An ELISA-based method for measurement of food-specific IgE antibody in mouse serum: an alternative to the passive cutaneous anaphylaxis assay

Passive cutaneous anaphylaxis (PCA) assay has been a gold standard method to measure allergen-specific IgE antibody (ASIgE Ab) levels in allergy mouse models. Many factors including stringent guidelines for laboratory animal use make PCA a difficult choice. Therefore, alternative methods are needed that can be readily applied for measurement of specific IgE antibody levels in mouse serum. Herein we describe a novel ELISA-based method that is more sensitive in comparison to PCA, IgE isotype-specific (because it has little cross-reactivity with IgG1 or IgG2a isotype) and highly reproducible (<10% inter- or intra-assay variation). Furthermore, we demonstrate the utility of this assay to measure specific IgE Ab against a variety of food extracts including chicken egg, peanut, almond, filbert/hazelnut and sweet potato. These findings are of particular interest to those who are seeking (i) to measure food-extract-specific IgE antibody in animal models and (ii) an alternative to the animal-based PCA method to measure mouse IgE antibodies.     

Ethinyl estradiol treats collagen-induced arthritis in DBA/1LacJ mice by inhibiting the production of TNF-alpha and IL-1beta

We previously demonstrated the therapeutic effects of ethinyl estradiol (EE), an orally active estrogen and a component of birth control pills, in encephalitogenic autoimmune encephalomyelitis (EAE). In this study, we report the effectiveness of EE in treating collagen-induced arthritis (CIA) induced with bovine type II collagen (bCII) in DBA/1LacJ mice, a CIA susceptible strain. Both low and high doses of EE notably suppressed clinical and histological signs of CIA in a dose-dependent manner compared to vehicle-treated controls. Oral treatment with EE decreased proliferation and secretion of pro-inflammatory factors, TNF-alpha IFN-gamma, MCP-1 and IL-6 by bCII peptide-specific T cells, production of bCII-specific IgG2a antibodies, and mRNA for cytokines, chemokines and chemokine receptors in joint tissue. This is the first report demonstrating effective treatment of joint inflammation and clinical signs of CIA with orally administered ethinyl estradiol, thus supporting its possible clinical use for treating rheumatoid arthritis in humans.     

Epstein-Barr virus association and ALK gene expression in anaplastic large-cell lymphoma

Anaplastic large cell lymphoma of T/null-cell type (ALCL) is associated with a characteristic genetic abnormality t(2;5) that results in the NPM-ALK chimeric gene and the protein product derived thereof. In 10% to 20% of ALCLs, the translocation partners of the ALK gene are genes other than NPM (variant translocations). ALK gene expression limited to the cytoplasm implies a variant translocation. In this study, we have investigated 46 cases of ALCL for expression and localization of ALK protein and its association with Epstein-Barr virus (EBV) (by hybridization to EBV-encoded nuclear RNA-1 [EBER-1] and immunostaining for LMP-1). ALCL patients with a null cell phenotype were significantly younger as compared with those of T-cell phenotype (mean age: 28 years v 42 years; P =.018). Sixteen of 46 ALCL cases (34%) were ALK positive. ALK-positive patients were significantly younger (mean age: 25 years for those with both cytoplasmic and nuclear staining; 22 years for those with exclusive cytoplasmic staining; and 41 years for those negative for the ALK gene; P =.023). EBER-1 was detected in 9 of 46 cases (20%), and LMP-1 expression was noted in 5 of them. By polymerase chain reaction analysis, all EBV-associated cases that were investigated showed type I EBV. Whereas 2 of 23 T-cell ALCLs (9%) were EBER-1+, and 7 of 23 null-cell ALCLs (30%) showed EBV association (P =.057). EBV association was seen in 20% of ALK-negative cases, in 0% of cases with ALK gene expression in both nucleus and cytoplasm, and in 60% of cases with ALK gene expression exclusively in the cytoplasm (P =.02). Further, although ALK-positive-EBER-1+ cases were LMP-1 negative, ALK-negative-EBER-1+ cases were LMP-1 positive. Our study raises the question whether EBV might have an etiological role in the evolution of ALCLs that lack classical t(2;5).     

Identification of medically important yeasts using PCR-based detection of DNA sequence polymorphisms in the internal transcribed spacer 2 region of the rRNA genes

Identification of medically relevant yeasts can be time-consuming and inaccurate with current methods. We evaluated PCR-based detection of sequence polymorphisms in the internal transcribed spacer 2 (ITS2) region of the rRNA genes as a means of fungal identification. Clinical isolates (401), reference strains (6), and type strains (27), representing 34 species of yeasts were examined. The length of PCR-amplified ITS2 region DNA was determined with single-base precision in less than 30 min by using automated capillary electrophoresis. Unique, species-specific PCR products ranging from 237 to 429 bp were obtained from 92% of the clinical isolates. The remaining 8%, divided into groups with ITS2 regions which differed by /=99%. Seven clinical isolates contained ITS2 sequences that did not agree with their phenotypic identification, and ITS2-based phylogenetic analyses indicate the possibility of new or clinically unusual species in the Rhodotorula and Candida genera. This work establishes an initial database, validated with over 400 clinical isolates, of ITS2 length and sequence polymorphisms for 34 species of yeasts. We conclude that size and restriction analysis of PCR-amplified ITS2 region DNA is a rapid and reliable method to identify clinically significant yeasts, including potentially new or emerging pathogenic species.     

Application of 16S rRNA gene sequencing to identify Bordetella hinzii as the causative agent of fatal septicemia

We report on the first case of fatal septicemia caused by Bordetella hinzii. The causative organism exhibited a biochemical profile identical to that of Bordetella avium with three commercial identification systems (API 20E, API 20 NE, and Vitek GNI+ card). However, its cellular fatty acid profile was not typical for either B. avium or previously reported strains of B. hinzii. Presumptive identification of the patient's isolate was accomplished by traditional biochemical testing, and definitive identification was achieved by 16S rRNA gene sequence analysis. Phenotypic features useful in distinguishing B. hinzii from B. avium were production of alkali from malonate and resistance to several antimicrobial agents.     

The views of U.S. medical school deans toward academic primary care.

PURPOSE: To understand the views of U.S. medical school deans about their primary care faculties. METHOD: In 2000, the authors mailed a questionnaire containing 43 multipart items to deans of 130 U.S. allopathic medical schools. The questionnaire assessed the deans' attitudes about and evaluation of primary care at their school and their school's efforts to strengthen it. Deans were asked to compare family medicine, general internal medicine, and general pediatrics with nonprimary care clinical departments at their schools. RESULTS: Of the 83 (64%) deans who responded, 82% reported their school had departments or divisions of family medicine, general internal medicine, and general pediatrics. Deans rated general internal medicine and general pediatrics higher than nonprimary care faculty on clinical expertise and productivity (p < .001) and family medicine equivalent to nonprimary care faculty. Deans rated all three primary care faculties superior to nonprimary care faculty for teaching skills (p < .001) and programs (p < .05), but lower than nonprimary care disciplines for research productivity (p < .01) and revenues (p < .001). They rated family medicine and general pediatrics lower for research skills (p < .001), but 73% of deans stated research was equally important for primary care and nonprimary care departments. Deans considered overall financial resources to be equivalent for primary care and nonprimary care departments, but 77% of deans felt primary care departments or divisions needed financial support from the medical school to survive. Most deans attempted to strengthen primary care by changing the curriculum to promote primary care and by providing financial support. CONCLUSIONS: Deans ranked primary care faculty high on clinical and teaching measures. Although they considered research to be an important activity for primary care faculty, they evaluated it low relative to nonprimary care departments.