Inhibition of TCR-mediated shedding of L-selectin (CD62L) on human and mouse CD4+ T cells by metalloproteinase inhibition: analysis of the regulation of Th1/Th2 function

The generation of a productive primary immune response is dependent on the ability of na?ve T lymphocytes to recirculate through peripheral lymph organs to encounter specific antigen. The process of na?ve CD4(+) T cell entry into lymph nodes correlates with cell surface expression of L-selectin (CD62L), which mediates early tethering and rolling events to endothelium prior to entry. Here, we demonstrate that surface expression of CD62L enhances CD4(+) T cell activation in vitro. The synthetic hydroxamate metalloproteinase inhibitor (BB-3103), specifically inhibits activation-induced shedding of CD62L from CD4(+) T cells by TCR cross-linking and lowers proliferation in part by reducing rapid tyrosine phosphorylation of zeta-associated protein 70 kDa (ZAP-70) and by increasing cytosolic free Ca(2+) concentration mobilization. BB-3103 also inhibited the proliferative response of both murine CD4(+) Th1 and Th2 subsets in vitro but the inhibitory effects were sustained only in Th2-type cells. Similarly, BB-3103 mediated prolonged inhibition of allergen-dependent peripheral T cell proliferation in atopic dermatitis patients but not in healthy controls. Analysis of CD62L expression on murine CD4(+) T cell subsets revealed that surface expression was maintained on Th1 cells but not Th2 cells. The differential effects of BB-3103 on primed effector CD4(+) T cells may provide new insights into generating therapeutic agents capable of redressing the Th2/Th1 imbalance in allergic diseases.     

Radiochromic film dosimetry of a low energy proton beam

In this work some dosimetric characteristics of MD-55-2 GafChromic films were studied in a low energy proton beam (21.5 MeV) directly in a water phantom. The nonlinearity of the optical density was quantified by a factor P(lin). A correction factor P(en), that accounts for optical density dependence on the energy, was empirically determined. The effects of detector thickness in depth dose measurements and of the film orientation with respect to beam direction were investigated. The results show that the MD-55-2 films provide dose measurements with the films positioned perpendicularly to the proton beam. A dosimetric formalizm is proposed to determine the dose to water at depth d, with films oriented perpendicularly to the beam axis. This formalism uses a calibration factor of the radiochromic film determined directly on the proton beam at a reference depth in water, and the P(lin) factor, that takes into account the nonlinearity of the calibration curve and the P(en) factor that, in turn takes into account the change of proton beam energy in water. The MD-55-2 films with their high spatial resolution and the quasiwater equivalent material are attractive, positioned perpendicularly along the beam axis, for the absolute dose determination of very small beam sizes and modulated proton beams.     

Involvement of leukotriene B4 and platelet-activating factor in cytokine priming of human polymorphonuclear leucocytes.

Recombinant human (rh) tumour necrosis factor (TNF) alpha and rh granulocyte-macrophage colony-stimulating factor (GM-CSF) prime human polymorphonuclear leucocytes (PMN) for increased superoxide anion (O2-) generation and for increased platelet-activating factor (PAF) biosynthesis and leukotriene B4 (LTB4) release. Both PAF and LTB4 are candidate mediators for the enhanced O2- generation in cytokine-primed PMN, since exogenous PAF or LTB4 primes PMN. We measured the generation and release of these mediators and examined their potential roles in cytokine priming using the PAF receptor antagonist, WEB 2086, and the inhibitor of 5-lipo-oxygenase, CGS 8515.rhTNF-alpha or rhGM-CSF, alone, increased PAF levels in PMN, but did not cause PAF release or LTB4 synthesis. N-formylmethionyl-leucyl-phenylalanine (FMLP) stimulated the release of detectable and biologically active amounts of both LTB4 and PAF in primed, but not in non-primed PMN. However, neither blockade of PAF receptors, nor inhibition of LTB4 synthesis influenced the priming of O2- generation by rhTNF-alpha or rhGM-CSF. Simultaneous pretreatment of PMN with WEB 2086 and CGS 8515 also failed to inhibit priming. Our results do not exclude a role for cell-associated PAF in the priming response, but indicate that the release of PAF and LTB4 do not mediate this phenomenon. The ability of cytokines to amplify the production and release of lipids may represent a mechanism to attract and localize the pro-inflammatory actions of stimulated PMN to regions where cytokine levels are also elevated.     

Cytoprotective effect of vitamin A and its clinical importance in the treatment of patients with chronic gastric ulcer

The effects of vitamin A were studied on the basal and maximal gastric secretory responses of 12 patients; and on healing in 60 patients with chronic gastric ulcer. The effect of vitamin A on ulcer healing was evaluated by a multiclinical, multicentre, randomized, prospective study in which the patients were divided into three groups. In group A the patients were treated with antacids only; in group B the patients were given antacids plus vitamin A (in doses of 3 X 50.000 U orally); and in group C the patients received antacids, vitamin A plus cyproheptadine (in doses of 3 X 4 mg orally). The treatment lasted four weeks. At the beginning and the end of treatment endoscopies were performed and ulcer sizes were measured planimetrically. Various other parameters such as ulcer index, antacid consumption and laboratory parameters were also evaluated during the four-week treatment. It was observed that: (i) vitamin A (given in doses of 100.000 U i.m.) decreased neither basal nor maximal gastric secretory responses; (ii) the number of patients with completely healed gastric ulcer was significantly higher (P less than 0.05) in groups B and C than in group A; (iii) the extent of ulcer reduction was significantly higher (P less than 0.01) in groups B and C than in group A; (iv) no significant changes were observed in ulcer index and antacid consumption during the four-week treatment in the different groups of patients; (v) the reduction of ulcer size was significantly greater (P less than 0.01) in the group treated with antacids plus vitamin A than in the group treated with antacids only, at two weeks of treatment.(ABSTRACT TRUNCATED AT 250 WORDS)     

The mechanism of the force response to stretch in human skinned muscle fibres with different myosin isoforms

Force enhancement during lengthening of an active muscle, a condition that normally occurs during locomotion in vivo , is attributed to recruitment of myosin heads that exhibit fast attachment to and detachment from actin in a cycle that does not imply ATP splitting. We investigated the kinetic and mechanical features of this cycle in Ca²⁺ activated single skinned fibres from human skeletal muscles containing different myosin heavy chain (MHC) isoforms, identified with single-fibre gel electrophoresis. Fibres were activated by using a new set-up that allows development of most of the tension following a temperature jump from 0–1°C to the test temperature (∼12°C). In this way we could prevent the development of sarcomere non-uniformity and record sarcomere length changes with a striation follower in any phase of the mechanical protocol. We found that: (i) fibres with fast MHC isoforms develop 40–70% larger isometric forces than those with slow isoforms, as a result of both a larger fraction of force-generating myosin heads and a higher force per head; (ii) in both slow and fast fibres, force enhancement by stretch is due to recruitment of myosin head attachments, without increase in strain per head above the value generated by the isometric heads; and (iii) the extent of recruitment is larger in slow fibres than in fast fibres, so that the steady force and power output elicited by lengthening become similar, indicating that mechanical and kinetic properties of the actin–myosin interactions under stretch become independent of the MHC isoform.     

Modulation of airway hyperresponsiveness by thiols in a murine in vivo model of allergic asthma

OBJECTIVE AND DESIGN: Since oxidative stress contributes to the pathogenesis of asthma, this study addressed the question whether supplementing the endogenous antioxidant, glutathione (GSH), would alleviate features of allergic asthma in the mouse. MATERIAL AND METHODS: Ovalbumin-sensitized mice received aerosols of the GSH-donors, glutathione-ethyl ester (GSEt) or N-acetylcysteine, before or during respiratory allergen challenges, or during methacholine challenges given one day after the last allergen challenge. Lung GSH levels were measured shortly after allergen or methacholine challenge. In addition, the effect of GSH supplements on airway hyperresponsiveness and inflammatory cell numbers in the airway lumen was assessed. RESULTS: GSEt decreased allergen-induced airway hyperresponsiveness when given in combination with methacholine. However, when given before or during allergen challenge, both GSH-donors failed to decrease the methacholine-induced airway contractility, change cell numbers in the airway lumen, or increase lung GSH levels. In addition, allergen challenges of sensitized mice did not decrease lung GSH levels. CONCLUSION: In contrast to guinea pigs and humans, allergen challenges in mice does not lead to acute oxidative stress.     

Improved detection of rhinoviruses by nucleic acid sequence-based amplification after nucleotide sequence determination of the 5' noncoding regions of additional rhinovirus strains

The isothermal nucleic acid sequence-based amplification (NASBA) system was applied for the detection of rhinoviruses using primers targeted at the 5' noncoding region (5' NCR) of the viral genome. The nucleotide sequence of the 5' NCRs of 34 rhinovirus isolates was determined to map the most conserved regions and design more appropriate primers and probes. The assay amplified RNA extracted from 30 rhinovirus reference strains and 88 rhinovirus isolates, it did not amplify RNA from 49 enterovirus isolates and other respiratory viruses. The assay allows one to discriminate between group A and B rhinoviruses. Sensitivities for the detection of group B and group A rhinoviruses was 20 and 200 50% tissue culture infective doses, respectively.     

Prevalence and incidence of antibodies to Borrelia burgdorferi and to tick-borne encephalitis virus in agricultural and forestry workers from Tuscany, Italy

The ticks Ixodes persulcatus and Ixodes ricinus are the main vectors of both Borrelia burgdorferi sensu lato and tick-borne encephalitis (TBE) virus in Eurasia. Borrelia burgdorferi is the cause of Lyme borreliosis, and TBE is a biphasic meningoencephalitis induced by an arbovirus belonging to the flavivirus family. The principal aims of the current investigation were (i) to determine the frequency of serological evidence of Borrelia burgdorferi sensu lato and TBE infections in healthy agricultural and forestry workers, (ii) to determine the incidence of seroconversion for antibodies against Borrelia burgdorferi sensu lato and TBE virus in Tuscan workers during a 1-year survey; and (iii) to assess the occupational risk for agricultural and forestry activities in a defined area (Tuscany, Italy). A total of 412 blood samples were taken from agricultural and forestry workers, and information on age, duration of employment, and history of tick bites was collected in a questionnaire to establish the risk factors for the diseases. Three hundred sixty-five blood donors from the same region served as controls. To estimate the rate of seroconversion, 176 of the agricultural and forestry workers were tested 1 year later. IgG and IgM antibodies against Borrelia burgdorferi sensu lato and TBE virus were detected in serum by an enzyme-linked immunosorbent assay and confirmed by Western blot analysis for Borrelia burgdorferi and by a test for inhibition of hemagglutination for TBE. Antibodies against Borrelia burgdorferi were more frequent among the workers than in the control group (7.8% vs. 4.9% in the IgG-IgM enzyme-linked immunosorbent assay and 7.03% vs. 3.56% in the confirmatory test). No seropositivity was observed for TBE virus. Eighteen of 176 subjects who underwent a second blood test developed specific antibodies against Borrelia burgdorferi within 1 year.     

Postnatal protein malnutrition affects play behavior and other social interactions in juvenile rats

The effects of postnatal protein malnutrition on juvenile social behaviors were investigated in male and female Wistar rats. During the lactation period (21 days), each litter (six male and two female pups) was provided with 16% (control) or 6% (low protein) casein diets. At weaning, the control group (W) continued to receive the 16% protein diet and the malnourished group was divided into two groups: one continuing to receive a 6% protein diet (malnourished group - M) and the other shifted to a 16% protein diet (previously malnourished group - PM). These conditions lasted until 38 days of age when the behavioral tests ended. To assess social interaction, pairs of rats of the same nutritional condition and same gender were placed in a familiar arena for 3 consecutive days. Playful social behavior (pin), nonplayful social behavior (anogenital sniff, walk-over, side-mount and allogroom) and nonsocial behavior (rear) were recorded in three 10-min sessions. Postnatal protein malnutrition significantly decreased playful social behavior, nonsocial behavior (rear) and nonplayful social behaviors such as side-mount and walk-over. Anogenital sniff and allogroom were increased by early malnutrition. Nutritional rehabilitation from weaning reversed the changes produced by protein malnutrition in nonplayful (side-mount, walk-over, anogenital sniff and allogroom) and nonsocial behaviors (rear) but increased playful social behavior (pin). Gender effects were observed only on side-mount (higher incidence in males) and walk-over (higher incidence in females) indicating that playful behavior and nonsocial behavior were not affected by sex. The present results suggest that behavioral differences described in adulthood may result from changes in social behaviors of juvenile rats induced by early protein malnutrition.