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.

Magnesium deficiency reveals the neurotoxicity of delta-9-tetrahydrocannabinol (THC) low doses in rats.

In the present study, muricide behaviour (MB) was studied in Long Evans rats in various situations. The MB pattern of each experimental group was compared, in 6 successive assays 1 hr-delayed to that of natural killer rats (NK). The percentage of NK rats was 11% in the strain used. In the 11 mg THC/kg b.w. treated naive rats, a significant additional percentage of rats (59%) became muricidal. The durations of the 3 MB phases were significantly increased as a result of an increased aggressiveness in the 1st assay but returned progressively to NK values on the 6th assay, in parallel with the physiological elimination of THC. This result indicates a true killing training in those non killer rats that became muricidal under THC. A severe magnesium deficiency induced by a 50 ppm magnesium-deficient diet induced 100% MB whereas a 150 ppm magnesium deficiency did not induce additional MB. In the severe deficiency, the MB pattern was rather similar to that of NK with the exception of the attack on the living mouse which was doubled probably because of magnesium-induced hyperexcitability responsible for a lower attack efficiency. In both 50 but also 150 ppm magnesium-deficient rats, a single injection of THC at low doses (2, 4 or 8 mg THC/kg b.w.) which is without aggressive effect in control rats, induced a 100% MB, the pattern of which was all the more severe as the magnesium deficiency was important or the THC dose higher. The pattern showed an important decrease in the two first phases and a dramatic increase in the attack on the dead mouse, suggesting that the combination of both treatments provoked severe central damage with a compulsive killing behavior. Consequently, it appears that a magnesium deficiency, even a moderate one, may aggravate the neurotoxicity of THC at low doses and, reciprocally, that low doses of THC may reveal the potential neurotoxicity of a moderate magnesium deficiency.

Comparison of a short irradiation (50 sec) by different wavelengths on audiogenic seizures in magnesium-deficient mice: evidence for a preventive neuroprotective effect of yellow.

Audiogenic seizures triggered by an acoustic stimulus of determined frequency and amplitude have been described in many laboratory animals in many circumstances including magnesium deficiency. This model, recently validated, was used, in DBA/2 mice, to study the preventive neuroprotective effect of 6 wavelengths of the visible spectrum used in Chromatotherapia* (lambda(max) 440, 484, 528, 572, 616 and 660 nm) at low irradiance. Each short illumination lasted 50 seconds and was followed by 20 minutes of darkness. It appeared that yellow fully protected 16 out of 17 mice from seizure occurrence. Green allowed the survival of 69% of mice but did not protect them from seizure occurrence. On the contrary, the other four colors (orange, red, purple and blue) failed to protect the mice and showed a tendency to accelerate their death. White color was not protective but allowed the difficult survival of 30% of mice. Darkness had no protective effect. These results even though surprising open a great field of investigation.

Structural alterations of the vascular wall in magnesium-deficient mice. A possible role of gelatinases A (MMP-2) and B (MMP-9).

Vascular alterations during magnesium deficiency have long been known but the implicated mechanisms have so far been poorly documented. In this preliminary assay, we compared the thoracic aortic histology in Swiss OF1 mice fed a severe magnesium-deficient diet (50 +/- 5 ppm) for 42 days to that of controls fed a standard diet (1700 +/- 100 ppm magnesium). It appeared (eosin-haematoxylin coloration) that, in magnesium-deficient mice, the aortic wall was thinner than in controls. Specific colorations of the two of main fibers vascular tissue (collagens and elastin) showed severe structural alterations of both components. These changes were consecutive to the expression of matrix metalloproteinases (MMP) -2 and -9 which were present as zymogens (inactive forms) in controls and supposed to be present in their active and inactive forms in magnesium-deficient mice (zymography). These changes which have not been reported so far would explain, at least in part, the sensitivity of magnesium-deficient mice to various stress or xenobiotics.

Synthesis and anticonvulsant and neurotoxic properties of substituted N-phenyl derivatives of the phthalimide pharmacophore.

A series of compounds including 4-amino (1), 3-amino (2), 4-nitro (3), 2-methyl-3-amino (4), 2-methyl-3-nitro (5), 2-methyl-4-amino (6), 2-methyl-4-nitro (7), 2-methyl-5-amino (8), 2-methyl-5-nitro (9), 2-methyl-6-amino (10), 2-methyl-6-nitro (11), 2,6-dimethyl (12), 2-methyl-3-carboxy (13), 2-methoxycarbonyl (14), 2-methyl-4-methoxy (15), 2,4-dimethoxy (16), 2-chloro-4-amino (17), and 2-chloro-4-nitro (18) N-phenyl substituents of phthalimide were evaluated along with N-[3-methyl-(2-pyridinyl)]phthalimide (19), N-(3-amino-2-methylphenyl)succinimide (20), and phenytoin for anticonvulsant and neurotoxic properties. Initial screening in the intraperitoneal (ip) maximal electroshock-induced seizure (MES) test and the subcutaneous pentylenetetrazol-induced seizure (scPtz) test in mice led to the selection of 1, 2, 4, 10, 12, 17, and 19 for oral MES evaluation in rats. The resultant ED(50) values for 4, 10, 17, and phenytoin were 8.0, 28.3, 5.7 and 29.8 mg/kg, respectively. In the batrachotoxin affinity assay, IC(50) values for 17 and phenytoin were 0.15 and 0.93 microM, respectively, and in the recently validated magnesium deficiency-dependent audiogenic seizure test, ED(50) values of 5.2 and 23 mg/kg were obtained for 17 and phenytoin, respectively. Electrophysiology studies on compound 17 point out its ability to (i) potentiate GABA-evoked current responses with a failure to directly activate the GABAA receptor and (ii) to affect, at 100 microM excitatory non NMDA, but not NMDA, receptors with a 25% block of kainate-evoked response. Electrophysiology measurements on voltage-gated sodium channels in N1E-115 neuroblastoma cells confirm voltage-dependent block of these channels by compound 17. In view of its interaction with multiple ion channels, one would predict that compound 17 might be active in a wide range of seizure models.

Magnesium deficiency-dependent audiogenic seizures (MDDASs) in adult mice: a nutritional model for discriminatory screening of anticonvulsant drugs and original assessment of neuroprotection properties.

A great many animal models for audiogenic seizures have been described. The extent to which these models may provide insight into neuroscience fields such as abnormal locomotor behavior (wild running), seizures and anticonvulsants, and neuroinsults and neuroprotectors is examined here by our study of magnesium deficiency-dependent audiogenic seizures (MDDASs) in adult mice. MDDASs were induced in all of the eight tested adult murine strains and are presented as a sequence of four successive components (latency, wild running, convulsion, and recovery phase periods). Compared with several classic seizure tests, the nutritional MDDAS model responded to low doses of prototype antiepileptic drugs (AEDs), including phenytoin (PHT), carbamazepine (CBZ), phenobarbital (PB), valproic acid (VPA), ethosuximide (ESM), and diazepam (DZP). Modulation by AEDs of the four components of MDDAS indicated that this seizure test was discriminatory, distinguishing between phenytoinergic (PHT, CBZ), GABAergic (PB, VPA, DZP), and ethosuximide (ESM) compounds. Suitability of the MDDAS test for evaluation of neuroprotective compounds was also examined: it showed partial (melatonin) and complete (WEB2170, an anti-PAF agent) reduction of recovery phase by non-anticonvulsant doses of test compounds. These neuroprotective responses were compared with neuroprotective potentials determined in a model of neonatal cerebral injury induced by focal injection of ibotenate (a glutamate analog). WEB2170 and melatonin reduced the size of lesions in white matter, but only WEB2170 protected cortical plate against ibotenate-induced lesions. In addition to the original neuroprotective behavior of WEB2170, studies on the neuroprotectors also supported GABAergic anticonvulsant activity of melatonin in the MDDAS test.

Effect of dietary magnesium on the susceptibility of mice to infection by protozoan parasites of the Apicomplexa and Mastigophora phyla.

Severe magnesium deficiency protects mice against infections by Plasmodium Spp. and Babesia hylomysci which invade mature erythrocytes. By contrast severe magnesium deficiency does not protect against parasite infections by P. berghei which invades reticulocytes, Toxoplasma gondii which invades macrophages, and Trypanosoma brucei which lives free in blood. The results indicate that the infectious response depends on the severity of magnesium deficiency and on the parasite species. The decrease in red blood cell magnesium and increased oxidant stress are possible explanations for the protective effect of magnesium deficiency.

Magnesium deficiency protects against Babesia hylomysci and mice become resistant to rechallenge with the parasite regardless of diet fed.

Mice were fed diets containing 960 mg (control), 100 mg (moderately Mg deficient) and 30 mg (severely Mg deficient) of Mg/kg. After 20 days, mice were inoculated with Babesia hylomysci (from Dr. Wery, Anvers, Belgium). Significant increases in RBC Mg levels were observed following infection. All the control and moderately deficient mice died from infection, whereas the severely Mg-deficient diet protected mice against infection, as shown by a decrease in parasitaemia and mortality. The decrease in RBC Mg, modifications in membrane properties and increased oxidant stress are possible explanations for the protective effect of severe Mg deficiency. When mice were maintained for 2 months after inoculation on a severely Mg-deficient diet and were then switched to a control diet, all survived and had low parasitaemias. After 1 month, these mice were rechallenged with B. hylomysci and 89% survived.

Magnesium deficiency affects malaria susceptibility in mice.

One hundred twenty mice were fed control and magnesium-(Mg) deficient diets containing 960 and 50 mg Mg/kg, respectively. After 12 days, mice were inoculated with several strains of Plasmodium (P). Parasitemias and survivals were monitored for 20 days after infection. The Mg-deficient diet protected mice against the nonlethal parasite P. chabaudi as shown by decreased parasitemia. All control mice infected with P. vinckei died from the infection. Mg-deficient mice had a much lower parasitemia and recovered from the infection. In mice infected with the lethal strain P. berghei, the parasitemia showed a biphasic response. In control mice, the first phase resulted in the death of 40% of the animals. After a spontaneous reduction of parasitemia, the surviving animals showed a second increase in parasitemia, which resulted in the death of the remaining animals. In the deficient group, all the animals survived the first phase of infection. By contrast, the evolution of the second phase was similar to that observed in controls. Examination of blood smears showed that the second phase corresponded to an invasion of young reticulocytes by the parasite. The results suggest that the protective effect of Mg deficiency is related to the decrease in erythrocyte Mg and to the prooxidative effect of this mineral deficiency in mature erythrocytes.

Protective effect of severe magnesium deficiency on Plasmodium chabaudi infection.

Mice were fed for 30 days on purified diets containing 50 (severely Mg deficient diet), 100 (moderately Mg deficient diet) and 1300 mg/kg (control diet). An additional group raised on stock UAR diet was also used for the experiment. The mice were maintained on the experimental diets for 12 days before being inoculated with P. chabaudi. Infection evolved similarly in mice fed the control purified diet, moderately Mg deficient diet and the stock diet whereas the severely Mg deficient diet induced a 50% decrease in malarial infection as shown by the decrease in the percentage of parasitized red blood cells (RBC). In control mice, RBC Mg values increased significantly during P. chabaudi infection; however RBC Mg values were significantly lower in Mg-deficient than in control animals.

Mechanism of increased erythrocyte membrane fluidity during magnesium deficiency in weanling rats.

The erythrocyte membrane was investigated in weanling male rats pair fed with magnesium-deficient and control diets for 8 days. Fluorescence polarization studies revealed a 15% increase in the fluidity of membranes from deficient rats. A similar increase in the fluidity of liposomes indicated that protein was not involved. The change was associated with decreased osmotic fragility of intact erythrocytes; the cells lost their biconcavity and had a flattened appearance with surface irregularities. Analysis of the membranes showed decreased amounts of magnesium, cholesterol, and sphingomyelin in the deficient group. The reduced ratios of cholesterol to phospholipid and sphingomyelin to phosphatidylcholine were consistent with the increased fluidity. Addition of physiological amounts of magnesium to the medium rigidified membranes incubated in tris(hydroxymethyl)-aminomethane buffer, and this was prevented by the presence of EDTA. Cross-incubation experiments with erythrocyte ghosts and plasma from the two groups of rats showed that magnesium-deficient plasma increased the fluidity of control ghosts and control plasma rigidified ghosts from magnesium-deficient rats. Addition of sufficient magnesium chloride to raise the magnesium content of deficient plasma to normal had no significant effect. These results show that the increased fluidity of the erythrocyte membrane in magnesium deficiency is due to physicochemical exchange with the plasma. Although magnesium can directly influence membrane fluidity, the change during its deficiency in vivo is mainly mediated indirectly via disturbances in lipid metabolism.

[Histopathology of the liver and kidney during malaria: relation to malaria-induced dyslipoproteinemia]. / Histopathologie du foie et du rein au cours du paludisme: rapports avec la dyslipoprotéinémie palustre.

A kinetic study concerning histologic and cytologic alterations during Plasmodium chabaudi infection of Swiss mice has been carried out. In liver, a reversible focal and non ischemic necrosis and a vascular congestion were observed together with an accumulation of malarial pigment. The endoplasmic reticulum cisternae and Golgi saccules of hepatocytes were highly distended. Hepatocyte microvilli in biliary canaliculi and in Disse' spaces were markedly less developed and less numerous than in normal liver. Intracytoplasmic lipid globules were found in large amount in hepatocytes before the peak of parasitaemia. Their number and size gradually diminished thereafter. Hepatocytic mitochondria showed important unspecific modifications probably in relation, at last partly, to the tissue anoxia. Some hepatocytic changes (intracytoplasmic lipid globules, enlargement of endoplasmic reticulum cisternae and Golgi saccules) were consistent with an increased synthesis of lipoproteins (VLDL). The kidney showed only minor histological and ultrastructural changes. However haemosiderin was observed in proximal tubules and in their bordering cells. The deposit of immune complex reported previously do not appear associated with tissular or cellular important alterations.

Alterations of lecithin-cholesterol acyltransferase activity during Plasmodium chabaudi rodent malaria.

In non fatal and synchronous P. chabaudi rodent malaria, we observed at the stage of parasitaemia peak, an alteration (50 % decrease) in LCAT activity. This decrease could be related partly to hepatic dysfunction, and mainly to circulating inhibitors released into blood from parasitized red blood cells at each end of a schizogonic cycle. This decrease in LCAT activity, at this step of the infection, accounts for part of the dyslipoproteinemia previously observed (i.e., increase in cholesterol and phospholipids into VLDL-LDL and decrease in the EC series and delayed conversion of Tg-rich lipoproteins into LDL-HDL. At a prepatent step of infection and after the parasitaemia peak, the alterations observed in LCAT activity, (respectively, increase and then decrease), would be related to similar changes in levels of cholesterol of HDL associated to complex changes in triacylglyceride transport and metabolism.

Influence of malaria on a pre-existing antibody response to heterologous antigens.

Swiss mice were immunized with various antigens: polyvinylpyrrolidone (PVP), human serum transferrin (HST), bovine serum albumin (BSA) or tetanus toxoid (TT). Fifteen days after the last injection of antigen, the mice were infected with Plasmodium chabaudi AJ. Two weeks after the beginning of malarial infection, parasitaemia became latent and total gammaglobulin levels, as well as IgG and IgM levels, were significantly increased. At that period the antigen-binding capacity (ABC) and the total antigen-binding Sites (Abt) were determined for PVP, HST and BSA. It appeared that the ABC and Abt of anti-HST or anti-BSA antibodies were lower than those of corresponding uninfected controls. With regard to anti-PVP antibodies, only the Abt was modified after infection, but not the ABC. The evolution of anti-TT Ab levels determined by a solid-phase assay with 125I-TT was as above with a marked decrease on and after the 4th week of infection. Injection of low-density lipoproteins (LDL) from day-9-infected mice to TT-immunized mice significantly reduced anti-TT antibody levels. At similar doses, LDL from normal mice did not induce an inhibitory effect. At mouse-equivalent-dose, LDL from infected mice revealed an inhibitory effect compared to LDL from uninfected controls. Our results suggest that LDL from P. chabaudi-infected mice can exert an immunoregulatory role and thus could explain part of the immune impairment observed during the infective process. Moreover, from the present data it might be postulated that the hypergammaglobulinaemia of P. chabaudi infection does not result from a parasite-potentiating effect on a pre-existing antibody response.

[Immunoglobulin-lipoprotein complexes during P. chabaudi infection]. / Complexes immunoglobulines-lipoproteines dans l'infection par Plasmodium chabaudi.

Immunoglobulin-lipoprotein complexes have been investigated in the course of P. chabaudi acute infection of Swiss mice. Serum ultracentrifugation in a sucrose density gradient was used to demonstrate the presence of immunoglobulins in the enriched lipoprotein fraction of density lower than 1100. The levels of lipoprotein-bound immunoglobulins (Ig-Lp) were measured using the rate-nephelometric Immuno-Chemistry-System. IgM-Lp and IgG-Lp were significantly increased at day 9 and reached a peak at day 13 post-infection. From day 13 to day 16 they dramatically decreased. This kinetic effect was similar to that of non-specific circulating immune complexes detected by the [125I]Clq binding assay. A radioimmuno-precipitation-PEG-assay (RIPEGAssay) with [125I]Lp revealed the highest Lp precipitation with day 13 post-infection mouse sera compared to controls. Similar results were recorded when control sera were 8 times as concentrated in order to obtain Ig levels identical in both control and experimental sera. Part of the lipoprotein-bound immunoglobulins were demonstrated to be anti-Lp antibodies by the way of the RIPEGAssay using F(ab')2 fragments from infected mouse sera. Moreover, the [125I]Clq bound to the Ig-Lp complexes isolated from day 13 mouse sera. The present data suggest that the alteration of lipid metabolism which characterizes malaria infection could lead to the complexation of lipoproteins to immunoglobulins.

Adipose tissue lipoprotein lipase activity in Plasmodium chabaudi and Plasmodium vinckei rodent malaria.

Adipose tissue lipoprotein lipase activity (LPL) falls dramatically during experimental infection of mice by Plasmodium chabaudi. The decrease in this activity accounts for the seric accumulation of triacyl-glyceride-rich lipoproteins (chylomicrons mainly and Very Low Density Lipoproteins (VLDL) partly). This loss of tissue LPL enzyme activity is moreover enhanced, in capillary vessels, by a simultaneous decrease in the activatory power of serum towards the enzyme. This phenomenon is mainly related to the presence of Very Low Density Lipoproteins. In the mouse VLDL apoprotein composition is supposed to be modified. On the contrary, in fatal infection of mice by P. vinckei the adipose tissue lipoprotein lipase (LPL) activity is normal or even increased.

Metabolism of lipoproteins in rodent malaria, relationship between lipolysis, steatosis and increased biosynthesis of V.L.D.L.

The kinetic study of the seric free fatty acids, total lipids and hepatic triacyglycerides had led us to conclude that the biosynthesis of T.A.G.-rich lipoproteins increases during malaria. It seems that the parasite induces a lipolysis of adipose tissue in order to meet its own needs for fatty acids and that the excess of the latter taken by the liver involves an increased synthesis of the V.L.D.L. The cis-vaccenic acid has also been analysed during the evolution of parasitaemia; these variations by themselves cannot explain the extra parasitic hemolysis.

[Morphology and infectivity of gametocytes of Plasmodium inui]. / Morphologie et infectivité des gamétocytes de Plasmodium inui.

The authors report biomorphological changes of Plasmodium inui gametocytes during the natural infection of spleenless Macaca fascicularis. The infection was controlled up to the 42th day by smears produced by pricks into their ears (P.O.) and on blood taken at the same time by A. stephensi (P.M.). The first oocystes appeared on the 8th day after the infection in mosquitoes fed on the monkey parasitised in the eight day after splenectomy, as gametocytaemia was not detectable yet in the P.O. smears. The infectivity of gametocytes is highest on the 13rd day for a 152/10(4) parasitaemia. The first sporozoites appeared on the 18th day at 25 degrees C after the infecting meal. The sporozoites were infecting for a second monkey. Four morphological types (O, I, II and III) gametocytes were identified. They corresponded to the same types previously described in rodent Plasmodium. The maximum of infectivity coincided with a sudden increase in mosquitoe macrogametocytaemia; on the other hand the infectivity was very weak during the following peek of macrogametocytaemia which corresponded however to the maximum of parasitaemia (1 318/10(4) 18 days after splenectomy). When infectivity was important, there was a greater number of type O and I gametocytes in P.M. than in P.O. Eventually, the behaviour of P. inui gametocytes is, for the mainly, almost the same as that of rodent Plasmodium. However, it is to be noted that the identification of the 4 morphological types in P. inui is easier in macrogametocytes than in microgametocytes. The reverse had been observed in rodent Plasmodium.