Pascal Cadot

Intermittent spontaneous breathing protects the rat diaphragm from mechanical ventilation effects

Objective:Short-term mechanical ventilation has been proven to reduce diaphragm force and fiber dimensions. We hypothesized that intermittent spontaneous breathing during the course of mechanical ventilation would minimize the effects of mechanical ventilation on diaphragm force and expression levels of transcription factors (MyoD and myogenin). Design:Randomized, controlled experiment. Setting:Animal basic science laboratory. Subjects:Male Wistar rats, weighing 350–500 g. Interventions:Anesthetized and tracheotomized rats were submitted to either 24 hrs of spontaneous breathing (SB, n = 5), 24 hrs of continuous controlled mechanical ventilation (CMV, n = 7), or controlled mechanical ventilation with intermittent spontaneous breathing: 60 mins every 5 hrs of mechanical ventilation repeated four times (ISB60, n = 8) or 5 mins every 5 hrs 55 mins of mechanical ventilation repeated four times (SB5, n = 9). They were compared with control animals free from intervention (C, n = 5). Measurements and Main Results:The profile of the diaphragm force-frequency curve of the controls and SB group was significantly different from that of the ISB and CMV groups; especially, the mean asymptotic force was less in the ISB and CMV compared with controls and SB. CMV resulted in a significant decrease in the diaphragm type I (−26%, p < .05 vs. C) and type IIx/b (−39%, p < .005 vs. C and SB) cross-sectional area, whereas this was not observed in the ISB groups. Diaphragm MyoD protein expression was significantly decreased after ISB60 (−35%, p < .0001 vs. C and SB) and even more after CMV (−73%, p < .0001 vs. others). The same pattern was observed with myogenin protein levels. Positive relationships between diaphragm MyoD and myogenin protein levels and diaphragm force were observed. Conclusions:The data demonstrated that intermittent spontaneous breathing during the course of mechanical ventilation may minimize the deleterious effect of controlled mechanical ventilation on diaphragm force, fiber dimensions, and expression of transcription factors.

Effects of Acute Administration of Corticosteroids during Mechanical Ventilation on Rat Diaphragm

RATIONALE Mechanical ventilation is known to induce ventilator-induced diaphragm dysfunction. Patients submitted to mechanical ventilation often receive massive doses of corticosteroids that may cause further deterioration of diaphragm function. OBJECTIVES To examine whether the combination of 24 hours of controlled mechanical ventilation with corticosteroid administration would exacerbate ventilator-induced diaphragm dysfunction. METHODS Rats were randomly assigned to a group submitted to 24 hours of controlled mechanical ventilation receiving an intramuscular injection of saline or 80 mg/kg methylprednisolone, a group submitted to 24 hours of spontaneous breathing receiving saline, or methylprednisolone and a control group. MEASUREMENTS AND MAIN RESULTS The diaphragm force-frequency curve was shifted downward in the mechanical ventilation group, but this deleterious effect was prevented when corticosteroids were administered. Diaphragm cross-sectional area of type I fibers was similarly decreased in both mechanical ventilation groups while atrophy of type IIx/b fibers was attenuated after corticosteroid administration. The mechanical ventilation-induced reduction in diaphragm MyoD and myogenin protein expression was attenuated after corticosteroids. Plasma cytokine levels were unchanged while diaphragm lipid hydroperoxides were similarly increased in both mechanical ventilation groups. Diaphragmatic calpain activity was significantly increased in the mechanical ventilation group, but calpain activation was abated with corticosteroid administration. Inverse correlations were found between calpain activity and diaphragm force. CONCLUSIONS A single high dose of methylprednisolone combined with controlled mechanical ventilation protected diaphragm function from the deleterious effects of controlled mechanical ventilation. Inhibition of the calpain system is most likely the mechanism by which corticosteroids induce this protective effect.

Purification and characterization of an 18-kd allergen of birch (Betula verrucosa) pollen: Identification as a cyclophilin

BACKGROUND Five birch pollen allergens have been identified so far. In a previous study we detected new birch pollen allergens with an isoelectric point in the range 9.0 to 9.3, present only in extracts prepared at controlled basic pH. OBJECTIVE The purpose of the current study was to purify and characterize those allergens. METHODS The target allergens were purified by ion exchange and hydrophobic interaction chromatography. Analyses were carried out by SDS-PAGE, isoelectric focusing, immunoblotting, and amino acid sequencing. The in vivo reactivity of the allergens was evaluated by skin testing. RESULTS An 18-kd protein, which we named Bet v 7, was purified. This 18-kd protein corresponded to 3 bands on isoelectric-focusing immunoblots that probably represent isoforms. On immunoblots up to 20.8% of birch pollen-allergic patients recognized those allergens. The clinical relevance of Bet v 7 was demonstrated by positive immediate-type skin testing on a patient allergic to birch pollen. Sequencing of an internal peptide yielded an amino acid sequence showing high homology with various plant cyclophilins. The rotamase activity of the protein, inhibited by cyclosporin A, further confirmed that Bet v 7 belongs to the group of cyclophilins. CONCLUSION We have purified a novel allergen of birch pollen, Bet v 7, belonging to the cyclophilin family. Because cyclophilins are highly conserved proteins over the phylogeny, we may postulate that Bet v 7 is a member of a new family of panallergens.

Contribution of regulatory T cells and effector T cell deletion in tolerance induction by costimulation blockade

Blocking of costimulatory signals for T cell activation leads to tolerance in several transplantation models, but the underlying mechanisms are incompletely understood. We analyzed the involvement of regulatory T cells (Treg) and deletion of alloreactive cells in the induction and maintenance of tolerance after costimulation blockade in a mouse model of graft-vs-host reaction. Injection of splenocytes from the C57BL/6 parent strain into a sublethally irradiated F1 offspring (C57BL/6 × C3H) induced a GVHR characterized by severe pancytopenia. Treatment with anti-CD40L mAb and CTLA4-Ig every 3 days during 3 wk after splenocyte injection prevented disease development and induced a long-lasting state of stable mixed chimerism (>120 days). In parallel, host-specific tolerance was achieved as demonstrated by lack of host-directed alloreactivity of donor-type T cells in vitro and in vivo. Chimerism and tolerance were also obtained after CD25+ cell-depleted splenocyte transfer, showing that CD25+ natural Treg are not essential for tolerance induction. We further show that costimulation blockade results in enhanced Treg cell activity at early time points (days 6–30) after splenocyte transfer. This was demonstrated by the presence of a high percentage of Foxp3+ cells among donor CD4+ cells in the spleen of treated animals, and our finding that isolated donor-type T cells at an early time point (day 30) after splenocyte transfer displayed suppressive capacity in vitro. At later time points (>30 days after splenocyte transfer), clonal deletion of host-reactive T cells was found to be a major mechanism responsible for tolerance.

Determination of independent risk factors and comparative analysis of diagnostic methods for immediate type latex allergy in spina bifida patients

Background Development of allergy to natural rubber latex in spina bifida patients is determined by several risk factors, such as age, number of interventions and atopic disease that are, however, interdependent. Furthermore, several diagnostic procedures have been analysed, but a comprehensive analysis of their diagnostic significance is lacking.

Uptake and presentation of malignant glioma tumor cell lysates by monocyte-derived dendritic cells

Malignant glioma of the CNS is a tumor with a very bad prognosis. Development of adjuvant immunotherapy is hampered by interindividual and intratumoral antigenic heterogeneity of gliomas. To evaluate feasibility of tumor vaccination with (autologous) tumor cells, we have studied uptake of tumor cell lysates by dendritic cells (DCs), and the T-cell stimulatory capacity of the loaded DCs. DCs are professional antigen-presenting cells, which have already been used as natural adjuvants to initiate immune responses in human cancer. An efficacious uptake of tumor cell proteins, followed by processing and presentation of tumor-associated antigens by the DCs, is indeed one of the prerequisites for a potent and specific stimulation of T lymphocytes. Human monocytes were differentiated in vitro to immature DCs, and these were loaded with FITC-labeled tumor cell proteins. Uptake of the tumor cell proteins and presentation of antigens in the context of both MHC class I and II could be demonstrated using FACS analysis and confocal microscopy. After further maturation, the loaded DCs had the capacity to induce specific T-cell cytotoxic activity against tumor cells. We conclude that DCs loaded with crude tumor lysate are efficacious antigen-presenting cells able to initiate a T-cell response against malignant glioma tumor cells.

House dust mite-specific T cells in healthy non-atopic children

Background T‐helper type 2 (Th2) cells play an important role in the pathogenesis of allergic diseases. Recent studies have demonstrated that allergen‐specific T cells can also be found in the blood of healthy individuals. Both IL‐10 and IFN‐γ might modulate the induction and maintenance of allergen‐specific tolerance.

Involvement of 4-1BB (CD137)–4-1BBligand interaction in the modulation of CD4+ T cell-mediated inflammatory colitis

4‐1BB ligand (4‐1BBL) expressed on antigen‐presenting cells interacts with 4‐1BB on activated T cells (especially CD8+ cells) and co‐stimulates the latter to secrete cytokines and to proliferate. The role of 4‐1BB−4‐1BBL interaction was studied here in a model of colitis based on naive CD4+ T cell transfer to SCID mice, a disease model in which CD8 cells do not take part. We found that CD4+ T cells from 4‐1BB‐deficient mice, after transfer in SCID mice, proliferated more rapidly compared to wild‐type CD4+ T cells. Mice reconstituted with naive CD4+ T cells from 4‐1BB‐deficient mice developed colitis, however, with a mixed Th1/Th2 response, in contrast to the Th1‐type response in mice reconstituted with wild‐type naive CD4+ T cells. Importantly, this altered cytokine response did not temper colitis severity. Although it has been reported previously that 4‐1BB co‐stimulation may contribute to regulatory T cell functioning, we found that CD4+CD25+ regulatory T cells from 4‐1BB‐deficient mice were perfectly able to prevent naive CD4+ T cell‐induced colitis. In conclusion, our data provide evidence that 4‐1BB−4‐1BBL interaction modulates the effector CD4+ T cell‐driven immune response and cytokine production in experimental colitis without affecting regulatory T cell function.

Inhalative occupational and ingestive immediate‐type allergy caused by chicory (Cichorium intybus)

We report a first case of occupational allergy to chicory (Cichorium intybus) in a vegetable wholesaler. Symptoms occurred after oral, cutaneous or inhalatory exposure. The patient also reported reactions after ingestion of botanically related endive (Cichorium endivia) and lettuce (Lactuca sativa.) We identified the responsible allergen by SDS‐PAGE and immunoblot to be a 48‐kDa protein, confined to the non‐illuminated parts of the plants. No cross‐reactivity was found with mugwort (Artemisia vulgaris) ryegrass (Lolium perenne) and birch (Betula verrucosa) pollen, which suggests that the vegetable is the primary allergenic material.

Influence of the pH of the extraction medium on the composition of birch (Betula verrucosa) pollen extracts.

Extracts from birch (Betula verrucosa) pollen were prepared at different pH, with constant pH monitoring and adjustment to preset values in the range 5.5‐8.5. The total protein content of these extracts was directly correlated with the pH. Coomassie brilliant blue‐stained isoelectric focusing and SDS‐PAGE gels and immunoblot analysis demonstrated qualitative differences: some proteins were lost while others appeared when pH was changed. At pH 8.5, formerly unknown birch pollen allergens were detected with pi 9, 9.10, and 9.30 by about 30% of birch pollen‐sensitive sera. Birch pollen extracts prepared at a pH close to neutrality, namely, 6.5 and 7.5, showed the greatest protein and different allergen diversity. Thus, extraction pH values are necessary to analyze the whole pattern of allergenic components in an extract.