Evaluation of enzyme-linked immunosorbent assay using crude Leishmania and recombinant antigens as a diagnostic marker for canine visceral leishmaniasis

The performances of ELISA assays with different antigen preparations, such as Leishmania amazonensis or L. chagasi lysates and the recombinant antigens rK-39 and rK-26, were compared using sera or eluates from dried blood collected on filter paper to detect anti-Leishmania antibodies in dogs from a visceral leishmaniasis-endemic area in Brazil. Of 115 IFAT-reactive dogs at 1:40 titre, 106 (92.2%) were positive in parasitological exams (skin and/or spleen). These animals were compared to healthy animals (n = 25), negative for IFAT at a titre of 1:40 and parasitological exams. The sensitivities of crude and recombinant antigens were similar and remarkably high for both sera and eluates (97-100%). Specificity was higher than 96% for sera and eluates for different antigens, except for L. chagasi antigen using eluates (88%). Concordance values among the tests were higher either for sera or eluates (J = 0.95-1.00). High concordances were observed between sera and eluates tested with different antigens (kappa = 0.93-0.97). Crude and recombinant antigens identified different clinical phases of canine leishmaniasis. These results show that eluates could be used in canine surveys to identify L. chagasi infection. Recombinant antigens added little when compared to crude antigen in identifying positive dogs. Cross-reactivity with other diseases whose distribution often overlaps VL-endemic areas is a limitation of crude antigen use however.

Identification of a genetic marker associated with the resistance to Schistosoma mansoni infection using random amplified polymorphic DNA analysis

In schistosomiasis, the host/parasite interaction remains not completely understood. Many questions related to the susceptibility of snails to infection by respective trematode still remain unanswered. The control of schistosomiasis requires a good understanding of the host/parasite association. In this work, the susceptibility/resistance to Schistosoma mansoni infection within Biomphalaria alexandrina snails were studied starting one month post infection and continuing thereafter weekly up to 10 weeks after miracidia exposure. Genetic variations between susceptible and resistant strains to Schistosoma infection within B. alexandrina snails using random amplified polymorphic DNA analysis technique were also carried out. The results showed that 39.8% of the examined field snails were resistant, while 60.2% of these snails showed high infection rates.In the resistant genotype snails, OPA-02 primer produced a major low molecular weight marker 430 bp. Among the two snail strains there were interpopulational variations, while the individual specimens from the same snail strain, either susceptible or resistant, record semi-identical genetic bands. Also, the resistant character was ascendant in contrast to a decline in the susceptibility of snails from one generation to the next.

Use of the pyrophosphatase activity as a reliable tonoplast marker in maize roots.

Membranes of maize (Zea mays L., cv LG 11) roots were fractionated by sucrose (in presence or absence of Mg2+) or dextran density gradient centrifugations and the locations of organelles were determined using marker enzymes. Latent UDPase was used as a Golgi marker, catalase for the peroxysomes, cytochrome c oxidase for the mitochondria, UDP-Gal-galactosyltransferase for the amyloplast membranes and NADH-cytochrome c reductase for the ER. Two markers were selected for the plasmalemma, the vanadate-sensitive ATPase and UDP-Glc-sterolglucosyltransferase. The distributions of the PPase and vacuolar ATPase were found to be similar after density gradient centrifugation. The PPase and vacuolar ATPase activities were clearly separated from almost all the other markers tested, however, a partial association of both activities with the ER cannot be completely ruled out. The PPase of maize roots is more active and easier to measure than the vacuolar ATPase and is therefore an excellent candidate for use as a tonoplast marker.

A novel neuro-muscular marker for the heavy subunit of neurofilament proteins, NF-H, and striated muscle myosin of Xenopus laevis.

A novel monoclonal antibody, M7, is described, that reacts on Western blots with the large subunit of the neurofilament triplet proteins (NF-H) and with striated muscle myosin of Xenopus laevis. Enzymatically digested neurofilament and myosin proteins revealed different immunoreactive peptide fragments on Western blots. Therefore, the antibody must react with immunologically related epitopes common to both proteins. Immunohistochemistry showed staining of large and small axons in CNS and PNS, and nerves could be followed into endplate regions of skeletal muscles. These muscles were characterized by a striated immunostaining of the M-lines. Despite the crossreactivity of M7 with NF-H and muscle myosin, this antibody may be a tool to study innervation of muscle fibers, and to define changes in the neuromuscular organization during early development and metamorphosis of tadpoles.

Tenascin-W is a specific marker of glioma-associated blood vessels and stimulates angiogenesis in vitro.

The microenvironment hosting a tumor actively participates in regulating tumor cell proliferation, migration, and invasion. Among the extracellular matrix proteins enriched in the stroma of carcinomas are the tenascin family members tenascin-C and tenascin-W. Whereas tenascin-C overexpression in gliomas is known to correlate with poor prognosis, the status of tenascin-W in brain tumors has not been investigated so far. In the present study, we analyzed protein levels of tenascin-W in 38 human gliomas and found expression of tenascin-W in 80% of the tumor samples, whereas no tenascin-W could be detected in control, nontumoral brain tissues. Double immunohistochemical staining of tenascin-W and von Willebrand factor revealed that tenascin-W is localized around blood vessels, exclusively in tumor samples. In vitro, the presence of tenascin-W increased the proportion of elongated human umbilical vein endothelial cells (HUVECs) and augmented the mean speed of cell migration. Furthermore, tenascin-W triggered sprouting of HUVEC spheroids to a similar extent as the proangiogenic factor tenascin-C. In conclusion, our study identifies tenascin-W as a candidate biomarker for brain tumor angiogenesis that could be used as a molecular target for therapy irrespective of the glioma subtype.-Martina, E., Degen, M., Rüegg, C., Merlo, A., Lino, M. M., Chiquet-Ehrismann, R., Brellier, F. Tenascin-W is a specific marker of glioma-associated blood vessels and stimulates angiogenesis in vitro.

Iron overload, an immunosuppression marker in HIV-infected patients.

Purpose: Iron overload (IO) has been associated with increased cardiovascular risk (CVR) and metabolic syndrome (MS) in the general population; both elevated CVR and MS are frequent in HIV- patients. Our aim was to analyze the prevalence of IO in a cohort of asymptomatic patients with HIV infection, and related factors. Methods: Cross-sectional study of a cohort of HIV outpatients in regular follow-up. Demographic, epidemiological, clinical, analytical and therapeutic data were collected. Patients completed a questionnaire about CVR factors and 10-year CV disease risk estimation (Framingham score), underwent a physical exam, and a fasting blood analysis. IO was defined as a plasma ferritin level higher than 200 m/L in women and 300 m/L in men. Results: 571 patients (446 men, 125 women), with a mean age of 43.2 years, sexual transmission of HIV in 68.5%, median CD4 count 474 cell/μL (IQR: 308-666), and 36.3% Aids cases 86.2% were on antiretroviral therapy (ART), and 74.8% of them had undetectable HIV viral load 14.6% met MS criteria, and mean CVR at 10 years was 6.67%. IO was detected in 11% of cases. Patients with IO were more immunosuppressed (CD4 count 369 vs 483/μL, p<0.0001), presented a higher prevalence of detectable HIV viral load (17.6% vs 8.9%; p<0.005), and of Aids cases (14.9% vs 8.7%; p<0.023), and lower plasma levels of cholesterol, HDLc and LDLc (154 vs 183, 34 vs 43, 93 vs 110 mg/dL, respectively; p<0.0001. In the multivariate analysis, the only related factor was CD4 count <350 cell/μL (OR 2.86, 95% CI 1.6-4.9; p<0.0001). IO was not associated with CVR nor with MS. Conclusions: IO is not uncommon in HIV patients, and it is only related with immunosuppression defined as CD4 count <350 cell/ mL, and in contrast to general population, it is not related with increased CVR nor with MS.

Pancreatic stone protein: a marker of organ failure and outcome in ventilator-associated pneumonia.

Background: Ventilator-associated pneumonia (VAP) is the most common hospital-acquired, life-threatening infection. Poor outcome and health-care costs of nosocomial pneumonia remain a global burden. Currently, physicians rely on their experience to discriminate patients with good and poor outcome. However, standardized prognostic measures might guide medical decisions in the future. Pancreatic stone protein (PSP)/regenerating protein (reg) is associated with inflammation, infection, and other disease-related stimuli. The prognostic value of PSP/reg among critically ill patients is unknown. The aim of this pilot study was to evaluate PSP/reg in VAP.Methods: One hundred one patients with clinically diagnosed VAP were assessed. PSP/reg was retrospectively analyzed using deep-frozen serum samples from VAP onset up to day 7. The main end point was death within 28 days after VAP onset.Results: Serum PSP/reg was associated with the sequential organ failure assessment score from VAP onset (Spearman rank correlation coefficient 0.49 P < .001) up to day 7. PSP/reg levels at VAP onset were elevated in nonsurvivors (n = 20) as compared with survivors (117.0 ng/mL [36.1-295.3] vs 36.3 ng/mL [21.0-124.0] P = .011). The areas under the receiver operating characteristic curves of PSP/reg to predict mortality/survival were 0.69 at VAP onset and 0.76 at day 7. Two PSP/reg cutoffs potentially allow for identification of individuals with a particularly good and poor outcome. Whereas PSP/reg levels below 24 ng/mL at YAP onset were associated with a good chance of survival, levels above 177 ng/mL at day 7 were present in patients with a very poor outcome.Conclusions: Serum PSP/reg is a biomarker related to organ failure and outcome in patients with VAP.

Antinucleosome antibodies as a marker of active proliferative lupus nephritis.

BACKGROUND: Antinucleosome autoantibodies were previously described to be a marker of active lupus nephritis. However, the true prevalence of antinucleosome antibodies at the time of active proliferative lupus nephritis has not been well established. Therefore, the aim of this study is to define the prevalence and diagnostic value of autoantibodies against nucleosomes as a marker for active proliferative lupus nephritis. STUDY DESIGN: Prospective multicenter diagnostic test study. SETTING & PARTICIPANTS: 35 adult patients with systemic lupus erythematosus (SLE) at the time of the renal biopsy showing active class III or IV lupus nephritis compared with 59 control patients with SLE. INDEX TEST: Levels of antinucleosome antibodies and anti-double-stranded DNA (anti-dsDNA) antibodies. REFERENCE TEST: Kidney biopsy findings of class III or IV lupus nephritis at the time of sampling in a study population versus clinically inactive or no nephritis in a control population. RESULTS: Increased concentrations of antinucleosome antibodies were found in 31 of 35 patients (89%) with active proliferative lupus nephritis compared with 47 of 59 control patients (80%) with SLE. No significant difference between the 2 groups with regard to number of positive patients (P = 0.2) or antibody concentrations (P = 0.2) could be found. The area under the receiver operating characteristic curve as a marker of the accuracy of the test in discriminating between proliferative lupus nephritis and inactive/no nephritis in patients with SLE was 0.581 (95% confidence interval, 0.47 to 0.70; P = 0.2). Increased concentrations of anti-dsDNA antibodies were found in 33 of 35 patients (94.3%) with active proliferative lupus nephritis compared with 49 of 58 control patients (84.5%) with SLE (P = 0.2). In patients with proliferative lupus nephritis, significantly higher titers of anti-dsDNA antibodies were detected compared with control patients with SLE (P < 0.001). The area under the receiver operating characteristic curve in discriminating between proliferative lupus nephritis and inactive/no nephritis in patients with SLE was 0.710 (95% confidence interval, 0.60 to 0.82; P < 0.001). CONCLUSIONS: Antinucleosome antibodies have a high prevalence in patients with severe lupus nephritis. However, our data suggest that determining antinucleosome antibodies is of limited help in the distinction of patients with active proliferative lupus nephritis from patients with SLE without active renal disease.

Assessment of particulate exposure and surface characteristics in association with urinary levels of 8-hydroxy-2'-deoxyguanosine, considered as marker of oxidative stress

Epidemiological studies have demonstrated that exposure to fine particles is associated to adverse health effects, including cancer, respiratory and cardiovascular diseases. However, mechanisms by which particles induce health effects remain unclear. According to one of the most investigated hypotheses, particles cause adverse effects through the production of reactive oxygen species (ROS), which are very hazardous compounds able to attack directly biological structures, including the DNA strand or the lipid bilayer of the cells. If the defense mechanisms, constituted of antioxidants, are not able to counter ROS, then these compounds will cause in the body a range of oxidation reactions called "oxidative stress". The aim of the present research project was to better understand mechanisms by which exposure to fine particles induces oxidative stress. The first point of this project was to check whether exposure to high levels of fine particles is directly linked to oxidative stress, and whether this oxidative stress is accompanied by the activation of the defense mechanisms (antioxidants). The second point was to study the role played by the particle surface characteristics in the oxidative stress process. For that purpose, a study was conducted in bus depots with the participation of 40 mechanics. First, occupational exposure to particles (PM4) and to other pollutants (NOx, O3) was measured over a two-day period. Then, urine samples of mechanics were collected in order to measure levels of 8-hydroxy-2'-deoxyguanosine (8OHdG) and antioxidants. 8OHdG is a molecule formed by the oxidation of DNA and allowing to assess the oxidative stress status of the mechanics. Finally, particles were collected on filters, and functional groups located on the particle surface were analyzed in the laboratory using a Knudsen flow reactor. This technique allows not only to quantify functional groups on the particle surface, but also to measure the reaction kinetics. Results obtained during the field campaign in bus depots showed that mechanics were exposed to rather low levels of PM4 (20-85 μg/m3) and of pollutants (NOx: 100-1000 ppb; O3: <15 ppb). However, despite this low exposure, urinary levels of the oxidative stress biomarker (8OHdG) increased significantly for non-smoking workers over a two-day period of shift. This oxidative stress was accompanied by an increase of antioxidants, indicating the activation of defense mechanisms. On the other hand, the analysis of functional groups on the particle surface showed important differences, depending on the workplace, the date and the activities of workers. The particle surface contained simultaneously antagonistic functional groups which did not undergo internal reactions (such as acids and bases), and was usually characterized by a high density of carbonyl functions and a low density of acidic sites. Reaction kinetics measured using the Knudsen flow reactor pointed out fast reactions of oxidizable groups and slow reactions of acidic sites. Several exposure parameters were significantly correlated with the increase of the oxidative stress status: the presence of acidic sites, carbonyl functions and oxidizable groups on the particle surface; reaction kinetics of functional groups on the particle surface; particulate iron and copper concentrations; and NOx concentration.

Marked increase of the astrocytic marker S100B in the cerebrospinal fluid of HIV-infected patients on LPV/r-monotherapy.

OBJECTIVE: To determine changes of cerebrospinal fluid (CSF) biomarkers of patients on monotherapy with lopinavir/ritonavir. DESIGN: The Monotherapy Switzerland/Thailand study (MOST) trial compared monotherapy with ritonavir-boosted lopinavir with continued therapy. The trial was prematurely stopped due to virological failure in six patients on monotherapy. It, thus, offers a unique opportunity to assess brain markers in the early stage of HIV virological escape. METHODS: : Sixty-five CSF samples (34 on continued therapy and 31 on monotherapy) from 49 HIV-positive patients enrolled in MOST. Using enzyme-linked immunosorbent assay, we determined the CSF concentration of S100B (astrocytosis), neopterin (inflammation), total Tau (tTau), phosphorylated Tau (pTau), and amyloid-β 1-42 (Aβ), the latter three indicating neuronal damage. Controls were CSF samples of 29 HIV-negative patients with Alzheimer dementia. RESULTS: In the CSF of monotherapy, concentrations of S100B and neopterin were significantly higher than in continued therapy (P = 0.006 and P = 0.013, respectively) and Alzheimer dementia patients (P < 0.0001 and P = 0.0005, respectively). In Alzheimer dementia, concentration of Aβ was lower than in monotherapy (P = 0.005) and continued therapy (P = 0.016) and concentrations of tTau were higher than in monotherapy (P = 0.019) and continued therapy (P = 0.001). There was no difference in pTau among the three groups. After removal of the 16 CSF with detectable viral load in the blood and/or CSF, only S100B remained significantly higher in monotherapy than in the two other groups. CONCLUSION: Despite full viral load-suppression in blood and CSF, antiretroviral monotherapy with lopinavir/ritonavir can raise CSF levels of S100B, suggesting astrocytic damage.

Pancreatic stone protein as a novel marker for neonatal sepsis.

PURPOSE: Early-onset sepsis (EOS) is one of the main causes for the admission of newborns to the neonatal intensive care unit. However, traditional infection markers are poor diagnostic markers of EOS. Pancreatic stone protein (PSP) is a promising sepsis marker in adults. The aim of this study was to investigate whether determining PSP improves the diagnosis of EOS in comparison with other infection markers. METHODS: This was a prospective multicentre study involving 137 infants with a gestational age of >34 weeks who were admitted with suspected EOS. PSP, procalcitonin (PCT), soluble human triggering receptor expressed on myeloid cells-1 (sTREM-1), macrophage migration inhibitory factor (MIF) and C-reactive protein (CRP) were measured at admission. Receiver-operating characteristic (ROC) curve analysis was performed. RESULTS: The level of PSP in infected infants was significantly higher than that in uninfected ones (median 11.3 vs. 7.5 ng/ml, respectively; p = 0.001). The ROC area under the curve was 0.69 [95 % confidence interval (CI) 0.59-0.80; p < 0.001] for PSP, 0.77 (95 % CI 0.66-0.87; p < 0.001) for PCT, 0.66 (95 % CI 0.55-0.77; p = 0.006) for CRP, 0.62 (0.51-0.73; p = 0.055) for sTREM-1 and 0.54 (0.41-0.67; p = 0.54) for MIF. PSP independently of PCT predicted EOS (p < 0.001), and the use of both markers concomitantly significantly increased the ability to diagnose EOS. A bioscore combining PSP (>9 ng/ml) and PCT (>2 ng/ml) was the best predictor of EOS (0.83; 95 % CI 0.74-0.93; p < 0.001) and resulted in a negative predictive value of 100 % and a positive predictive value of 71 %. CONCLUSIONS: In this prospective study, the diagnostic performance of PSP and PCT was superior to that of traditional markers and a combination bioscore improved the diagnosis of sepsis. Our findings suggest that PSP is a valuable biomarker in combination with PCT in EOS.

Use of the frc gene as a molecular marker to characterize oxalate-oxidizing bacterial abundance and diversity structure in soil.

Oxalate catabolism, which can have both medical and environmental implications, is performed by phylogenetically diverse bacteria. The formyl-CoA-transferase gene was chosen as a molecular marker of the oxalotrophic function. Degenerated primers were deduced from an alignment of frc gene sequences available in databases. The specificity of primers was tested on a variety of frc-containing and frc-lacking bacteria. The frc-primers were then used to develop PCR-DGGE and real-time SybrGreen PCR assays in soils containing various amounts of oxalate. Some PCR products from pure cultures and from soil samples were cloned and sequenced. Data were used to generate a phylogenetic tree showing that environmental PCR products belonged to the target physiological group. The extent of diversity visualised on DGGE pattern was higher for soil samples containing carbonate resulting from oxalate catabolism. Moreover, the amount of frc gene copies in the investigated soils was detected in the range of 1.64x10(7) to 1.75x10(8)/g of dry soil under oxalogenic tree (representing 0.5 to 1.2% of total 16S rRNA gene copies), whereas the number of frc gene copies in the reference soil was 6.4x10(6) (or 0.2% of 16S rRNA gene copies). This indicates that oxalotrophic bacteria are numerous and widespread in soils and that a relationship exists between the presence of the oxalogenic trees Milicia excelsa and Afzelia africana and the relative abundance of oxalotrophic guilds in the total bacterial communities. This is obviously related to the accomplishment of the oxalate-carbonate pathway, which explains the alkalinization and calcium carbonate accumulation occurring below these trees in an otherwise acidic soil. The molecular tools developed in this study will allow in-depth understanding of the functional implication of these bacteria on carbonate accumulation as a way of atmospheric CO(2) sequestration.

Evaluation of adding a second marker to overcome Staphylococcus aureus spa typing homoplasies.

The utility of sequencing a second highly variable locus in addition to the spa gene (e.g., double-locus sequence typing [DLST]) was investigated to overcome limitations of a Staphylococcus aureus single-locus typing method. Although adding a second locus seemed to increase discriminatory power, it was not sufficient to definitively infer evolutionary relationships within a single multilocus sequence type (ST-5).

Tartrate-Resistant Acid Phosphatase 5b: a Serum Marker of Bone Resorption

Bone is a physiologically dynamic tissue being constantly regenerated throughout life as a consequence of bone turnover by bone-resorbing osteoclasts and bone-forming osteoblasts. In certain bone diseases, such as osteoporosis, the imbalance in bone turnover leads to bone loss and increased fracture risk. Measurement of bone mineral density (BMD) predicts the risk of fracture, but also biochemical markers of bone metabolism have been suggested to be suitable for prediction of fractures and monitoring the efficacy of antiresorptive treatment. Tartrate-resistant acid phosphatase 5b (TRACP 5b) is an enzyme released from osteoclasts into the circulation, from where it can be detected kinetically or immunologically. Conventional assays for serum total TRACP were spectrophotometric and suffered from interference by other acid phosphatases and non-osteoclastic TRACP 5a isoform. Our aim was to develop novel immunoassays for osteoclastic TRACP 5b. Serum TRACP 5b levels were elevated in individuals with high bone turnover, such as children, postmenopausal women, patients with osteoporosis, Paget’s disease and breast cancer patients with bone metastases. As expected, hormone replacement therapy (HRT) in postmenopausal women decreased the levels of serum TRACP 5b. Surprisingly, the highest TRACP 5b levels were observed in individuals with rare autosomal dominant osteopetrosis type II (ADO2), which is characterized by high BMD and fracture risk with simultaneously elevated levels of deficient osteoclasts. In ADO2 patients, elevated levels of serum TRACP 5b were associated with high fracture frequency. It is likely that serum TRACP 5b reflects the number of inactive osteoclasts in ADO2. Similar results supporting the hypothesis that TRACP 5b would reflect the number of osteoclasts instead of their activity were observed with cultured osteoclasts and in animal models. Novel TRACP 5b immunoassays may prove to be of value either as independent or combinatory tools with other bone metabolic markers and BMD measurements in clinical practice and bone research.

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.