[Potentiation of Delta9-tetrahydrocannabinol (THC) effects by magnesium deficiency in the rat]. / Potentialisation des effets du Delta9-tétrahydrocannabinol (THC) par la carence magnésique chez le rat.

Aggressive behavior can be classified into three major categories: defense, social attack and predatory behavior. The predatory behavior of rats, which prompts them to prey on mice (muricidal behavior) may be induced by injection of high doses (11 mg/kg) of Delta9-tetrahydrocannabinol (THC) or by acute magnesium deficiency. We have studied the effect of a single injection of low doses of THC (2, 4 or 8 mg/kg) in rats with a severe (50 ppm magnesium diet) or moderate (150 ppm) magnesium deficiency. The combination of moderate magnesium deficiency with low doses of THC induced muricidal behavior in all the rats and an increase in aggressiveness at the doses of 4 or 8 mg/kg of THC. Hyperaggressiveness increased with magnesium deficiency severity. Serotonin is probably involved in aggressiveness induced by both moderate magnesium deficiency and low doses of THC, but implication of other neurotransmitters and magnesium deficiency-induced alterations of CB1-and/or CB2-receptor expression are not excluded.

Effect of Mg-deficiency on endothelial cell allogeneic activation in a model of isolated perfused mouse lung.

Following vascularized organ allotransplantation, an early intragraft inflammatory process is initiated by adhesion molecule-ligand interaction between recipient blood leukocytes and graft endothelial cells (EC). We have previously shown that chronic hypomagnesemia did not induce any inflammatory process in the lung, hence neither EC activation, nor lung remodelling. In the present study we have investigated the effects of allogeneic blood perfusion on lungs from magnesium-deficient mice in our experimental model of isolated mouse lung. After 3h of isogeneic or allogeneic perfusion, no inflammatory process was detected by histochemical examination of lung tissue; the mRNA levels of the adhesion molecules E-selectin, ICAM-1 and VCAM-1, and of the pro-inflammatory cytokines TNF-alpha and IL-2 in lung tissue, determined by reverse transcriptase-polymerase chain reaction (RT-PCR), were similar, and the expression of E-selectin and I-Ab antigen on EC by immunohistochemical staining was undetectable. All of these markers were shown to be dramatically increased after allogeneic perfusion of lung from magnesium-non deficient mice. Our results clearly show that allogeneic perfusion of lungs from magnesium-deficient mice cannot induce EC activation or lung inflammation, indicating that hypomagnesemia in donors does not constitute an additional risk for allograft outcome and might allow to lighten the recipient's immunosuppressive treatment.

Severe Mg-deficiency is not associated with endothelial cell activation in mouse lung.

Several experimental and clinical studies suggest that the lungs are a specific target of Mg-hypomagnesemia, which is a common side effect of cyclosporin A therapy. Due to the possible effect of hypomagnesemia on lung allograft function, the aim of this study was to evaluate endothelial cell (EC) activation and tissue remodelling (apoptosis) in the lungs from mice fed Mg-deficient diets. Immunocytochemical examinations did not reveal any inflammatory process in Mg-deficient mice, infiltration of leukocytes (CD45+ cells), expression of I-Ab class II molecules, E-selectin or ICAM-1 on ECs, and apoptotic cells. Quantification of mRNAs for E-selectin, ICAM-1 and VCAM-1, which are the most pertinent adhesins expressed by ECs, and for the cytokines TNFalpha and IL-2, demonstrated that severe Mg-deficiency does not result in EC activation. The balance between the up-regulation of G-CSF-R and CCL4 genes, and the down-regulation of the OPN gene shown by the cDNA microarray technique might be responsible for the absence of development of an inflammatory response, lung EC activation, and lung remodelling. However, we can hypothesize that severe Mg deficiency results in a latent inflammatory status of the lungs, which might be expressed following immune stresses, like transplantation conditions.

A new actimetry-based test of photic sensitization in a murine photosensitive magnesium depletion model.

Photic hypersensitivity may induce various signs of nervous hypersensitivity, including diurnal cephalalgias, anxiety, dyssomnia, seizures, fatigue and/or myalgias. The patients usually present both dishabituation and generalization in response to repetitive light stimuli instead of habituation as in normal subjects. These clinical manifestations appear when light intensity is maximum (daytime; spring and summer) in magnesium-depleted patients with hypofunction of the biological clock. The best photic hypersensitivity management involves darkness therapy, either darkness per se or darkness-mimicking agents. To detect efficiently the best drugs that may be used in the treatment of disorders due to photosensitive magnesium depletion, we are proposing a simple and reproducible actimetry-based test in a murine photosensitive magnesium depletion model. Photostimulation using a stroboscope (100 J, 50 Hz) was performed on magnesium-deficient and control mice. It led to habituation with a decreased activity in response to intermittent light stimulation in control mice, whereas it induced in magnesium-deficient mice both sensitization (or potentiation), with nervous hyperexcitability, and generalization, involving sound hypersensitivity, after visual stimulation. In preclinical evaluation, this test provides a valuable animal model to study the neuroprotective effect of drugs in photosensitive syndromes, which often associate sensitization and generalization to various stimuli.

Cytokine deviation induced by intrathymic injection of staphylococcal enterotoxin B (SEB).

We have previously shown that intrathymic (i.t.) injection of staphylococcal enterotoxin B (SEB) to mice induces both T cell clonal deletion and IL-2-dependent anergy. In the present study, we have used a quantitative RT-PCR to demonstrate that i.t. administration of SEB induced a significant decrease in the levels of the IL-2 and IFN-gamma mRNAs in total splenocytes, from day 7 to day 28 post-injection. I.t. SEB injection also induced a significant increase in the levels both of IL-10 and TGF-beta mRNAs on day 7, leading to a significant enhance in the IL-10 + TGF-beta/IL-2 + IFN-gamma mRNA ratio on days 7 and 28. By contrast, IL-10 and TGF-beta mRNAs were unchanged after intraperitoneal (i.p.) or subcutaneous (s.c.) SEB injections, although both IL-2 and IFN-gamma mRNA levels were decreased. The cytokine mRNA ratio was enhanced on days 7 and 28 after i.p. injection. Interestingly, a cytokine mRNA ratio of a least 10 in favour of IL-10 plus TGF-beta mRNAs was correlated with the hyporesponsive state observed in vitro after i.t. and i.p. injections. Our results clearly demonstrate that i.t. SEB administration induces a switch from Th1-type to Th2-type cytokine expression in the spleen. The deviation from IL-2 plus IFN-gamma towards IL-10 plus TGF-beta expression could be responsible for the immunoregulatory effect exerted upon SEB-reactive T cells, which is characterized by an IL-2-dependent, specific anergy in vitro. Moreover, it highlights the crucial role of the route of SEB injection in the pattern of cytokine expression.

Kinetic analysis of microchimerism induced by intrathymic injection of allogeneic splenocytes in mice.

Allograft survival facilitated by intrathymic (i.t.) injection of allogeneic cells have shown that modifications of T-cell development induce specific tolerance. One hypothesis is that the resulting microchimerism may play a role in preparing the host immune system for the allograft. To investigate whether the deliberate introduction of allogeneic splenocytes into the thymus of adult mice allows the establishment of a lasting donor/recipient microchimerism, a full allogeneic mouse system (H-2 and Mls) with additional sex mismatch was used. Male cells injected into female mice were detected using an optimized nested-polymerase chain reaction which specifically amplifies the SRY gene with a sensitivity of 1/10(4). After i.t. injection, donor cells were observed early both in the lymph nodes and spleen (75 and 25% of mice, respectively). They were still present on day 6, although preferentially in the thymus (100% of mice) than in the lymph nodes (50% of mice) or in the spleen (22% of mice). After intraperitoneal (i.p.) or subcutaneous (s.c.) injection, donor cells were early (2 h) but transiently detected in the thymus, since on day 6 they were detected in 0 and 17% of mice after i.p. and s.c. injection, respectively. Kinetics of donor-cell detection was similar both in the spleen and lymph nodes with a clear decrease in the percentage of mice with donor-cell detection between day 2 and day 6 (20 and 17% of positive mice for the spleen after i.p. and s.c. injections, respectively--20 and 33% of positive mice for the lymph nodes after i.p. and s.c. injections, respectively). Our results clearly show that i.t. injection of allogeneic splenocytes induces a microchimerism which is both more lasting and detected in a higher percentage of mice than by the i.p. and s.c. routes, both at the central (thymus) and peripheral (spleen) levels.

Intrapulmonary production of RANTES during rejection and CMV pneumonitis after lung transplantation.

RANTES (regulated upon activation, normally T expressed and secreted) is a chemoattractant for macrophages, memory T lymphocytes, and eosinophils. We investigated whether intrapulmonary production of the chemokine RANTES contributes to the recruitment of immune cells during lung transplantation complications. RANTES concentration was measured in bronchoalveolar lavage (BAL) fluids using an ELISA assay. It was significantly higher during CMV pneumonitis (36.2 +/- l6 pg/ml, n=12, P=0.031) and allograft rejection (31.1 +/- 8.5 pg/ml, n=27, P=0.013) than in patients without complications (9.1 +/- 2.3 pg/ml, n=22). At least some of the RANTES was produced by lung macrophages: BAL macrophages cultured for 24 hr spontaneously released larger amount of RANTES during CMV pneumonitis (140 +/- 53 pg/ml, n=8, P=0.002) and allograft rejection (84 +/- 44 pg/ml, n=11, P=0.037) than in control patients (15.2 +/- 6.5 pg/ml, n=21). Moreover, macrophages in transbronchial biopsies were labeled by an anti-RANTES mAb. RANTES production by BAL macrophages was followed in 2 patients with CMV pneumonitis. It remained high as long as CMV-induced cytopathic effects or clinical symptoms were present, but it returned to baseline as the infection was controlled. These results suggest that the intrapulmonary production of the chemokine RANTES by activated macrophages contributes to the intrapulmonary accumulation of immune cells during complications of lung transplantation.

Experimental tracheal and tracheoesophageal allotransplantation. Paris-Sud University Lung Transplantation Group.

We investigated the effects of allograft perfusion with a preservative technique and of combined thyrotracheoesophageal implantation on airway epithelium of long segments of thyrotracheal grafts allotransplanted on their own vascular pedicles into immunosuppressed pigs. Four groups of five animals each underwent heterotopic (into the neck) thyrotracheal (group 1) and thyrotracheoesophageal (group 2) and orthotopic thyrotracheal (group 3) and thyrotracheoesophageal (group 4) allotransplantation. Allograft revascularization included (1) interposition of donor right subclavian artery--incorporating the inferior thyroid artery--to recipient right carotid artery (end-to-end fashion) and (2) end-to-side anastomosis of donor anterior vena cava to recipient right external jugular vein. All thyrotracheoesophageal blocks were harvested after inferior thyroid artery perfusion with 4 degrees C Euro-Collins solution. The overall lengths of tracheal and esophageal grafts were 10.7 +/- 2.7 cm and 13.4 +/- 3.6 cm, respectively. In the heterotopic groups, all allografts were viable and histologically normal at postmortem examination and the incidence and severity of airway ischemia and rejections (at equal residual levels of cyclosporine) were not different between groups 1 and 2. In the orthotopic groups, the first two pigs died of airway collapse with histologically normal grafts. In the remaining pigs, temporary airway stenting was inserted and allografts remained viable and histologically intact for their entire length 30 days after transplantation. Transplanted tracheal smooth muscles had concentration-dependent contractions and relaxations similar to those of nontransplanted (native) tracheas. This study documents the feasibility of allotransplanting long tracheal and esophageal segments on their own vascular pedicles and demonstrates that allograft preservation and thyrotracheoesophageal transplantation are equally effective in minimizing airway ischemia. Thyrotracheoesophageal transplantation does not enhance recipient alloimmune response compared with thyrotracheal transplantation alone.

Heterotopic pig model for direct revascularization and venous drainage of tracheal allografts. Paris-Sud University Lung Transplantation Group.

A macrosurgical technique of thyrotracheal harvesting and direct revascularization with and without venous drainage in a heterotopic thyrotracheal and immunosuppressed allograft in the pig model is described. Harvesting included en bloc cervicothoracic exenteration of the aortic arch and its supraortic trunks, anterior vena cava, jugular veins, subclavian vessels, thyroid gland, cervicothoracic trachea, and esophagus. This technique conserves the tracheal arterial supply provided by either the right or left subclavian artery, directly or indirectly via the inferior thyroid artery, and venous return provided by the anterior vena cava, directly or indirectly via the descending cervical vein. In recipients, implantation included (1) arterial end-to-end anastomoses of the proximal and postscalenic stumps of donor's subclavian artery to the proximal and prescalenic stumps of recipient's subclavian artery; (2) end-to-side venous anastomosis of the donor's anterior vena cava to the recipient's brachiocephalic venous trunk; and (3) heterotopic implantation of the proximal and distal orifices of the grafted trachea into the neck. Ten adult Large White pigs underwent direct revascularization of a thyrotracheal allograft with (n = 6, group 1) and without (n = 4, group 2) venous drainage. All grafts of group 2 exhibited a venous infarction, extensive inferior thyroid artery thrombosis, and ischemic and suppurative thyrotracheal necrosis 1 to 2 days after transplantation. In group 1, the length of the grafted trachea and number of rings were 9.75 +/- 1.5 cm and 22.1 +/- 3.3, respectively; ischemic time was 236.3 +/- 338.3 minutes. Group 1 pigs were put to death 4 (n = 4) and 3 (n =2) weeks after transplantation. All tracheal grafts had histologically normal airway epithelium; isolated areas of necrotic ischemia of the chorion and submucosa lasted for the first 7 days after transplantation but disappeared after epithelial regeneration. Premortem angiograms showed that all vascular anastomoses were patent. Grafts were histologically normal at postmortem examinations and all but one had no rejection. This large animal model demonstrates that long tracheal allografts might be transplanted by means of this direct revascularization and venous drainage technique.

Transforming growth factor beta in normal human lung: preferential location in bronchial epithelial cells.

BACKGROUND: Transforming growth factor beta (TGF-beta) is an immunomodulatory cytokine regulating the proliferation and differentiation of various cell types. It also contributes to the maintenance of tissue architecture by influencing the production of extracellular matrix components. TGF-beta has been detected in bronchoalveolar lavage fluid from normal human lung, but the nature and distribution of cells containing TGF-beta in this organ remain unknown. METHODS: Fourteen normal human lung specimens were studied by immunohistochemistry with a monoclonal antibody recognizing TGF-beta 1, TGF-beta 2 and TGF-beta 3. RESULTS: TGF-beta was detected in all cases. Bronchial epithelial cells contained the largest amounts of TGF-beta. In these cells the staining was brightest at the apical pole. Macrophages and smooth muscle cells also contained TGF-beta, although less than epithelial cells. No TGF-beta was detected in other cell populations, including endothelial cells, fibroblasts, and pneumocytes. CONCLUSIONS: The bronchial epithelial compartment appears to be the main location of TGF-beta in the normal human lung, suggesting that this cytokine has a pivotal role in the immunological properties of the bronchial mucosa.

Effect of fusafungine on adherence of Haemophilus influenzae type b to human epithelial cells in vitro.

Haemophilus influenzae, a normal host of the nasopharynx of humans, may become a pathogen. The first step of infection is adherence to epithelial cells of the nasopharynx through glycopeptidic adhesins, or pili. Adherence to human epithelial cells in continuous lines, HeLa and Hep2, of 8 piliated strains of Haemophilus influenzae isolated from human infections of the respiratory tract was studied in vitro in the presence of fusafungine, a local bacteriostatic antibiotic. When the bacteria were grown in the presence of 0.5 x the MIC, fusafungine afforded 45-75% of adherence inhibition, but this inhibitory effect did not parallel the MICs. In contrast, no significant effect could be observed either when epithelial cells were exposed to 0.5 x the MIC before use in the adherence assay, or when this assay was performed in the presence of 0.5 x the MIC of fusafungine. The partial adherence inhibition observed suggests that fusafungine interacts with the bacterial binding sites but that other mechanisms may contribute to the inhibitory process. This effect of fusafungine should prevent but not eradicate colonization of the nasopharyngeal mucosa by Haemophilus influenzae and may account for the therapeutic efficacy reported in infections of the respiratory tract due to Haemophilus influenzae.

[In vitro evaluation of antimicrobial activity of fusafungine]. / Evaluation in vitro de l'activité antimicrobienne de la fusafungine.

Fusafungine is an antibiotic extracted from the fungus Fusarium laterium WR strain 437. The antimicrobial activity of fusafungine was determined on strains from laboratory collections and clinical isolates; since fusafungine is not soluble in the classical media, the usual techniques had to be modified. MICs for all the Gram-positive cocci and bacteria tested, aerobic, or anaerobic, were below 30 mcg/ml. The antimicrobial activity included Mycoplasma pneumoniae (MIC less than 18 mcg/ml) and Streptococcus mutans (MIC less than 30 mcg/ml). An antifungal activity was shown for most of the Candida albicans tested (MIC less than 32 mcg/ml) and for different Nocardia sp. (MIC less than 13 mcg/ml). Moreover, fusafungine induced neither acquired resistance nor cross-resistance towards the antibiotics classically used in therapy.

Immunity to tetanus: tetanus antitoxin and anti-BIIb in human sera.

Immunity to tetanus was investigated in 157 individuals aged between 1 and 77 years using, for the evaluation of both tetanus antitoxin and antibody to fragment BIIb (anti-BIIb), an easily performed ELISA that gave reproducible results. Among these people 72% were protected against tetanus (tetanus antitoxin titres greater than 0.06 IU ml-1). Anti-BIIb titres greater than 0.15 U ml-1 were found in 75% of the males vs 57% of the females (P less than 0.02) with marked variations according to age. Furthermore, 13 out of 41 individuals with antitoxin titres less than 0.06 IU ml-1 (not protected against tetanus) were found to have anti-BIIb titres greater than 0.15 U ml-1. These data raise questions as to the significance and protective effect of anti-BIIb against tetanus.

Relation between protective potency and specificity of antibodies in sera of tetanus immunized individuals.

Out of 157 human sera analyzed for antitetanus antibody content by ELISA, 13 turned out to contain only anti-BIIb antibodies, of which 8 proved to be neutralizing. Of these, the 3 sera 303, 306 and 312 together with a commercially available standard preparation of human antitetanus immunoglobulins were further analyzed as to their antibody composition by ELISA using plates sensitized with either the toxoid or various tetanus toxin-derived fragments. It was verified that the protective potency of these antisera was related mainly to their anti-BIIb antibody content. Adsorption experiments confirmed that anti-BIIb antibodies were primarily involved in toxin neutralization, although the presence of high levels of both anti-alpha and anti-Ibc antibodies could confer neutralizing capacity on the sera. A rabbit antiserum raised with the BIIb fragment resulted in a neutralizing antiserum that allowed us to calibrate the ELISA with the anti-BIIb antibodies as International Units.