Enteroadherent Escherichia coli as a cause of diarrhea among children in Mexico.

Enteropathogenic Escherichia coli (EPEC) often exhibits localized adherence or diffuse adherence to HEp-2 cells. We recently provided evidence that HEp-2 cell-adherent or enteroadherent E. coli (EAEC) not belonging to EPEC serogroups was the cause of diarrhea among U.S. travelers to Mexico. In the present study, we looked for EAEC and EPEC in stool specimens from 154 children with acute diarrhea and 137 well children seen at several outpatient clinics in Guadalajara, Mexico. EAEC showing localized adherence (EAEC-L) was isolated from 13.0% of the patients and 0.7% of the controls (P less than 0.0001). EAEC showing diffuse adherence (EAEC-D) was recovered from 20.8% of the patients and 7.3% of the controls (P less than 0.001). EPEC was isolated from 4.5 and 6.7% of the patients and controls, respectively. Among all enteropathogens, only enterotoxigenic E. coli occurred as commonly (21.4%) as EAEC-D and EAEC-L did in children with diarrhea. Of the EAEC-L strains isolated from children with diarrhea, 20% belonged to recognized EPEC serogroups, and 3.1% of EAEC-D strains belonged to recognized EPEC serogroups. This study suggests that EAEC may be an important pediatric enteropathogen in Mexican children with diarrhea and further supports the observation that adherence to HEp-2 cells may be a marker of virulence independent of EPEC serogroup among E. coli strains.     

Cutaneous papillary squamous cell carcinoma. Report of three new cases and review of the literature

Squamous cell carcinoma is one of the most common malignant cutaneous tumors. Several histological variants have been described; the papillary subtype is one of the most infrequent, with only four cases having being reported previously. We report three new cases of this unusual variant of cutaneous squamous cell carcinoma, review the literature and consider the main differential diagnoses. Polymerase chain reaction performed in our three cases did not demonstrate human papilloma virus infection.     

Green fluorescent protein expressed by a recombinant vaccinia virus permits early detection of infected cells by flow cytometry

We have tested Green Fluorescent Protein (GFP) expressed by a vaccinia virus recombinant as a marker for viral infection. Virus recombinants expressing either wild-type GFP, or a Ser₆₅ to Thr mutated version (GFP-S65T) were used to infect cultured cells, and the appearance of fluorescence was followed during infection by flow cytometry. Although both versions were detectable in infected cells, GFP-S65T gave up to 26-fold brighter fluorescence than wild-type GFP when excited by an argon laser beam (488 nm). In addition, GFP-S65T fluorescence appeared earlier, and infected cells could be detected above background as soon as 1 h after infection. We have used this construct to infect porcine peripheral blood lymphocytes, and show its usefulness to study virus tropism when used in combination with cell-type specific markers. Thus, GFP provides a direct, fast and convenient way to monitor infection by flow cytometry.     

Molecular detection of Histoplasma capsulatum var. capsulatum in human clinical samples

We developed a seminested PCR for the diagnosis of histoplasmosis that amplifies a portion of the Histoplasma capsulatum H antigen gene. This assay is highly sensitive and specific, being able to detect genomic material corresponding to less than 10 yeast cells without cross-reaction against other bacterial or fungal pathogens.     

Engineering porcine arteries: effects of scaffold modification

Techniques have been developed to culture bovine or porcine vascular cells on polyglycolic acid (PGA) scaffolds to form engineered vessels. Previously, it was shown that smooth muscle cells (SMCs) that were in close proximity to PGA remnants after 8 weeks of culture had lower expression of SMC markers of differentiation and were more mitotic compared with SMCs that were distant from polymer residuals. Modifications of PGA were explored as a means to minimize residual polymer fragments after culture. To hasten degradation, polymer was treated with heat, NaOH, or gamma-irradiation. Differential scanning calorimetry, mass and tensile strength degradation, and inherent viscosity were used to assess polymer characteristics. When polymer was maintained in aqueous conditions, tensile strength of treated PGA degraded to zero within 3 weeks for each treatment. Engineered vessel constructs cultured on NaOH and gamma-treated polymer displayed smooth muscle alpha-actin throughout the vessel wall. Scaffold treatment impacted graft morphology, cellular differentiation, and mechanical integrity.     

A rapid and easy method for multiple endocrine neoplasia type 1 mutation detection using conformation-sensitive gel electrophoresis

Until now, the study of the multiple endocrine neoplasia type 1 (MEN1) gene in patients suspected of having the disease was expensive and laborious due to the large size of the gene. We have optimized the conformation-sensitive gel electrophoresis (CSGE) technique to analyze by four rather simple multiplex PCR reactions, and a single electrophoresis run, the entire coding region of the MEN1 gene, plus the exon–intron boundaries. This improvement of the CSGE technique was confirmed as an effective procedure for screening for the MEN1 gene by detecting ten previously known MEN1 gene mutations and four polymorphisms. The MEN1 gene of 12 patients with unknown mutations was then screened, and an abnormal CSGE profile was identified in 10/12 cases. Subsequent DNA sequencing demonstrated 3 of them to be novel mutations (E45K, 4479delACAG, 6073insC) and 7 to have been previously reported; in the remaining 2 patients, we confirmed the absence of any alteration of the coding sequence of MEN1. Mutation screening of the MEN1 gene using CSGE was demonstrated to be a fast, simple, and inexpensive method to study patients suspected of having MEN1 disease. Received: November 29, 2001 / Accepted: January 28, 2002     

Patterns of reactivity to lipid transfer proteins of plant foods and Artemisia pollen: an in vivo study

BACKGROUND: Lipid transfer proteins (LTPs) are major allergens of Rosaceae fruits in the Mediterranean area. IgE-cross-reactivity has been demonstrated in vitro among LTPs from peach, apple, chestnut and Artemisia pollen. The aim of this study was to evaluate the reactivity to LTPs from peach, apple, chestnut and Artemisia pollen by means of skin prick tests (SPTs). METHODS: Forty-seven patients allergic to peach (peach group), 20 patients sensitized to Artemisia pollen with no food allergies (Artemisia group), and 12 control subjects were skin tested with fresh peach, as well as with whole extracts and purified LTPs of peach, apple, chestnut and Artemisia pollen. RESULTS: The rates of positive SPTs for peach, apple, chestnut and Artemisia LTPs were, respectively, 91, 77, 23, and 36% in the peach group, and 30, 5, 15 and 40% in the Artemisia group. No response was observed in the control subjects. SPTs with peach LTP strongly correlated with SPTs conducted with fresh peach. In the peach group, the most frequent pattern of reactivity to LTPs was the combination peach-apple (45%), followed by peach-apple-Artemisia-chestnut (21%). Significant correlations were found between peach and apple LTPs, and between Artemisia and chestnut LTPs. Positive SPTs to chestnut LTP were only observed in patients with positive SPTs to Artemisia LTP. All the patients with positive case histories to chestnut reacted to chestnut LTP. CONCLUSIONS: LTPs are plant panallergens with different patterns of cross-reactivity. They are major allergens of Rosaceae fruits and seem to be involved in allergic reactions to unrelated foodstuffs such as chestnut, probably through sensitization to the cross-reactive Artemisia LTP. Rosaceae LTPs could be useful tools for in vivo diagnosis of Rosaceae fruit allergy.     

Optimal heparin surface concentration and antithrombin binding capacity as evaluated with human non-anticoagulated blood in vitro

Contact between blood and a biomaterial surface takes place in many applications and is known to activate the coagulation and complement systems. Heparin surface coatings have been shown to reduce blood activation upon contact with artificial surfaces. To establish the optimal heparin surface concentration, blood was incubated in a tubing loop model at 37 degrees C. The tubing was coated with different surface concentrations of heparin and rotated at three different velocities. We demonstrate that the blood compatibility of a surface with regard to coagulation, complement, and platelet activation can be improved by increasing the heparin surface concentration in the 6-12 pmol antithrombin/cm2 concentration interval. The binding of factor H is not influenced by the increased heparin surface concentration, suggesting that this factor is not the primary regulator of complement on heparin surfaces. In addition, the heparin coating has no effect on the complement activation that occurs on gas surfaces in extracorporeal circuits.     

Effects of dexamethasone and transient malnutrition on rabbits infected with aerosolized Mycobacterium tuberculosis CDC1551

Malnutrition is common in the developing world, where tuberculosis is often endemic. Rabbits infected with aerosolized Mycobacterium tuberculosis that subsequently became inadvertently and transiently malnourished had compromised cell-mediated immunity comparable to that of the rabbits immunosuppressed with dexamethasone. They had significant leukopenia and reduced delayed-type hypersensitivity responses. Malnutrition dampened cell-mediated immunity and would interfere with diagnostic tests that rely on intact CD4 T-cell responses.