Glutathione levels in and total antioxidant capacity of Candida sp. cells exposed to oxidative stress caused by hydrogen peroxide

INTRODUCTION: The capacity to overcome the oxidative stress imposed by phagocytes seems to be critical for Candida species to cause invasive candidiasis. METHODS: To better characterize the oxidative stress response (OSR) of 8 clinically relevant Candida sp., glutathione, a vital component of the intracellular redox balance, was measured using the 5,5'-dithiobis-(2-nitrobenzoic acid (DTNB)-glutathione disulfide (GSSG) reductase reconversion method; the total antioxidant capacity (TAC) was measured using a modified method based on the decolorization of the 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic) acid radical cation (ABTS*+). Both methods were used with cellular Candida sp. extracts treated or not with hydrogen peroxide (0.5 mM). RESULTS: Oxidative stress induced by hydrogen peroxide clearly reduced intracellular glutathione levels. This depletion was stronger in Candida albicans and the levels of glutathione in untreated cells were also higher in this species. The TAC demonstrated intra-specific variation. CONCLUSIONS: Glutathione levels did not correlate with the measured TAC values, despite this being the most important non-enzymatic intracellular antioxidant molecule. The results indicate that the isolated measurement of TAC does not give a clear picture of the ability of a given Candida sp. to respond to oxidative stress.

Changes in the TBARs content and superoxide dismutase, catalase and glutathione peroxidase activities in the lymphoid organs and skeletal muscles of adrenodemedullated rats

Thiobarbituric acid reactant substances (TBARs) content, and the activities of glucose-6-phosphate dehydrogenase (G6PDh), citrate synthase (CS), Cu/Zn- and Mn-superoxide dismutase (SOD), catalase, and glutathione peroxidase (GPX) were measured in the lymphoid organs (thymus, spleen, and mesenteric lymph nodes (MLN)) and skeletal muscles (gastrocnemius and soleus) of adrenodemedullated (ADM) rats. The results were compared with those obtained for sham-operated rats. TBARs content was reduced by adrenodemedullation in the lymphoid organs (MLN (28%), thymus (40%) and spleen (42%)) and gastrocnemius muscle (67%). G6PDh activity was enhanced in the MLN (69%) and reduced in the spleen (28%) and soleus muscle (75%). CS activity was reduced in all tissues (MLN (75%), spleen (71%), gastrocnemius (61%) and soleus (43%)), except in the thymus which displayed an increment of 56%. Cu/Zn-SOD activity was increased in the MLN (126%), thymus (223%), spleen (80%) and gastrocnemius muscle (360%) and was reduced in the soleus muscle (31%). Mn-SOD activity was decreased in the MLN (67%) and spleen (26%) and increased in the thymus (142%), whereas catalase activity was reduced in the MLN (76%), thymus (54%) and soleus muscle (47%). It is particularly noteworthy that in ADM rats the activity of glutathione peroxidase was not detectable by the method used. These data are consistent with the possibility that epinephrine might play a role in the oxidative stress of the lymphoid organs. Whether this fact represents an important mechanism for the establishment of impaired immune function during stress remains to be elucidated.

Glucose-6-phosphate dehydrogenase and glutathione reductase activity in methemoglobin reduction by methylene blue and cyst amine: study on glucose-6-phosphate dehydrogenase-deficient individuals, on normal subjects and on riboflavin-treated subjects

The authors have standardized methods for evaluation of the activity of the glucose-6-phosphate dehydrogenase and of glutathione reductase. The general principle of the first method was based on methemoglobin formation by sodium nitrite followed by stimulation of the glucose-6-phosphate dehydrogenase with methylene blue. Forty six adults (23 males and 23 females) were studied. Subjects were not G6PD deficient and were aged 20 to 30 years. The results showed that methemoglobin reduction by methylene blue was 154.40 and 139.90 mg/min (p<0.05) for males and females, respectively, in whole blood, and 221.10 and 207.85 mg/min (n.s.), respectively, in washed red cells. These data showed that using washed red cells and 0.7g% sodium nitrite concentration produced no differences between sexes and also shortened reading time for the residual amount of methemoglobin to 90 minutes. Glutathione reductase activity was evaluated on the basis of the fact that cystamine (a thiol agent) binds to the SH groups of hemoglobin, forming complexes. These complexes are reversed by the action of glutathione reductase, with methemoglobin reduction occurring simultaneously with this reaction. Thirty two adults (16 males and 16 females) were studied. Subjects were not G6PD deficient and were aged 20 to 30 years. Methemoglobin reduction by cystamine was 81.27 and 91.13 mg/min (p<0.01) for males and females, respectively. These data showed that using washed red cells and 0.1 M cystamine concentration permits a reading of the residual amount of methemoglobin at 180 minutes of incubation. Glutathione reductase activity was evaluated by methemoglobin reduction by cystamine in 14 females before and after treatment with 10 mg riboflavin per day for 8 days. The results were 73.69 and 94.26 jug/min (p<0.01) before and after treatment, showing that riboflavin treatment increase glutathione reductase activity even in normal individuals. Three Black G6PD-deficient individuals (2 males and 1 female) were also studied. The G6PD and glutathione reductase were partially activated, the change being more intense in males. On the basis of race and of the laboratory characteristics observed, it is possible to suggest that the G6PD deficiency of these individuals is of the African type and that the female is heterozygous for this deficiency. Analysis of the results as a whole permitted us to conclude that the methods proposed here were efficient for evaluating the activity of the glucose-6-phosphate dehydrogenase and of glutathione reductase. The latter is dependent on the pentose pathway, which generates NADPH, and on riboflavin, a FAD precursor vitamin.

Reduced schedule of human anti rabies immunization with Fuenzalida & Palacios vaccine

The present study evaluates the humoral and cellular immune responses in 35 volunteers submited to short antirabies vaccination schedules with the Fuenzalida & Palacios vaccine based on the administration of doses on non consecutive days. The volunteers were divided into two groups. The first group received a total number of five doses given on days 0, 4, 7, 20 and 35. The other group received four doses, the first one being a double dose given on day 0 and than three other single doses on days 7, 20 and 35. The evaluation of humoral immune response was carried out by serum neutralization (SN) and indirect immunofluorescense (IIF) tests, while the cellular immune response was evaluated by lymphoblastic transformation assay (LTA) and skin test (ST). According to our results these reduced schedules elicited early and effective humoral and cellulafimmune responses to rabies antigen suggesting that new reduced schedules should be extensively studied in order to give the proper bases to the proposition of changes in the current long-term schedule.

Reduced schedule of human anti-rabies immunization with Fuenzalida & Palacios vaccine: additional data

It was reevaluated a reduced schedule for anti-rabies post-exposure immunization with newborn mice nervous tissue vaccine (Fuenzalida 8c Palacios) in a group of 30 non exposed volunteers. The vaccine was administered by intramuscular injections on days zero, 2, 4, 16 and 27, in the deltoid area. Antibody levels were determinated by a simplified serum neutralization microtest on days zero, 16 and 37. On days 16 and 37 the antibody levels of the whole group was >0.5 IU/ml and >1.0 IU/ml, respectively. The cell mediated immunity was precociously detected (on day 4) by the delayed type hipersensitivity skin test. Our results show that this reduced schedule elicited an early and effective humoral and cellular immune response. However it is necessary other studies with larger groups of vaccinees in order to obtain definitive conclusion.

Isoniazid acetylating phenotype in patients with paracoccidioidomycosis and its relationship with serum sulfadoxin levels, glucose-6-phosphate dehydrogenase and glutathione reductase activities

The authors evaluated the isoniazid acetylating phenotype and measured hematocrit, hemoglobin, glucose-6-phosphate dehydrogenase and glutathione reductase activities plus serum sulfadoxin levels in 39 patients with paracoccidioidomycosis (33 males and 6 females) aged 17 to 58 years. Twenty one (53.84%) of the patients presented a slow acetylatingphenotype and 18(46.16%) a fast acetylating phenotype. Glucose-6-phosphate- dehydrogenase (G6PD) acti vity was decreased in 5(23.80%) slow acetylators and in 4(22.22%) fast acetylators. Glutathione reductase activity was decreased in 14 (66.66%) slow acetylators and in 12 (66.66%) fast acetylators. Serum levels of free and total sulfadoxin Were higher in slow acetylator (p < 0.02). Analysis of the resultspermitted us to conclude that serum sulfadoxin levels are related to the acetylatorphenotype. Furthermore, sulfadoxin levels were always above 50 µg/ml, a value considered therapeutic. Glutathione reductase deficiency observed in 66% of patients may be related to the intestinal malabsorption of nutrients, among them riboflavin, a FAD precursor vitamin, inpatients with paracoceidioidomycosis.

Reduced Progression of Cardiac Allograft Vasculopathy with Routine Use of Induction Therapy with Basiliximab

AbstractIntroduction:Cardiac allograft vasculopathy (CAV) is a major limitation for long-term survival of patients undergoing heart transplantation (HT). Some immunosuppressants can reduce the risk of CAV.Objectives:The primary objective was to evaluate the variation in the volumetric growth of the intimal layer measured by intracoronary ultrasound (IVUS) after 1 year in patients who received basiliximab compared with that in a control group.Methods:Thirteen patients treated at a single center between 2007 and 2009 were analyzed retrospectively. Evaluations were performed with IVUS, measuring the volume of a coronary segment within the first 30 days and 1 year after HT. Vasculopathy was characterized by the volume of the intima of the vessel.Results:Thirteen patients included (7 in the basiliximab group and 6 in the control group). On IVUS assessment, the control group was found to have greater vessel volume (120–185.43 mm3 vs. 127.77–131.32 mm3; p = 0.051). Intimal layer growth (i.e., CAV) was also higher in the control group (27.30–49.15 mm3 [∆80%] vs. 20.23–26.69 mm3[∆33%]; p = 0.015). Univariate regression analysis revealed that plaque volume and prior atherosclerosis of the donor were not related to intima growth (r = 0.15, p = 0.96), whereas positive remodeling was directly proportional to the volumetric growth of the intima (r = 0.85, p < 0.001).Conclusion:Routine induction therapy with basiliximab was associated with reduced growth of the intima of the vessel during the first year after HT.

Reduced capacity of peritoneal exudate cells for oxidative killing of Schistosoma mansoni schistosomula

Peritoneal exudate cells from mice infected with Schistosoma mansoni (S-PEC) can kill schistosomula in vitro in the presence of immune serum. S-PEC produce a low level of respiratory burst, and schistosomula mortality in their presence is not reduced when exogenous antioxidants are added, suggesting that with S-PEC, oxidative killing is not important. Hydrogen peroxide (H2O2) and superoxide production by S-PEC, and cells from BCG and thioglycollate (THGL) injected non-infected mice, non-specifically stimulated with opsonized zymosan, were measured. Levels of H2O2 produced by S-PEC were significantly lower than BCG or THGL PEC, and were below the H2O2 threshold for schistosomula killing. This resulted in lower levels of cell-mediated killing of schistosomula in vitro by S-PEC than by BCG or THGL PEC. Superoxide levels, however, were similar between the three cell populations. The efficiency of PEC to kill schistosomules in vitro correlated with H2O2 rather than superoxide levels. The lower tolerance of schistosomula, compared to adult S. mansoni to GSH depleting agents increases their sensitivity to oxidative attack and resulted in higher levels of cell-mediated killing in vitro.

Glutathione S-transferases of 28kDa as major vaccine candidates against schistosomiasis

For the development of vaccine strategies to generate efficient protection against chronic infections such as parasitic diseases, and more precisely schistosomiasis, controlling pathology could be more relevant than controlling the infection itself. Such strategies, motivated by the need for a cost-effective complement to existing control measures, should focus on parasite molecules involved in fecundity, because in metazoan parasite infections pathology is usually linked to the output of viable eggs. In numerous animal models, vaccination with glutathione S-transferases of 28kDa has been shown to generate an immune response strongly limiting the worm fecundity, in addition to the reduction of the parasite burden. Recent data on acquired immunity directed to 28GST in infected human populations, and new development to draw adapted vaccine formulations, are presented.

Reduced protective effect of Plasmodium berghei immunization by concurrent Schistosoma mansoni infection

Studies on concomitant schistosomiasis and human and experimental malaria have shown a variation in the immunospecific response, as well as an increase in the severity of both parasitoses. In the present study, a murine co-infection model was used to determine the effects of a co-infection with Schistosoma mansoni and Plasmodium berghei on the protective immunity acquired by repeated malarial infections and subsequent curative treatment with chloroquine. Our results have demonstrated that, compared to an infection with P. berghei only, the co-infection increases the malarial parasitaemia and decreases the survival rate. Indeed, mice that were immunized by infection and treatment with drug displayed no mortality whereas co-infected mice showed a reduced protective efficacy of immunization against P. berghei (mortality > 60%). Interestingly, this high mortality rate was not associated with high levels of parasitaemia. Our findings support the idea of a suppressive effect of a Schistosoma co-infection on the anti-malarial protection by immunization. This result reveals a possible drawback of the development of anti-malarial vaccines, especially considering the wide endemic areas for both parasitoses.

Immune response to Leishmania (Leishmania) chagasi infection is reduced in malnourished BALB/c mice

Protein-energy malnutrition and micronutrient deficiencies may down-regulate immune response and increase morbidity and mortality due to infection. In this study, a murine model was used to study the effects of protein, iron and zinc deficiencies on the immune response to Leishmania (Leishmania) chagasi infection. Mice were initially fed a standard diet or with a diet containing 3% casein but deficient in zinc and iron. After malnutrition was established, mice were inoculated with L. chagasiand sacrificed four weeks later in order to evaluate liver and spleen parasite loads and serum biochemical parameters. Significant decreases in liver and spleen weight, an increase in the parasite loads in these organs and decreases in serum protein and glucose concentrations in malnourished animals were observed. Furthermore, the production of interferon-gamma by spleen cells from infected malnourished mice stimulated by Leishmaniaantigen was significantly lower compared with that in control diet mice. These data suggest that malnutrition alters the immune response to L. chagasiinfection in the BALB/c model and, in association with the effects on biochemical and anatomical parameters of the host, favored increases in the parasite loads in the spleens and livers of these animals.

Reduced susceptibility to vancomycin and biofilm formation in methicillin-resistant Staphylococcus epidermidis isolated from blood cultures

This study aimed to correlate the presence of ica genes, biofilm formation and antimicrobial resistance in 107 strains of Staphylococcus epidermidis isolated from blood cultures. The isolates were analysed to determine their methicillin resistance, staphylococcal cassette chromosome mec (SCCmec) type, ica genes and biofilm formation and the vancomycin minimum inhibitory concentration (MIC) was measured for isolates and subpopulations growing on vancomycin screen agar. The mecA gene was detected in 81.3% of the S. epidermidis isolated and 48.2% carried SCCmec type III. The complete icaADBC operon was observed in 38.3% of the isolates; of these, 58.5% produced a biofilm. Furthermore, 47.7% of the isolates grew on vancomycin screen agar, with an increase in the MIC in 75.9% of the isolates. Determination of the MIC of subpopulations revealed that 64.7% had an MIC ≥ 4 μg mL-1, including 15.7% with an MIC of 8 μg mL-1 and 2% with an MIC of 16 μg mL-1. The presence of the icaADBC operon, biofilm production and reduced susceptibility to vancomycin were associated with methicillin resistance. This study reveals a high level of methicillin resistance, biofilm formation and reduced susceptibility to vancomycin in subpopulations of S. epidermidis. These findings may explain the selection of multidrug-resistant isolates in hospital settings and the consequent failure of antimicrobial treatment.

Ecophysiological response of Sorghum halepense populations to reduced rates of nicosulfuron

The control and regrowth after nicosulfuron reduced rate treatment of Johnsongrass (Sorghum halepense L. Pers.) populations, from seven Argentinean locations, were evaluated in pot experiments to assess if differential performance could limit the design and implementation of integrated weed management programs. Populations from humid regions registered a higher sensibility to reduced rates of nicosulfuron than populations from subhumid regions. This effect was visualised in the values of regression coefficient of the non-linear models (relating fresh weight to nicosulfuron rate), and in the time needed to obtain a 50% reduction of photosynthesis rate and stomatal conductance. The least leaf CO2 exchange of subhumid populations could result in a lower foliar absorption and translocation of nicosulfuron, thus producing less control and increasing their ability to sprout and produce new aerial biomass. The three populations from subhumid regions, with less sensibility to nicosulfuron rates, presented substantial difference in fresh weight, total rhizome length and number of rhizome nodes, when they were evaluated 20 week after treatment. In consequence, a substantial Johnsongrass re-infestation could occur, if rates below one-half of nicosulfuron labeled rate were used to control Johnsongrass in subhumid regions.

Stalk and sucrose yield in response to nitrogen fertilization of sugarcane under reduced tillage

The objective of this work was to evaluate the agroindustrial production of sugarcane (millable stalks and sucrose yield) after successive nitrogen fertilizations of plant cane and ratoons in a reduced tillage system. The experiment was carried out at Jaboticabal, SP, Brazil, on a Rhodic Eutrustox soil, during four consecutive crop cycles (March 2005 to July 2009). Plant cane treatments consisted of N-urea levels (control, 40, 80, and 120 kg ha-1 N + 120 kg ha-1 P2O5 and K2O in furrow application). In the first and second ratoons, the plant cane plots were subdivided in N-ammonium nitrate treatments (control, 50, 100, and 150 kg ha-1 N + 150 kg ha-1 K2O as top dressing over rows). In the third ratoon, N fertilization was leveled to 100 kg ha-1 in all plots, including controls, to detect residual effects of previous fertilizations on the last crop's cycle. Sugarcane ratoon was mechanically harvested. A weighing truck was used to evaluate stalk yield (TCH), and samples were collected in the field for analysis of sugar content (TSH). Increasing N doses and meteorological conditions promote significant responses in TCH and TSH in cane plant and ratoons, in the average and accumulated yield of the consecutive crop cycles.