Resistance Exercise Restores Endothelial Function and Reduces Blood Pressure in Type 1 Diabetic Rats

Background: Resistance exercise effects on cardiovascular parameters are not consistent. Objectives: The effects of resistance exercise on changes in blood glucose, blood pressure and vascular reactivity were evaluated in diabetic rats. Methods: Wistar rats were divided into three groups: control group (n = 8); sedentary diabetic (n = 8); and trained diabetic (n = 8). Resistance exercise was carried out in a squat device for rats and consisted of three sets of ten repetitions with an intensity of 50%, three times per week, for eight weeks. Changes in vascular reactivity were evaluated in superior mesenteric artery rings. Results: A significant reduction in the maximum response of acetylcholine-induced relaxation was observed in the sedentary diabetic group (78.1 ± 2%) and an increase in the trained diabetic group (95 ± 3%) without changing potency. In the presence of NG-nitro-L-arginine methyl ester, the acetylcholine-induced relaxation was significantly reduced in the control and trained diabetic groups, but not in the sedentary diabetic group. Furthermore, a significant increase (p < 0.05) in mean arterial blood pressure was observed in the sedentary diabetic group (104.9 ± 5 to 126.7 ± 5 mmHg) as compared to that in the control group. However, the trained diabetic group showed a significant decrease (p < 0.05) in the mean arterial blood pressure levels (126.7 ± 5 to 105.1 ± 4 mmHg) as compared to the sedentary diabetic group. Conclusions: Resistance exercise could restore endothelial function and prevent an increase in arterial blood pressure in type 1 diabetic rats.

Combination Therapy for the Cardiovascular Effects of Perinatal Lead Exposure in Young and Adult Rats

Background: Combination therapy can play a significant role in the amelioration of several toxic effects of lead (Pb) and recovery from associated cardiovascular changes. Objective: To investigate the effects of combination therapy on the cardiovascular effects of perinatal lead exposure in young and adult rats Methods: Female Wistar rats received drinking water with or without 500 ppm of Pb during pregnancy and lactation. Twenty-two- and 70-day-old rat offspring who were or were not exposed to Pb in the perinatal period received meso-dimercaptosuccinic acid (DMSA), L-arginine, or enalapril and a combination of these compounds for 30 additional days. Noradrenaline response curves were plotted for intact and denuded aortas from 23-, 52-, 70-, and 100-day-old rats stratified by perinatal Pb exposure (exposed/unexposed) and treatment received (treated/untreated). Results: Systolic blood pressure was evaluated and shown to be higher in the 23-, 52-, 70-, and 100-day age groups with Pb exposure than in the corresponding control age groups: 117.8 ± 3.9*, 135.2 ± 1.3*, 139.6 ± 1.6*, and 131.7 ± 2.8*, respectively and 107.1 ± 1.8, 118.8 ± 2.1, 126.1 ± 1.1, and 120.5 ± 2.2, respectively (p < 0.05). Increased reactivity to noradrenaline was observed in intact, but not denuded, aortas from 52-, 70-, and 100-day-old exposed rats, and the maximum responses (g of tension) in the respective Pb-exposed and control age groups were as follows: 3.43 ± 0.16*, 4.32 ± 0.18*, and 4.21 ± 0.23*, respectively and 2.38 ± 0.33, 3.37 ± 0.13, and 3.22 ± 0.21, respectively (p < 0.05). Conclusions: All treatments reversed the changes in vascular reactivity to noradrenaline in rats perinatally exposed to Pb. The combination therapy resulted in an earlier restoration of blood pressure in Pb-exposed rats compared with the monotherapies, except for enalapril therapy in young rats. These findings represent a new approach to the development of therapeutic protocols for the treatment of Pb-induced hypertension.

Effects of One Resistance Exercise Session on Vascular Smooth Muscle of Hypertensive Rats

AbstractBackground:Hypertension is a public health problem and increases the incidence of cardiovascular diseases.Objective:To evaluate the effects of a resistance exercise session on the contractile and relaxing mechanisms of vascular smooth muscle in mesenteric arteries of NG-nitro L-arginine methyl ester (L-NAME)-induced hypertensive rats.Methods:Wistar rats were divided into three groups: control (C), hypertensive (H), and exercised hypertensive (EH). Hypertension was induced by administration of 20 mg/kg of L-NAME for 7 days prior to experimental protocols. The resistance exercise protocol consisted of 10 sets of 10 repetitions and intensity of 40% of one repetition maximum. The reactivity of vascular smooth muscle was evaluated by concentration‑response curves to phenylephrine (PHEN), potassium chloride (KCl) and sodium nitroprusside (SNP).Results:Rats treated with L-NAME showed an increase (p < 0.001) in systolic blood pressure (SBP), diastolic blood pressure (DBP) and mean arterial pressure (MAP) compared to the initial period of induction. No difference in PHEN sensitivity was observed between groups H and EH. Acute resistance exercise reduced (p < 0.001) the contractile response induced by KCl at concentrations of 40 and 60 mM in group EH. Greater (p < 0.01) smooth muscle sensitivity to NPS was observed in group EH as compared to group H.Conclusion:One resistance exercise session reduces the contractile response induced by KCl in addition to increasing the sensitivity of smooth muscle to NO in mesenteric arteries of hypertensive rats.

Acute endotoxemia inhibits microvascular nitric oxide-dependent vasodilation in humans.

Nitric oxide (NO) is crucial for the microvascular homeostasis, but its role played in the microvascular alterations during sepsis remains controversial. We investigated NO-dependent vasodilation in the skin microcirculation and plasma levels of asymmetric dimethylarginine (ADMA), a potent endogenous inhibitor of the NO synthases, in a human model of sepsis. In this double-blind, randomized, crossover study, microvascular NO-dependent (local thermal hyperemia) and NO-independent vasodilation (post-occlusive reactive hyperemia) assessed by laser Doppler imaging, plasma levels of ADMA, and l-arginine were measured in seven healthy obese volunteers, immediately before and 4 h after either a i.v. bolus injection of Escherichia coli endotoxin (LPS; 2 ng/kg) or normal saline (placebo) on two different visits at least 2 weeks apart. LPS caused the expected systemic effects, including increases in heart rate (+43%, P < 0.001), cardiac output (+16%, P < 0.01), and rectal temperature (+1.4°C, P < 0.001), without change in arterial blood pressure. LPS affected neither baseline skin blood flow nor post-occlusive reactive hyperemia but decreased the NO-dependent local thermal hyperemia response, l-arginine, and, to a lesser extent, ADMA plasma levels. The changes in NO-dependent vasodilation were not correlated with the corresponding changes in the plasma levels of ADMA, l-arginine, or the l-arginine/ADMA ratio. Our results show for the first time that experimental endotoxemia in humans causes a specific decrease in endothelial NO-dependent vasodilation in the microcirculation, which cannot be explained by a change in ADMA levels. Microvascular NO deficiency might be responsible for the heterogeneity of tissue perfusion observed in sepsis and could be a therapeutic target.

Extracellular serine-proteinases isolated from Streptomyces alboniger: Partial characterization and effect of aprotinin on cellular structure

Streptomyces alboniger ATCC 12461 grown in brain heart infusion (BHI) medium produced two extracellular serine-proteinases, denoted SP I and SP II, which were purified by ammonium sulfate precipitation and aprotinin-agarose affinity chromatography. SP I was purified 88,9-fold and SP II 66,7- fold, with 33.4% and 10.4% yield, respectively. The optimum pH for the proteinases activity, using a-N-p-tosyl-L-arginine-methyl ester (TAME) as substrate, was 9-10 and the optimum temperature was 37ºC. The proteolytic activity of SP I and SP II was inhibited by aprotinin and SP I was partially inhibited by leupeptin, both serine-proteinase inhibitors. S. alboniger growth in BHI-liquid medium decreased when 5 mg/ml, 10 mg/ml of aprotinin was used, being completely inhibited with 20 mg/ml and 40 mg/ml. At the ultrastructural level, aprotinin-treated S. alboniger cells showed swelling of the bacterial body and condensation of the genetic material, probably related to the inhibition of its growth.

Hypoxic contraction of small pulmonary arteries from normal and endotoxemic rats: fundamental role of NO.

The present study was aimed at examining the role of nitric oxide (NO) in the hypoxic contraction of isolated small pulmonary arteries (SPA) in the rat. Animals were treated with either saline (sham experiments) or Escherichia coli lipolysaccharide [LPS, to obtain expression of the inducible NO synthase (iNOS) in the lung] and killed 4 h later. SPA (300- to 600-micrometer outer diameter) were mounted as rings in organ chambers for the recording of isometric tension, precontracted with PGF2alpha, and exposed to either severe (bath PO2 8 +/- 3 mmHg) or milder (21 +/- 3 mmHg) hypoxia. In SPA from sham-treated rats, contractions elicited by severe hypoxia were completely suppressed by either endothelium removal or preincubation with an NOS inhibitor [NG-nitro-L-arginine methyl ester (L-NAME), 10(-3) M]. In SPA from LPS-treated rats, contractions elicited by severe hypoxia occurred irrespective of the presence or absence of endothelium and were largely suppressed by L-NAME. The milder hypoxia elicited no increase in vascular tone. These results indicate an essential role of NO in the hypoxic contractions of precontracted rat SPA. The endothelium independence of HPV in arteries from LPS-treated animals appears related to the extraendothelial expression of iNOS. The severe degree of hypoxia required to elicit any contraction is consistent with a mechanism of reduced NO production caused by a limited availability of O2 as a substrate for NOS.

Subcellular localization of an intracellular serine protease of 68 kDa in Leishmania (Leishmania) amazonensis promastigotes

Here we report the subcellular localization of an intracellular serine protease of 68 kDa in axenic promastigotes of Leishmania (Leishmania) amazonensis, using subcellular fractionation, enzymatic assays, immunoblotting, and immunocytochemistry. All fractions were evaluated by transmission electron microscopy and the serine protease activity was measured during the cell fractionation procedure using a-N-r-tosyl-L-arginine methyl ester (L-TAME) as substrate, phenylmethylsulphone fluoride (PMSF) and L-1-tosylamino-2-phenylethylchloromethylketone (TPCK) as specific inhibitors. The enzymatic activity was detected mainly in a membranous vesicular fraction (6.5-fold enrichment relative to the whole homogenate), but also in a crude plasma membrane fraction (2.0-fold). Analysis by SDS-PAGE gelatin under reducing conditions demonstrated that the major proteolytic activity was found in a 68 kDa protein in all fractions studied. A protein with identical molecular weight was also recognized in immunoblots by a polyclonal antibody against serine protease (anti-SP), with higher immunoreactivity in the vesicular fraction. Electron microscopic immunolocalization using the same polyclonal antibody showed the enzyme present at the cell surface, as well as in cytoplasmic membranous compartments of the parasite. Our findings indicate that the internal location of this serine protease in L. amazonensis is mainly restricted to the membranes of intracellular compartments resembling endocytic/exocytic elements.

mitoKATP channel activation in the postanoxic developing heart protects E-C coupling via NO-, ROS-, and PKC-dependent pathways.

Whereas previous studies have shown that opening of the mitochondrial ATP-sensitive K(+) (mitoK(ATP)) channel protects the adult heart against ischemia-reperfusion injury, it remains to be established whether this mechanism also operates in the developing heart. Isolated spontaneously beating hearts from 4-day-old chick embryos were subjected to 30 min of anoxia followed by 60 min of reoxygenation. The chrono-, dromo-, and inotropic disturbances, as well as alterations of the electromechanical delay (EMD), reflecting excitation-contraction (E-C) coupling, were investigated. Production of reactive oxygen species (ROS) in the ventricle was determined using the intracellular fluorescent probe 2',7'-dichlorofluorescin (DCFH). Effects of the specific mitoK(ATP) channel opener diazoxide (Diazo, 50 microM) or the blocker 5-hydroxydecanoate (5-HD, 500 microM), the nitric oxide synthase (NOS) inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME, 50 microM), the antioxidant N-(2-mercaptopropionyl)glycine (MPG, 1 mM), and the PKC inhibitor chelerythrine (Chel, 5 microM) on oxidative stress and postanoxic functional recovery were determined. Under normoxia, the baseline parameters were not altered by any of these pharmacological agents, alone or in combination. During the first 20 min of postanoxic reoxygenation, Diazo doubled the peak of ROS production and, interestingly, accelerated recovery of ventricular EMD and the PR interval. Diazo-induced ROS production was suppressed by 5-HD, MPG, or L-NAME, but not by Chel. Protection of ventricular EMD by Diazo was abolished by 5-HD, MPG, L-NAME, or Chel, whereas protection of the PR interval was abolished by L-NAME exclusively. Thus pharmacological opening of the mitoK(ATP) channel selectively improves postanoxic recovery of cell-to-cell communication and ventricular E-C coupling. Although the NO-, ROS-, and PKC-dependent pathways also seem to be involved in this cardioprotection, their interrelation in the developing heart can differ markedly from that in the adult myocardium.

Skin hyperemia induced by local heating : why is it blunted on repeat stimulations ?

Background: In human skin, local heating produces local vasodilatation, a response termed thermal hyperemia. Thermal hyperemia is largely mediated by nitric oxide (NO). It is blunted on repeat stimulations applied to the same skin spot, a phenomenon termed desensitization. As this phenomenon could reflect a desensitization in the vasodilator effects of NO, we investigated whether a prior exposure to exogenous NO would result in an attenuated vasodilatory response to a subsequent thermal challenge. Methods: Thirteen healthy young men were studied. Skin blood flow (SkBF) was mesured on forearm skin with laser Doppler imaging. Exposure to exogenous NO was carried out by iontophoresis of sodium nitroprusside (SNP), a donor of NO. A local thermal stimulus (temperature step from 34 to 41°C maintained for 30 minutes) was applied with temperature-controlled chambers. We tested the influence of a previous transient exposure to exogenous NO on : 1) thermal hyperemia and 2) the response to a second identical exposure to exogeneous NO. Results: Thermal hyperemia (plateau SkBF at 30 minutes minus SkBF at 34°C) obtained on a site preexposed to exogenous NO two hours before was lower than obtained on a site preexposed to iontophoretic current only (mean±SD 395±139 perfusion units [PU] vs 540±79 PU ; p<0.01). When repeated on the same skin site two hours after the first one, exposure to exogenous NO led to a blunted vasodilatory response (298±121 PU vs 394±92 PU), although this difference was not statistically significant (p≈0.09). Conclusion: In forearm human skin, prior exposure to exogenous NO partially inhibits thermal hyperemia. These data support that desensitization of thermal hyperemia depends on a downregulation of the NO-cGMP pathway, possibly downstream from the endogenous production of NO.

Synthesis and transport of creatine in the CNS: importance for cerebral functions.

J. Neurochem. (2010) 10.1111/j.1471-4159.2010.06935.x Abstract Apart of its well known function of 'energetic buffer' through the creatine/phosphocreatine/creatine kinase system allowing the regeneration of ATP, creatine has been recently suggested as a potential neuromodulator of even true neurotransmitter. Moreover, the recent discovery of primary creatine deficiency syndromes, due to deficiencies in l-arginine : glycine amidinotransferase or guanidinoacetate methyltransferase (the two enzymes allowing creatine synthesis) or in the creatine transporter, has shed new light on creatine synthesis, metabolism and transport, in particular in CNS which appears as the main tissue affected by these creatine deficiencies. Recent data suggest that creatine can cross blood-brain barrier but only with a poor efficiency, and that the brain must ensure parts of its needs in creatine by its own endogenous synthesis. Finally, the recent years have demonstrated the interest to use creatine as a neuroprotective agent in a growing number of neurodegenerative diseases, including Parkinson's and Huntington's diseases. This article aims at reviewing the latest data on creatine metabolism and transport in the brain, in relation to creatine deficiencies and to the potential use of creatine as neuroprotective molecule. Emphasis is also given to the importance of creatine for cerebral function.

Metallothionein-1 and nitric oxide expression are inversely correlated in a murine model of Chagas disease

Chagas disease, caused by Trypanosoma cruzi, represents an endemic among Latin America countries. The participation of free radicals, especially nitric oxide (NO), has been demonstrated in the pathophysiology of seropositive individuals with T. cruzi. In Chagas disease, increased NO contributes to the development of cardiomyopathy and megacolon. Metallothioneins (MTs) are efficient free radicals scavengers of NO in vitro and in vivo. Here, we developed a murine model of the chronic phase of Chagas disease using endemic T. cruzi RyCH1 in BALB/c mice, which were divided into four groups: infected non-treated (Inf), infected N-monomethyl-L-arginine treated (Inf L-NAME), non-infected L-NAME treated and non-infected vehicle-treated. We determined blood parasitaemia and NO levels, the extent of parasite nests in tissues and liver MT-I expression levels. It was observed that NO levels were increasing in Inf mice in a time-dependent manner. Inf L-NAME mice had fewer T. cruzi nests in cardiac and skeletal muscle with decreased blood NO levels at day 135 post infection. This affect was negatively correlated with an increase of MT-I expression (r = -0.8462, p < 0.0001). In conclusion, we determined that in Chagas disease, an unknown inhibitory mechanism reduces MT-I expression, allowing augmented NO levels.

Chronic nitric oxide synthase inhibition and carotid artery distensibility in renal hypertensive rats.

The goal of the present study was to examine the viscoelastic properties of the carotid artery in genetically identical rats exposed to similar levels of blood pressure sustained by different mechanisms. Eight-week old male Wistar rats were examined 2 weeks after renal artery clipping (two-kidney, one clip [2K1C] Goldblatt rats, n = 53) or sham operation (n = 49). One half of the 2K1C and sham rats received the nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME, 1.48 mmol/L) in their drinking water for 2 weeks after the surgical procedure. Mean blood pressure increased significantly in the 2K1C-water (182 mm Hg), 2K1C-L-NAME (197 mm Hg), and sham-L-NAME (170 mm Hg) rats compared with the sham-water rats (127 mm Hg). Plasma renin activity was not altered by L-NAME but significantly enhanced after renal artery clipping. A significant and similar increase in the cross-sectional area of the carotid artery was observed in L-NAME and vehicle-treated 2K1C rats. L-NAME per se did not modify cross-sectional area in the sham rats. There was a significant upward shift of the distensibility-pressure curve in the L-NAME- and vehicle-treated 2K1C rats compared with the sham-L-NAME rats. L-NAME treatment did not alter the distensibility-pressure curve in the 2K1C rats. These results demonstrate that the mechanisms responsible for artery wall hypertrophy in renovascular hypertension are accompanied by an increase in arterial distensibility that is not dependent on the synthesis of nitric oxide.

3-amino-1,2,4-triazole inhibits macrophage NO synthase.

Murine macrophages activated by interferon-gamma and lipopolysaccharide become leishmanicidal through a process involving L-arginine-derived nitrogen oxidation products. Both nitrite secretion and parasite killing by activated macrophages were inhibited by 3-amino-1,2,4-triazole as well as the related compound, 3-amino-1,2,4-triazine. Moreover, NO synthase activity in cytosolic extracts of activated cells was inhibited by both compounds. 4-amino-1,2,4-triazole, an isomer of 3-amino-1,2,4-triazole, was without effect. Our results suggest that besides its known inhibitory effect on catalases and peroxidases, 3-amino-1,2,4-triazole is an inhibitor of NO synthase. The resemblance between the tautomeric form of 3-amino-1,2,4-triazole and the guanidino group of L-arginine, the natural substrate for NO synthase, might be responsible for the observed inhibition.

Study of the proteolytic activity of the paracaspase MALT1 in T cell activation

Summary : Several signalling cascades are initiated through the triggering of the T cell receptor (TCR) by an antigenic peptide expressed at the surface of an antigen presenting cell. These pathways lead to morphological changes controlling T cell adhesiveness and migration to the site of infection, and to the activation of transcription factors that regulate key genes for the proper development of the immune response. Amongst them, the nuclear factor xB (NF-κB) is the subject of intense research since more than twenty years because deregulated NF-κB signalling in lymphocytes can lead to immunodeficiency, autoimmunity or lymphomas. Therefore, the understanding of the molecular mechanisms regulating NF-κB activation is important for the development of new therapeutics aimed at treating various diseases. In T lymphocytes, a complex composed of CARMAI, BCL10 and MALT1 relays signals from TCR proximal events to NF-κB activation. Gene translocations of the BCL10 or MALTI genes or oncogenic mutations affecting CARNA 1 result in constitutive NF-κB activation and are related to the development of certain forms of lymphomas. MALT1 contains acaspase-like domain, but it is unknown whether this domain is proteolytically active. In this study, we found that MALT1 has arginine-directed proteolytic activity. We showed that the proteolytic activity of MALT 1 is key to TCR-induced NF-κB activation and production of interleukin 2. We identified BCL 10 as a MALT 1 substrate, and we showed that its cleavage regulates T cell adhesion to the extracellular matrix protein fibronectin. Furthermore, we identified caspase 10 as another substrate of MALT1. caspase 10 is a close homologue of caspase 8 and is known to be involved in the induction of apoptosis upon Fast or TRAIL stimulation. We showed that caspase 10 is important for TCR-induced NF-κB activation and interleukin 2 production, identifying for the first time a non apoptotic function for caspase 10. These data provide evidence for previously uncharacterized roles of MALT 1 and BCL 10 in the regulation of T cell adhesion and of caspase 10 in the activation of lymphocytes, and allow a better understanding of the molecular mechanisms of T lymphocyte activation. Since the proteolytic activity of MALT1 is essential to T cell activation, it suggests that the targeting of this activity may be relevant for the development of immunomodulatory or anticancer drugs. Résumé : De nombreuses voies de signalisation sont initiées via la stimulation des récepteurs des cellules T (TCR) par un peptide antigénique exprimé à la surface d'une cellule présentatrice d'antigènes. Ces cascades de signalisation produisent des changements morphologiques qui contrôlent l'adhésion des cellules T et leur migration vers le site d'infection. Elles contrôlent également l'activation de facteurs de transcription qui régulent la transcription de gènes importants pour la réponse immunitaire. Parmi ces derniers, le facteur nucléaire KB (NF-κB) joue un rôle essentiel, puisqu'une régulation aberrante de son activité dans les lymphocytes peut causer des immunodéficiences, des maladies autoimmunes ou des lymphomes. C'est pour cela que la compréhension des mécanismes moléculaires qui contrôlent l'activation de NF-κB est donc importante pour le développement de nouvelles thérapies. Un complexe contenant les protéines CAIZMAI, BCL10 et MALT1 transmet, dans les lymphocytes T, le signal du TCR vers l'activation de NF-κB. Des translocations des gènes qui codent pour BCL10 et MALTI et des mutations affectant la fonction de CARNAI ont été liées au développement de certaines formes de lymphomes. MALTI contient un domaine qui ressemble au domaine catalytique présent dans les caspases, mais il n'est pas connu si ce domaine a une activité protéolytique. Dans cette étude, nous avons découvert que MALTI est une protéase qui a une spécificité pour les acides aminés basiques comme l'arginine. Nous montrons que l'activité protéolytique de MALTI est importante pour l'activation de NF-κB et la production d'interleukine 2 après stimulation du TCR. Nous avons observé que BCL10 est clivé par MALTI pendant l'activation des lymphocytes T, et que ce clivage est impliqué dans la régulation de l'adhésion des lymphocytes T à la fibronectin, une protéine de la matrice extracellulaire. De plus, nous avons identifié que la caspase 10, qui a une grande homologie avec la caspase 8 et qui jusqu'à maintenant est connue pour son rôle dans l'induction de la mort cellulaire en réponse à une stimulation par Fast ou par TRAIL, est également un substrat de MALT 1. En montrant que la caspase 10 est nécessaire à l' activation de NF-icB et à la production de l'interleukine 2 après stimulation du TCR, nous décrivons pour la première fois une fonction non apoptotique de la caspase 10. Ces résultats décrivent de nouveaux rôles pour MALT1 et BCL10 dans le contrôle de l'adhésion des lymphocytes T et de la caspase 10 pour l'activation des lymphocytes T. Puisque l'activité protéolytique de MALT1 est essentielle pour l'activation des lymphocytes T, nous suggérons que cibler cette activité protéolytique de MALT 1 pourrait amener de nouvelles possibilités de traitement de maladies où une activation aberrante des lymphocytes est impliquée.

Arginase as a marker of disease severity in human leishmaniasis

The leishmaniases are a group of diseases transmitted by the bite of Leishmania infected female phlebotomine sand flies. The diseases occur in different forms: localized, diffuse and muco-cutaneous leishmaniasis, and visceral leishmaniasis (VL). Inside macrophages, the main host cells of the obligate intracellular Leishmania parasites, nitric oxide synthase and arginase can regulate parasite killing or growth. In experimental leishmaniasis, we previously reported that non-healing disease is associated with higher arginase activity at site of pathology, correlating with local suppression of T cell function. To test whether these data translate to human leishmaniasis, the following study was initiated: I first tested the hypothesis that local suppression of T cell responses observed in persistent CL is associated with arginase induced L-arginine depletion. The results showed that arginase activity is increased at site of pathology compared to peripheral blood mononuclear cells (PBMCs) of LCL patients and intact skin of healthy controls. The phenotype of arginase expressing cells was identified in both compartments as CD15+ CD14|0W low-density granulocytes (LDGs). Finally, high arginase activity at site of pathology observed in cutaneous lesions of patients coincides with downregulation of CD3Ç, CD4 and CD8 molecules in CD4+ and CD8+ T cells at site of pathology. We concluded that increased arginase levels in lesions of LCL patients might contribute to CL pathogenesis by impairing T cell effector function at site of pathology. Next, it was tested whether arginase, an enzyme associated with immunosuppression, is higher in patients with VL and contributes to impaired T cell function through depletion of L- arginine. The results showed that higher level of arginase activity in the PBMC coincides with active phase of VL. Cells expressing arginase in PBMCs were also found to be LDGs. Importantly, increased arginase activity and frequency of degranulated neutrophils coincided with lower plasma L-arginine levels. Furthermore, downregulation of CD3Ç, in T cells correlated with low plasma arginine levels. VL/HIV co-infection is a frequently reported leishmaniasis complication in Ethiopia associated with poor prognosis, with up to 40% mortality rate and high relapse rate. Arginase activity was significantly increased in PBMCs and plasma of VL patients co-infected with HIV than in those having VL alone. Similarly, cells expressing arginase in PBMCs were found to be LDGs. In summary, the results presented here show that increased arginase activity is a marker of disease severity in human leishmaniasis with and without HIV; further, these results suggest that arginase mediated L-arginine depletion may inhibit T cell function and contribute to impaired control of infection. - Les leishmanioses sont un groupe de maladies transmises par la piqûre de mouches des sables femelles, appelées phlébotomes, ayant été infectées par Leishmania. Les maladies se manifestent sous différentes formes: la leishmaniose cutanée localisée, la leishmaniose diffuse et mucocutanée et la leishmaniose viscérale (LV). A l'intérieur des macrophages, les principales cellules hôtes des parasites, l'oxyde nitrique synthase et l'arginase, peuvent contrôler, soit la mort du parasite, soit sa croissance. Pour la leishmaniose expérimentale, nous avons déjà rapporté que le développement de lesions qui ne guérissent pas est associé à une activité plus grande d'arginase au site d'infection, en corrélation avec la suppression locale de la fonction des cellules T. Pour vérifier si ces données pouvaient s'appliquer à la leishmaniose humaine, j'ai d'abord vérifié l'hypothèse selon laquelle la suppression locale des réponses des cellules T observée dans la CL persistante, est associée à la la diminution de L- arginine induite par l'arginase. Les résultats ont montré que l'activité arginase est augmentée au site d'infection, par rapport aux cellules mononucléées du sang périphérique (CMSP) de patients LCL et à la peau intacte des contrôles sains. Le phénotype de cellules exprimant l'arginase a été identifié dans les deux compartiments comme des granulocytes CD15+ et CD 14" de basse densité (LDG). Enfin, l'activité arginase élevée au site de la pathologie, observée dans les lésions cutanées de patients, coïncide avec la reduction dde l'expression des molécules CD3Ç, CD4 et CD8 dans les cellules T CD4+ et CD8+ au site de pathologie . Nous avons conclu que l'augmentation des niveaux d'arginase dans les lésions de patients LCL pourrait contribuer à la pathogenèse de la CL, en altérant la fonction effectrice des celllules T au site de la pathologie. Ensuite, nous avons vérifié si l'arginase, une enzyme associée à l'immunosuppression, était plus élevée chez les patients atteints de VL et si elle contribuait à la mauvaise fonction des cellules T par la depletion en L-arginine. Les résultats ont montré qu'un niveau plus élevé de l'activité arginase dans les PBMC correspond à la phase active de la VL. Les cellules exprimant l'arginase dans les CMSP se sont révélées à être de type LDG . Il est important de souligner que l'augmentation de l'activité arginase et la fréquence des neutrophiles dégranulés a coïncidé avec des niveaux inférieurs de L-arginine plasmatique. En outre, la suppression de CD3Ç dans les cellules T correlle avec de faibles niveaux d'arginine plasmatique . Il a été fréquement rapporté que la co-infection VL/VIH est une complication de la leishmaniose en Ethiopie, associée à un mauvais prognostic, un taux de mortalité pouvant atteindre 40% et un pourcentage élevé de rechutes. L'activité de l'arginase a beaucoup plus augmentée dans les CMSP et le plasma de patients atteints de VL et co-infectés par le VIH, que chez ceux seulement attaints de VL. De même, les cellules exprimant l'arginase dans les CMSP sont aussi des LDG. En résumé, les résultats présentés ici montrent que l'augmentation de l'activité de l'arginase est un marqueur de gravité de la la leishmaniose humaine, avec ou sans VIH ; en outre, ces résultats suggèrent que la déplétion de L-arginine par l'arginase pourrait inhiber la fonction des cellules T et contribuer à un contrôle réduit de l'infection. - Les Leishmanioses sont des maladies parasitaires transmises par la piqûre d'une mouche des sables femelle (phlébotome) infectée par Leishmania. La maladie se manifeste sous différentes formes cliniques : la leishmaniose viscérale, une maladie progressive mortelle en l'absence de traitement, la leishmaniose muco-cutanée (MCL), la leishmaniose cutanée diffuse (LCD ) maladie mutilante, qui peut être de longue durée et la leishmaniose cutanée localisée maladie dont on guérit mais laissant une cicatrice inesthétique à vie. La maladie est largement répandue, elle affecte les populations les plus pauvres dans 98 pays et 350 millions de personnes à risque. Globalement on estime à 500.000 les nouveaux cas de la forme viscérale et 1-1.5 million ceux de la leishmaniose cutanée. La leishmaniose est fortement endémique en Ethiopie et se manifeste dans les formes viscérale et cutanée. Le parasite Leishmania infecte et se multiplie dans les cellules du système immunitaire, principalement les macrophages. Les macrophages sont capables de tuer le parasite Leishmania s'ils reçoivent des instructions correctes de la part d'autres cellules du système immunitaire, les lymphocytes. Les macrophages expriment deux enzymes importants, appelés oxide nitrique synthase inductible (iNOS ) et l'arginase, qui sont respectivement associés à la promotion de la mort du parasite et la multiplication. L'enzyme iNOS présent dans les macrophages métabolise l'arginine afin de générer de l'oxyde d'azote (NO) , une molécule effectrice nécessaire pour tuer le parasite . Au contraire, lorsque les macrophages sont activés d'une certaine manière conduisant à l'augmention de la régulation de l'arginase, ils métabolisent l'arginine en polyamines qui favorisent la croissance du parasite. Au cours du développement de la leishmaniose, les lymphocytes ne parviennent pas à transmettre aux macrophages les signaux nécessaires pour tuer le parasite. Les mécanismes cellulaires qui sont la cause de ce défaut, ne sont pas bien compris. En utilisant des modèles animaux, nous avons montré la régulation à la hausse de l'arginase au site de la pathologie, qui s'est traduit par l'altération de la fonction effectrice des lymphoctes. Nous avons initié des études de leishmaniose humaine en Ethiopie afin d'identifier le rôle de l'arginase dans la sévérité de la maladie. Nos résultats montrent, que l'arginase est fortement augmentée dans la lésion des patients CL, et dans le sang des patients VL et ceux co-infectés par VL / VIH. Le niveau d' arginase régulée à la hausse coincide avec l'expression inférieure d'une molécule de signalisation dans les lymphocytes, qui est essentielle à leur bon fonctionnement. En VL actif, l'augmentation d'arginase se traduit par la diminution de l'arginine qui est indispensable à la synthèse de NO et au bon fonctionnement des lymphocytes. Ainsi, l'incapacité des lymphocytes à envoyer des signaux adéquats aux macrophages pourrait être due à la suppression de l'arginine.