Ancient T-independence of mucosal IgX/A: gut microbiota unaffected by larval thymectomy in Xenopus laevis.

Many studies address the influence of the gut microbiome on the immune system, but few dissect the effect of T cells on gut microbiota and mucosal responses. We have employed larval thymectomy in Xenopus to study the gut microbiota with and without the influence of T lymphocytes. Pyrosequencing of 16S ribosomal RNA genes was used to assess the relative abundance of bacterial groups present in the stomach, small and large intestine. Clostridiaceae was the most abundant family throughout the gut, while Bacteroidaceae, Enterobacteriaceae, and Flavobacteriaceae also were well represented. Unifrac analysis revealed no differences in microbiota distribution between thymectomized and unoperated frogs. This is consistent with immunization data showing that levels of the mucosal immunoglobulin IgX are not altered significantly by thymectomy. This study in Xenopus represents the oldest organisms that exhibit class switch to a mucosal isotype and is relevant to mammalian immunology, as IgA appears to have evolved from IgX based upon phylogeny, genomic synteny, and function.

CCAAT box binding protein NF-Y facilitates in vivo recruitment of upstream DNA binding transcription factors.

NF-Y binds a CCAAT motif found in many eukaryotic polymerase II-dependent promoters. In the HLA-DRA promoter it has been demonstrated that stereo-specific alignment between this motif and the upstream elements X1 and X2 is required for activation. To study the underlying mechanism for this requirement, a panel of transfected cell lines that maintained integrated, wild-type and mutant promoters were analyzed by in vivo genomic footprinting. Cell lines harboring a mutated CCAAT element exhibited a loss of interactions at the CCAAT site, as expected, and no transcriptional activity. Most importantly, mutation of the CCAAT sequence nearly abolished in vivo binding at the X1 and X2 sites, while mutations of X1 and X2 had little effect on CCAAT box binding. However, X1 and X2 binding was interdependent. In vitro, X1 binding activities are known to be stabilized by NF-Y binding. Interaction between NF-Y and X box binding proteins was demonstrated by reciprocal co-immunoprecipitation in the absence of DNA and co-affinity purification in the presence of DNA. Collectively, these studies indicate that occupancy of the CCAAT element represents an early event affecting other protein-DNA interactions and suggest that NF-Y stabilizes and interacts with X box factors to mediate this function. These findings may represent a common theme among promoters containing a CCAAT element.

Evaluation of a new method for cardiovascular reasoning.

OBJECTIVE: Evaluate the accuracy of the detailed diagnostic reasoning of the Heart Failure Program incorporating a new mechanism to handle temporal relationships and severity constraints. DESIGN: Tools were developed to summarize diagnoses and automatically generate evaluation forms. Five expert cardiologists were asked to review the reasoning of the program, with two analyzing each case. Cases were gathered retrospectively for diversity and difficulty and 26 randomly selected cases were evaluated. The underlying issues were identified and classified. RESULTS: Both reviewers rated the first diagnosis correct in 25% of the cases and at least one rated it wrong in 10%. Analyzing the detailed reasoning, 137 issues were raised, about 5.3 per case. Of these, 53% were possible concerns raised by one reviewer. Of the 5.3 issues per case, 2.5 were attributable to controversies, misunderstandings, or mistakes; 1 was due to the overly simplistic representation of the summaries; and 1.8 were issues related to the program. CONCLUSION: Overall, the program is capable of providing high-quality detailed diagnostic hypotheses for complex cardiovascular cases. The results highlight several issues: 1) the difficulty of effectively summarizing hypotheses, 2) the nature of a physician's causal explanation, and 3) some problems in evaluating detailed diagnostic reasoning. The mistakes the program made imply that some additional refinement is needed but that the reasoning mechanisms developed can support the appropriate reasoning. The appropriate next step is a prospective evaluation addressing the program's usefulness.

Summarization of complex causal diagnostic hypotheses.

The Heart Disease Program produces detailed causal diagnostic hypotheses for patients with cardiovascular diseases. This poster discusses our experience with summarizing these hypotheses for the physician. The basic approach is to merge the nodes of the structure indicating causal mechanism into the more important nodes. Analysis of the results shows that to generate effective summaries the identification of syndromes is very important, the definitions of the labels need to be carefully enforced, the causality of diseases should be supported by evidence and not just probability, and the sense of causal order must be carefully preserved.

Development of a knowledge base for diagnostic reasoning in cardiology.

This paper reports on a formative evaluation of the diagnostic capabilities of the Heart Failure Program, which uses a probability network and a heuristic hypothesis generator. Using 242 cardiac cases collected from discharge summaries at a tertiary care hospital, we compared the diagnoses of the program to diagnoses collected from cardiologists using the same information as was available to the program. With some adjustments to the knowledge base, the Heart Failure Program produces appropriate diagnoses about 90% of the time on this training set. The main reasons for the inappropriate diagnoses of the remaining 10% include inadequate reasoning with temporal relations between cause and effect, severity relations, and independence of acute and chronic diseases.

Chronic aortic regurgitation: prognostic value of left ventricular end-systolic dimension and end-diastolic radius/thickness ratio.

The prognostic value of preoperative echocardiographic data was assessed in 32 patients who underwent aortic valve replacement for chronic aortic regurgitation. All patients had preoperative studies and were followed up prospectively for 1 to 6 years after surgery. Postoperatively, 25 patients (Group A) achieved a normal left ventricular end-diastolic dimension and a significant regression of myocardial hypertrophy; 7 patients (Group B) had persistent left ventricular enlargement. During the follow-up period, the patients in Group A had fewer symptoms and used fewer medications than those in Group B. Moreover, survival at 4 years appeared to be better in Group A (96%) than in Group B (71%); two patients in Group B died with congestive heart failure; there were no such deaths in Group A. Preoperatively, a left ventricular dimension at end-diastole (DED) larger than 3.8 cm/m2 body surface area, a dimension at end-systole (DES) greater than 2.6 cm/m2 body surface area, an end-diastolic radius/wall thickness ratio (R/Th) greater than 3.8 or a product of R/Th and left ventricular systolic pressure (P X R/Th) exceeding 600 are predictive of a Group B result. If end-systolic dimension is greater than 2.6 and P X R/Th is greater than 600, all Group B patients can be identified; all but one patient in Group A had an end-systolic dimension less than 2.6 and P X R/Th less than 600. It is concluded that patients with chronic aortic regurgitation who are at risk of persistent postoperative left ventricular enlargement (with associated cardiac symptoms and reduced survival) can be identified by preoperative echocardiography.