surgical residents closely. As a consequence of the reduction of duty hours with the limitation of an 80-hour work-week (averaged over 4 weeks), we were interested in the impact of these restrictions on surgical (volume) experience since its first year of implementation (2003--2004). Therefore, we evaluated the surgical volume of general surgical services since the implementation of the ACGME duty-hour restrictions and compared this volume with that of previous years without these duty limits. METHODS: The Biostatistical Management Section of the ACGME implemented prospective analysis of categorized data for total surgical procedures and Chief Resident cases. The study interval included all resident surgical procedures completed from 1997 to 2004. We were interested particularly in evaluating trends and outcomes after the first year of successful full compliance of the 80-hour work week. Specific evaluations included the impact on surgical programs for total major procedures and Chief Resident cases requisite for application to the American Board of Surgery. RESULTS: The average number of total major procedures for both resident and program averages were noted to increase steadily through the academic years of evaluation (1997--2001). A sharp decrease was evident in the total major procedures for the academic year 2001--2002 that relates to a correction of the biostatistical database implemented by the ACGME to correct a system conversion that began in the academic year 2001--2002. Despite significant changes to the system data mappings, beginning in the academic year 2001, this reduction is explained by the total counted surgeries as major that were eliminated in a revised counting methodology. It was evident on evaluation of the average (of averages) for major surgical procedures per resident (per program) in academic years 1997 to 2004 that the number of procedures was not statistically different in the academic years evaluated when compared with the year for implementation of duty-hour standards (2003--2004). Data analysis further indicates that the average procedures (per resident) performed as Chief Resident in general surgery remained stable from 1997 to 2004; the use of tiered t tests comparing Chief Resident averages (per program) for the academic years 2002--2003 versus 2003--2004 indicated that data remained consistent and confirmed no statistical variance in volumes during this interval (P=0.43). Because some general surgery programs have exceptions for duty-hour requirements (n=15) to allow an 88-hour week averaged over 4 weeks, these differences were of interest to evaluate programs with and without these duty-hour exceptions. Preliminary data with these limited parameters of evaluation suggest no detrimental outcomes related to the duty-hour restrictions for total major procedures per resident or for surgical procedures as Chief Residents for programs with and without these approved exceptions. CONCLUSIONS: RRCs that evaluate general surgery and surgical specialties have responded aggressively and professionally to implement the duty-hour standards per the ACGME. This brief report should be considered an interim communication to evaluate the surgical experience impact for programs currently under the restriction of duty-hour limits. The data provided in the first year of evaluation since the implementation of the 80-hour work-week restriction policy suggest that there has been no significant change in the overall surgical experience for major procedures (per resident), nor has there been a negative impact on Chief Resident surgical experience. A continuum of the prospective evaluation process is required by the RRC-S and other surgical specialties to ensure that requisite surgical volume is maintained throughout the entire 5 years of clinical surgery.

Antisense strategy against PDGF B-chain proves effective in preventing experimental liver fibrogenesis.

Hepatic stellate cells (HSCs) and transdifferentiated myofibroblasts are the principal producers of excessive extracellular matrix in liver fibrosis and cirrhosis. Activation of HSC is regulated by several cytokines and growth factors, including platelet-derived growth factor B-chain (PDGF-B), a potent mitogen for HSC, and overexpressed during hepatic fibrogenesis. Previous studies showed that MAPK and phosphatidylinositol 3' kinase are key signaling pathways involved in PDGF-induced stimulation of HSC. Based on the involvement of PDGF-B in fibrogenesis, reducing ligand stimulation of proliferative cytokine- or growth factor receptors interfering with receptor signaling therefore presents an interesting strategy for hepatic fibrosis prevention or interruption. We therefore generated an adenoviral vector serotype 5 (Ad5) expressing an antisense mRNA of the PDGF B-chain (Ad5-CMV-asPDGF) for application in an experimentally induced liver fibrogenesis model. The transgene clearly showed the ability to down-regulate endogenous PDGF B-chain and PDGFRbeta mRNA in culture-activated HSC and rat livers. The asPDGF mRNA also attenuates experimental liver fibrogenesis indicated by reduced levels of alpha-SMA and collagen type I expression.

Analysis of polymorphic TGFB1 codons 10, 25, and 263 in a German patient group with non-syndromic cleft lip, alveolus, and palate compared with healthy adults.

BACKGROUND: Clefts of the lip, alveolus, and palate (CLPs) rank among the most frequent and significant congenital malformations. Leu10Pro and Arg25Pro polymorphisms in the precursor region and Thr263Ile polymorphism in the prodomain of the transforming growth factor beta1 (TGF-beta1) gene have proved to be crucial to predisposition of several disorders. METHODS: In this study, polymorphism analysis was performed by real-time polymerase chain reaction (LightCycler) and TGF-beta1 levels determined by enzyme-linked immunosorbent assay. RESULTS: Only 2/60 Caucasian non-syndromic patients with CLP (3.3%) carried the Arg25Pro and another 2/60 patients (3.3%) the Thr263Ile genotypes, whereas, in a control group of 60 healthy Caucasian blood donors, these heterozygous genotypes were more frequent 16.7% having Arg25Pro (10/60; p < 0.035) and 10,0% having Thr263Ile (6/60), respectively. TGF-beta1 levels in platelet-poor plasma of heterozygous Arg25Pro individuals were lower than those of homozygous members (Arg25Arg) in the latter group, but this discrepancy narrowly failed to be significant. Although polymorphisms in codon 10 and 25 were associated with each other, no difference was found between patients and controls concerning the Leu10Pro polymorphism. CONCLUSIONS: The genetic differences in codons 25 and 263 suggest that TGF-beta1 could play an important role in occurrence of CLP, however, functional experiments will be required to confirm the mechanisms of disturbed development.

Dominant-negative soluble PDGF-beta receptor inhibits hepatic stellate cell activation and attenuates liver fibrosis.

Hepatic fibrogenesis is a consequence of hepatic stellate cells that become activated and transdifferentiate into a myofibroblastic phenotype with the ability to proliferate and synthesize large quantities of extracellular matrix components. In this process, platelet-derived growth factor (PDGF) is the most potent stimulus for hepatic stellate cell proliferation and migration, and is overexpressed during active hepatic fibrogenesis. This cytokine binds to the PDGF receptor type beta, activates Ras and sequentially propagates the stimulatory signal sequentially via phosphorylation of Raf-1, MEK and the extracellular-signal regulated kinases ERK1/ERK2. Hepatic injury is associated with both increased autocrine PDGF signaling and upregulation of PDGF receptor. In this study, we report that a dominant-negative soluble PDGF-beta receptor consisting of a chimeric IgG containing the extracellular portion of the PDGF receptor type beta blocks HSC activation and attenuates fibrogenesis induced by ligation of the common bile duct in rats. In culture-activated hepatic stellate cells, the soluble receptor blocks phosphorylation of endogenous PDGF receptor, phosphorylation of the ERK1/EKR2 signal and reduces proliferative activities of HSC. In vivo, both the delivery of the purified soluble PDGF antagonist and the administration of adenoviruses expressing the artificial transgene were able to reduce significantly the expression of collagen and alpha-smooth muscle actin. Our results demonstrate that PDGF plays a critical role in the progression and initiation of experimental liver fibrogenesis, and suggest that early anti-PDGF intervention should have a therapeutical impact on the treatment of liver fibrogenesis.

Inhibitory effect of soluble PDGF-beta receptor in culture-activated hepatic stellate cells.

Following liver injury, hepatic stellate cells undergo phenotypic transformation with acquisition of myofibroblast-like features, characterized by increased cell proliferation, motility, contractility, and extracellular matrix production. Activation of hepatic stellate cells is regulated by several cytokines and growth factors, including platelet-derived growth factor B-chain, a potent mitogen for HSC, overexpressed during hepatic fibrogenesis. This pleiotropic mediator exerts cellular effects by binding to specific receptors, inducing receptor dimerization and tyrosine-autophosphorylation. Activated receptor phosphotyrosines recruit signal transduction molecules, initiating various signaling pathways. We produced a soluble PDGFbeta-receptor (sPDGFRbeta) consisting of an extracellular domain connected to the IgG-Fc part of human immunoglobulin heavy chain. This soluble, chimeric receptor inhibits PDGF signaling and PDGF-induced proliferation in culture-activated hepatic stellate cells. Furthermore, sPDGFR decreased collagen type I (alphaI) mRNA expression and inhibits autocrine-looping in PDGF-BB mRNA production. In summary, sPDGFRbeta clearly shows effective inhibitory properties in early HSC activation, suggesting potential therapeutic impact for anti-PDGF intervention in liver fibrogenesis.

Graduates' perceptions of their clinical competencies in allergy and immunology: results of a survey.

PURPOSE: The Accreditation Council for Graduate Medical Education (ACGME) and the American Board of Medical Specialties (ABMS) have identified six areas of general competency. This study surveyed graduates of allergy and immunology training programs about their perceived clinical competency and the adequacy of their subspecialty training. METHOD: In August 2000 and May 2001, a questionnaire was mailed to 373 physicians who had completed a fellowship in allergy and immunology in the United States between 1995 and 2001. Physicians were asked to rate the perceived importance and adequacy of their training in, and their level of competency for, 57 general competencies and subspecialty-specific competencies and procedures. RESULTS: A total of 253 physicians responded (68%). All items in the six ACGME/ABMS general competencies had high ratings (>/= 90%) for perceived importance. One item in the practice-based learning area had low ratings for adequacy of training (57%) and intermediate for competency (75%). Two items in the system-based practice area had low ratings for training (65% and 67%) and intermediate for competency (86% and 88%). Generally, core specialty-specific items (allergic rhinitis, asthma, and urticaria) had high ratings (>/= 90%) for importance, training, and competency. Without exception, items with ratings of less than 70% for adequacy of training also had ratings of less than 90% for competency. CONCLUSION: The general competencies were considered important, but training in system-based practice and practice-based learning may be deficient. Although self-perceived competency in core areas of allergy and immunology was high, weaknesses in training and self-perceived competency in selected areas were identified.