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.     

NADPH oxidase priming and p47phox phosphorylation in neutrophils from synovial fluid of patients with rheumatoid arthritis and spondylarthropathy

Superoxide anion (O2°-)production by neutrophil NADPH oxidase participates in arthritic joint lesion formation. Proinflammatory cytokines such as tumor necrosis factor (TNF), interleukin 8 (IL-8) and granulocyte/macrophage-colony stimulating factor (GM-CSF) have a priming effect on neutrophil NADPH oxidase activity. NADPH oxidase activation is dependent on phosphorylation of p47phox, a cytosolic component of the enzyme. We studied O2°-production and p47phox phosphorylation in synovial fluid (SF) from patients with rheumatoid arthritis (RA) and spondylarthropathy (SpA) according to TNF, IL-8 and GM-CSF levels. O2°-production by neutrophils isolated from SF of all the arthritis patients (RA and SpA) was higher than that of circulating resting neutrophils and when stimulated with fMLP or PMA. In addition, p47phox was partially phosphorylated in SF neutrophils compared to circulating neutrophils. High levels of TNF and IL-8 (but not GM-CSF) are detected in patient's SF (compared to circulating blood levels). TNF levels were significantly higher in RA than in SpA SF. These results suggest that increased NADPH oxidase activity could be involved in arthritic joint inflammation through increased p47phox phosphorylation. This could be the result of the presence of high levels of priming agents such as TNF and IL-8 but not GM-CSF.     

Chemical constituents of diesel exhaust particles induce IL-4 production and histamine release by human basophils

BACKGROUND: An epidemiologic relationship between airway allergic diseases and exposure to atmospheric pollutants has been demonstrated and suggested to be one factor in the increasing prevalence of asthma. Diesel exhaust particles (DEPs) have been shown to participate in the development of allergic airway inflammation, in which the targets include macrophages, B and T cells, epithelial cells, and mast cells. In addition to the adjuvant effect of DEPs on total and allergen-specific IgE production, DEPs also act to induce chemokines and cytokines and may play a key role in primary sensitization. OBJECTIVE: DEPs have been shown to increase local IL-4-containing Kit(+) cells soon after in vivo nasal challenge. The aim of this study was to examine the effects of DEPs on human basophils, a key source of IL-4. METHODS: Peripheral blood leukocytes from allergic and control subjects were cultured in the presence of organic extracts of DEP (DEPex) with or without allergen. The cultures were analyzed for IL-4-containing cells by using multiparameter flow cytometry, IL-4 secretion with ELISA, and histamine release. RESULTS: Basophils, when exposed in vitro to DEPex, expressed IL-4 and released histamine significantly (P <.01) more than with antigen activation. DEPex did not synergize with allergen in cytokine production and histamine release. DEPex-induced basophil IL-4 expression peaked at 2 hours and persisted through 20 hours, in contrast to allergen-induced IL-4, which was transient. The effect of DEPex on basophil cytokine expression and histamine release was dose dependent and occurred with cells from both allergic and nonallergic subjects. DEPex induced IL-4 expression and histamine release in highly enriched basophil populations, suggesting it acts directly on basophils. Other peripheral blood leukocytes, including T cells, did not contribute to this cytokine expression. Preincubation with N-acetylcysteine completely abrogated DEPex-driven basophil IL-4 expression. CONCLUSIONS: Basophils are a direct target for DEPex, inducing IL-4 expression and histamine release in an IgE-allergen independent fashion. N-acetylcysteine inhibition of DEPex-driven IL-4 expression provides evidence that generation of reactive oxygen species is required for the effects observed. The capability of DEPex to activate basophils in both allergic and nonallergic subjects suggests a potential role of this pollutant in the increasing prevalence of allergic diseases.     

Antithrombotic properties of trillium coated connectors

Trillium coating (Medtronic Inc., Minneapolis, MN) offers, in addition to the presence of heparin, endothelium-like properties of its negatively charged surface. Its thromboresistant properties on coated connectors are tested here and compared with uncoated standard connectors, as well as with the Carmeda BioActive surface (CBAS) heparin surface coating. A partial cardiopulmonary bypass bovine model (body weight 68 +/- 5 kg) was selected, and the surfaces were exposed to the blood stream (pump flow 3.5 L/min) for up to 350 minutes without systemic heparinization. Thereafter, another set of samples was exposed to stagnant blood for 20 minutes. Besides hemodynamic, hematologic, and biochemical analyses, the macroscopic appearance of 45 blood exposed surface samples were graded semiquantitatively on a scale of 0 to 10: no macroscopic deposits = grade 0, one spot (1 mm diameter) = grade 1, two spots = grade 2, five or more spots = grade 5, 10% of the surface covered with clots = grade 6, 100% covered = grade 10. When exposed to blood flow, Trillium and CBAS coatings showed a statistically significant (p = 0.03) better thromboresistance (score: 0 +/- 0 for both) than uncoated connectors (score: 0.8 +/- 1.5) in this nonheparinized model. The same holds true when the connectors were exposed to stagnant blood (score: 0 +/- 0 for both coatings vs 4.3 +/- 2.8 for controls; p = 0.03). Therefore, Trillium coating exhibits significant antithrombotic properties that outperform standards for connectors used in clinical perfusion.     

The relative contribution of direct and indirect antigen recognition pathways to the alloresponse and graft rejection depends upon the nature of the transplant

In this study, we measured direct and indirect T-cell alloresponses mediated by CD4(+) and CD8(+) T cells in three mouse transplantation models: skin, cornea, and retina. We show that the contribution of direct and indirect antigen recognition pathways to the alloresponse to fully allogeneic grafts varies depending upon the nature of the tissue/organ transplanted. The implications of this finding for understanding the cellular mechanisms by which rejection is mediated in different transplant models are discussed.     

Electronic implementation of guidelines in the EsPeR system: a knowledge specification method

Despite initiatives to standardize methods for the development of clinical guidelines, several barriers hinder their integration in daily clinical practice: failure to fulfil quality criteria, poor effectiveness of their dissemination. Computerization of guidelines can favor their dissemination. The initial step of computerization is the knowledge specification from the text of the guideline. We describe the method of knowledge specification, which is used in EsPeR (Personalized Estimate of Risks), a web-based decision support system in preventive medicine, which allows, for a given person, to estimate risks and access recommendations, based on clinical profile. This method is based on a structured and systematic analysis of text allowing detailed specification of a decision tree. We use decision tables to validate the decision algorithm and decision trees to specify this algorithm, along with elementary messages of recommendation. Editing tools are used to facilitate the process of validation and the workflow between expert physicians and computer scientists. Applied to eleven different guidelines, the method allows a quick and valid computerization and integration in the EsPeR system. The method used for computerization could help to define a framework usable at the initial step of guideline development in order to produce guidelines ready for electronic implementation.     

Randomized, double-blind, placebo-controlled study of montelukast for treating perennial allergic rhinitis.

BACKGROUND: Perennial allergic rhinitis (PAR) is a persistent allergic inflammation of the upper respiratory tract due to year-round allergen exposure. OBJECTIVE: To evaluate the leukotriene receptor antagonist montelukast for the treatment of PAR. METHODS: Protocol 265 was a 2-arm study performed during the winter. After a placebo run-in period, adults with perennial allergen sensitivity and active symptoms of PAR were randomized to receive 10 mg of montelukast (n=1002) or placebo (n=990) once daily during a 6-week, double-blind, active-treatment period. The primary end point was the daytime nasal symptoms score, defined as the average of scores for nasal congestion, rhinorrhea, and sneezing rated daily by patients. RESULTS: Statistically significant improvements in PAR symptoms were seen in patients treated with montelukast. Their daytime nasal symptoms scores were reduced during treatment compared with those of the placebo group: the difference between treatments in least squares mean change from baseline was -0.08 (95% confidence interval [CI], -0.12 to -0.04; P < .001). Montelukast treatment also improved global evaluations of allergic rhinitis by patients and Rhinoconjunctivitis Quality of Life Questionnaire scores: differences vs the placebo group were -0.15 (95% CI, -0.27 to -0.04; P < .01) and -0.15 (95% CI, -0.24 to -0.06; P < .001), respectively. Other end points that showed statistically significant improvement with montelukast treatment were nighttime symptoms and each of the 4 nasal symptoms (congestion, rhinorrhea, sneezing, and itching). The treatment effects of montelukast were stable and persistent during the entire 6 weeks of treatment. CONCLUSION: Montelukast provided statistically significant relief of PAR symptoms during 6 weeks of treatment.     

Effect of zidovudine resistance mutations on virologic response to treatment with zidovudine or stavudine,each in combination with lamivudine and indinavir

The authors studied the effect of zidovudine (ZDV) resistance mutation on virologic response to treatment with ZDV or stavudine (d4T) each in combination with lamivudine and indinavir. Viral genotyping was performed on plasma HIV-1 RNA at study entry and concerned 155 patients previously treated with ZDV, didanosine, or zalcitabine and enrolled in the NOVAVIR (Agence National de Recherche sur le SIDA [ANRS] 073) trial. Three virologic responses were investigated: early response (<50 copies/mL at week 24), late response (<500 copies/mL at week 80), and virologic failure (two HIV-1 RNA >5000 copies/mL). Patients were classified as resistant or susceptible to ZDV according to the ANRS algorithm. Plasma viral RNA from 123 of 155 patients had two or more ZDV resistance mutations. The number of ZDV resistance mutations was positively correlated with the duration of prior antiviral therapy (p <.001). At week 24, 74% and 77% of patients with virus classified as resistant were responders in the d4T and ZDV arm, respectively. Similar results were found at week 80. Virologic failure was reached in 7 of 24 patients with virus classified as susceptible and in 26 of 131 patients with resistant virus (p =.29). In the ZDV arm, patients classified as resistant had longer times to virologic failure than those classified as susceptible (p =.003). In conclusion, sustained virologic response despite presence of ZDV resistance mutations implies that these mutations do not preclude an early and durable response to treatment with a potent three-drug regimen in these patients. Patients susceptible to ZDV had lower median mean corpuscular volumes and lower random indinavir levels, suggesting that adherence was the main reason for failure.     

Ocular counterrolling induced by centrifugation during orbital space flight

During the 1998 Neurolab mission (STS-90), four astronauts were exposed to interaural centripetal accelerations (Gy centrifugation) of 0.5g and 1g during rotation on a centrifuge, both on Earth and during orbital space flight. Subjects were oriented either left-ear out or right-ear out, facing or back to motion. Binocular eye movements were measured in three dimensions using a video technique. On Earth, tangential centrifugation that produces 1g of interaural linear acceleration combines with gravity to tilt the gravitoinertial acceleration (GIA) vector 45° in the roll plane relative to the head vertical, generating a summed vector of 1.4g. Before flight, this elicited mean ocular counterrolling (OCR) of 5.7°. Due to the relative absence of gravity during flight, there was no linear acceleration along the dorsoventral axis of the head. As a result, during in-flight centrifugation, gravitoinertial acceleration was strictly aligned with the centripetal acceleration along the interaural axis. There was a small but significant decrease (mean 10%) in the magnitude of OCR in space (5.1°). The magnitude of OCR during postflight 1g centrifugation was not significantly different from preflight OCR (5.9°). Findings were similar for 0.5g centrifugation, but the OCR magnitude was approximately 60% of that induced by centrifugation at 1g. OCR during pre- and postflight static tilt was not significantly different and was always less than OCR elicited by centrifugation on Earth for an equivalent interaural linear acceleration. In contrast, there was no difference between the OCR generated by in-flight centrifugation and by static tilt on Earth at equivalent interaural linear accelerations. These data support the following conclusions: (1) OCR is generated predominantly in response to interaural linear acceleration; (2) the increased OCR during centrifugation on Earth is a response to the head dorsoventral 1g linear acceleration component, which was absent in microgravity. The dorsoventral linear acceleration could have activated either the otoliths or body-tilt receptors that responded to the larger GIA magnitude (1.4g), to generate the increased OCR during centrifugation on Earth. A striking finding was that magnitude of OCR was maintained throughout and after flight. This is in contrast to most previous postflight OCR studies, which have generally registered decreases in OCR. We postulate that intermittent exposure to artificial gravity, in the form of the centripetal acceleration experienced during centrifugation, acted as a countermeasure to deconditioning of this otolith-ocular orienting reflex during the 16-day mission. Electronic Publication