Characterization of wild-type recombinant Bet v 1a as a candidate vaccine against birch pollen allergy

BACKGROUND: We describe the production in Escherichia coli as a recombinant protein of clinical grade wild-type Bet v 1a (rBet v 1a), to be used as a candidate vaccine against birch pollen allergy. METHODS: This recombinant protein was purified by hydrophobic interaction and ion exchange chromatography and characterized by SDS-PAGE, immunoprint and circular dichroism in parallel with natural Bet v 1 (nBet v 1) purified from a birch pollen extract. We also compared rBet v 1 and nBet v 1 for their capacity to induce histamine release from basophils and to stimulate T lymphocyte proliferation. RESULTS: rBet v 1a appears in SDS-PAGE as an 18-kDa monomeric protein, whereas purified nBet v 1 comprises a mixture of isoforms (resolving as three distinct bands and six spots after 1-dimensional and 2-dimensional electrophoresis, respectively). Both recombinant and natural purified Bet v 1 molecules are recognized by IgE from birch pollen-allergic patients as well as anti-Bet v 1 murine monoclonal antibodies, suggesting that the recombinant protein is correctly folded in a native configuration. Circular dichroism analysis confirmed that the two Bet v 1 molecules exhibit similar 3-dimensional structures, even if rBet v 1a appears more compact and stable in thermodenaturation/renaturation experiments. Both rBet v 1 and nBet v 1 induce the degranulation of sensitized basophils and proliferation of Bet v 1-specific T lymphocytes in a similar manner. CONCLUSIONS: On the basis of these structural and biological properties, rBet v 1a is a valid candidate vaccine against birch pollen allergy, currently evaluated in humans.     

Evaluation of mast cell activation syndromes: impact of pathology and immunohistology

Mast cell activation syndromes (MCAS) are clinically defined disease states with a largely unknown morphological background. Since mastocytosis may be associated with MCAS, it is crucial in every patient to document or exclude mastocytosis by appropriate histological, molecular, and serological investigations of tissues/organs that are commonly involved in mastocytosis like skin, mucosa of the gastrointestinal tract and bone marrow. Accordingly, histopathological investigation including immunohistological stains is crucial to reach the final diagnosis in such patients and to classify MCAS into primary MCAS, which can present with or without evidence of overt mastocytosis, or secondary MCAS, where an underlying disease with or without tissue inflammation is detected. Cases without evidence of mastocytosis, monoclonal mast cells, or any underlying disease should be termed idiopathic MCAS. When the activating point mutant KIT D816V is detectable but criteria for diagnosis of mastocytosis are not completely met, a so-called (mono)clonal MCAS as a subvariant of primary MCAS should be diagnosed.     

Biochemomechanics of Cerebral Vasospasm and its Resolution: II. Constitutive Relations and Model Simulations

Cerebral vasospasm is a poorly understood clinical condition that appears to result from complex biochemical and biomechanical processes that manifest as yet another example of vascular growth and remodeling. We submit that mathematical modeling holds great promise to help synthesize diverse types of data and thereby to increase our understanding of vasospasm. Toward this ultimate goal, we present constitutive relations and parametric studies that illustrate the potential utility of a new theoretical framework that combines information on wall mechanics, hemodynamics, and chemical kinetics. In particular, we show that chemical and mechanical mediators of cellular and extracellular matrix turnover can differentially dominate the progression and resolution of vasospasm. Moreover, based on our simulations, endothelial damage can significantly alter the time-course and extent of vasospasm as can impairment of autoregulation. Although the present results are consistent with salient features of clinically reported vasospasm, and thus provide some new insight, we suggest that most importantly they reveal areas of pressing need with regard to the collection of additional experimental data. Without appropriate data, our understanding of cerebral vasospasm will remain incomplete. Address correspondence to J. D. Humphrey, Department of Biomedical Engineering, Texas A&M University, 337 Zachry Engineering Center, 3120 TAMU, College Station, TX 77843-3120, USA. Electronic mail: jhumphrey@tamu.edu

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Tobacco as an allergen in bronchial disease.

BACKGROUND: Skin testing and sera measurements have verified the existence of tobacco specific IgE. However, the few published studies on this matter report conflicting results concerning their clinical significance. OBJECTIVE: To verify if a specific clinical allergenic response against tobacco might be possible in allergenic and nonallergenic bronchial diseases. METHODS: We performed a cross-sectional observational case-control analysis on 180 patients with asthma, chronic obstructive pulmonary disease (COPD), and bronchial carcinoma and controls who were randomly chosen. Skin prick tests and serum specific IgE to tobacco and related allergens, bronchial challenge with cigarettes and tobacco extract, patch tests with tobacco and nicotine, sodium dodecyl sulfate-polyacrylamide gel electrophoresis immunoblotting, and Enzyme AllergoSorbent Test (EAST) inhibition were performed. RESULTS: Twenty-eight patients had positive tobacco skin prick test results. The association among positive skin prick test results, IgE, and bronchial challenge was strong (P < .001). Tobacco sensitivity was higher in patients with pollen asthma than in patients with COPD and carcinoma and negative in patients with intrinsic asthma and controls. A positive bronchial challenge result was related to the length of habit (P < .001) and the tobacco index in patients who had stopped smoking (P < .001). Delayed bronchial and patch response was more common in patients with COPD (P < .001). Tobacco IgE response (EAST) was related to sensitivity to Lolium perenne (rye grass) pollen (P < .001) but not to other vegetables that belong to the Solanaceae family. EAST inhibition showed cross-reactivity between tobacco and Lolium pollen. CONCLUSIONS: Tobacco may be responsible for a specific IgE response. Patients with pollen asthma were those with more positive responses to tobacco due to cross-reactivity between Lolium and tobacco allergens.     

High early uterine vascular resistance values increase the risk of adverse pregnancy outcome independently from placental VEGF and VEGFR1 reactivities

ObjectiveFrom data in the literature, we hypothesized that high vascular resistance values in the uterine arteries at the end of the first trimester would increase adverse pregnancy outcomes and therefore might be accompanied by changes in VEGF/VEGFR1 immunoreactivities.Study designIn our university hospital 82 women (Study I n = 62 and Study II n = 20) were divided into two groups according to their uterine vascular resistance values. Uterine vascular resistance values were measured in the 10–13th weeks of gestation by color-Doppler ultrasonography. Women were divided into low and high vascular resistance groups. In the prospective follow-up study (Study I) the data of the pregnancy outcome were recorded. In cross-sectional study (Study II), VEGF and VEGFR1 immunoreactivities were measured on the tissue samples from women who underwent termination of pregnancy.ResultsIn the high vascular resistance group (PI > 2.3), the probability of adverse pregnancy outcome was significantly higher (40.0% vs. 12.8%). No differences in VEGF and VEGFR1 immunoreactivities were observed between groups. In both groups, intense VEGF immunoreactivity was observed in the maternal glandular epithelium and in the decidual cells. Weak reactivity was observed in the villous trophoblast. VEGFR1 immunoreactivity was intense in all regions.ConclusionsOur data suggest that high vascular resistance values in the first trimester are independent from VEGF/VEGFR1 immunoreactivities and markedly increase the probability of adverse pregnancy outcomes. This may be used for early screening of pregnant women in the first trimester.     

Telomere length analysis on cord blood cells by the flow-FISH method

Telomerase is the enzyme responsible for synthesizing telomeric repeats at the ends of chromosomes to maintain telomere length. Recent studies have suggested that telomere shortening may serve as a surrogate marker of the progression of malignant disorders and seems to be accelerated in allogeneic bone marrow transplant recipients. In this study, the results of the telomere length of nine cord blood mononuclear cell samples are presented. Telomere length was measured by the flow-FISH method, using a peptide nucleic acid probe. The proportion of cord blood cell subsets (CD19/CD34/CD3) was also evaluated. The telomere length of the internal control 1301 cell line was estimated to be 100%. The mean telomere length of cord blood cells was 18.5 +/- 3.9%, compared with the internal control. The progenitor CD34+ cells were detected as 2.6 +/- 0.7% in the lymphoid gate measured. Linear correlation analysis did not find any connection between the cell subsets (CD3+, CD34+, CD19+) and the telomere length. The findings confirm that the telomere flow-FISH method is sufficient for estimation of the telomere length. Assessment of the current procedures of collection, manipulation, and ex vivo expansion of cord blood cells in terms of their effect on telomere shortening might be important.