Nitroblue Tetrazolium Test Research Articles

Antioxidant efficiency of lycopene on oxidative stress - induced damage in bovine spermatozoa.

BackgroundLycopene (LYC) is a natural carotenoid with powerful reactive oxygen species (ROS) scavenging activities. The aim of this study was to investigate if lycopene has the ability to reverse ROS-mediated alterations to the motility, viability and intracellular antioxidant profile of bovine spermatozoa subjected to ferrous ascorbate (FeAA). Spermatozoa were washed out of fresh bovine semen, suspended in 2.9 % sodium citrate and subjected to LYC treatment (0.25, 0.5, 1 or 2 mmol/L) in the presence or absence of FeAA (150 μmol/L FeSO4 and 750 μmol/L ascorbic acid) during a 6 h in vitro culture. Spermatozoa motion characteristics were assessed using the SpermVision™ computer-aided sperm analysis (CASA) system. Cell viability was examined with the metabolic activity (MTT) assay, ROS generation was quantified via luminometry and the nitroblue-tetrazolium (NBT) test was applied to quantify the intracellular superoxide formation. Cell lysates were prepared at the end of the in vitro culture to investigate the intracellular activity of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) as well as the concentrations of glutathione (GSH) and malondialdehyde (MDA).ResultsFeAA treatment led to a reduced spermatozoa motility (P < 0.001), viability (P < 0.001) and a decline of the antioxidant capacity of spermatozoa (P < 0.001) but increased the ROS generation (P < 0.001), superoxide production (P < 0.001) and lipid peroxidation (P < 0.001). LYC administration resulted in a preservation of the spermatozoa motion parameters (P < 0.001), mitochondrial activity (P < 0.001) and antioxidant characteristics (P < 0.001 with respect to SOD; P < 0.01 in relation to CAT; P < 0.05 as for GPx and GSH) with a concentration range of 1 and 2 mmol/L LYC revealed to be the most effective.ConclusionsOur results suggest that LYC exhibits significant ROS-scavenging and antioxidant properties which may prevent spermatozoa alterations caused by oxidative stress, and preserve the functionality of male reproductive cells.

Protective Effects of α-tocopherol on the Activity and Antioxidant Profile of Bovine Spermatozoa Subjected to Ferrous Ascorbate-Induced Oxidative Stress

As spermatozoa are highly vulnerable to oxidative stress development, in vitro antioxidants offer an additional line of defense to the male reproductive system against oxidative insults. α‑tocopherol (α-TOC) is the most abundant form of vitamin E identified in the seminal plasma and spermatozoa membranes, being able to terminate numerous oxidative chain reactions causing substantial damage to biomolecules vital for sperm survival. This study was designed to shed more light on the in vitro effects of α‑tocopherol with respect to the vitality and intracellular antioxidant profile of bovine spermatozoa subjected to ferrous ascorbate-induced oxidative stress. Spermatozoa were washed out from 50 bovine ejaculates, suspended in 2.9 % sodium citrate and subjected to α-TOC treatment (10, 50, 100 and 500 μmol/L) in the presence or absence of ferrous ascorbate (FeAA; 150 μmol/L FeSO4 and 750 μmol/L ascorbic acid) during a 6h in vitro culture. Spermatozoa motion parameters were assessed using the SpermVision™ computer-aided sperm analysis (CASA) system. Cell viability was examined with the metabolic activity (MTT) assay, and the nitroblue-tetrazolium (NBT) test was applied to quantify the intracellular superoxide formation. Cell lysates were prepared at the end of the experiments in order to assess the intracellular activity of superoxide dismutase (SOD), catalase (CAT), as well as glutathione (GSH) and malondialdehyde (MDA) concentrations. Treatment with FeAA reduced both spermatozoa motility parameters (P &lt; 0.001) as well as viability (P &lt; 0.05 with respect to Time 0 h; P &lt; 0.01 in case of Time 2 h and P &lt; 0.001 in relation to Time 6 h), decreased the antioxidant parameters of the samples (P &lt; 0.001 in case of SOD; P &lt; 0.01 with respect to CAT and GSH) but increased the superoxide production (P &lt; 0.01 in case of Time 0h and P &lt; 0.001 with respect to Times 2 h and 6 h) and lipid peroxidation (P &lt; 0.001). α‑TOC administration resulted in a preservation of the spermatozoa motility characteristics (P &lt; 0.001 with respect to 500 μmol/L α-TOC), viability (P &lt; 0.001 in case of 500 μmol/L α-TOC and P &lt; 0.05 with respect to 100 μmol/L α-TOC) and antioxidant profile (P &lt; 0.01 related to the impact of 500 μmol/L α-TOC on the SOD activity; P &lt; 0.05 in relation to CAT; P &lt; 0.01 with respect GSH; 100-500 μmol/L α-TOC), with 500 μmol/L α-TOC being the most effective. Our results suggest that α‑tocopherol possesses significant antioxidant properties that may prevent the deleterious effects caused by free radicals to spermatozoa, and extend the fertilization potential of male reproductive cells.

The anthraquinones rubiadin and its 1-methyl ether isolated from Heterophyllaea pustulata reduces Candida tropicalis biofilms formation

BackgroundCandida tropicalis is increasingly becoming among the most commonly isolated pathogens causing fungal infections with an important biofilm-forming capacity. PurposeThis study addresses the antifungal effect of rubiadin (AQ1) and rubiadin 1-methyl ether (AQ2), two photosensitizing anthraquinones (AQs) isolated from Heterophyllaea pustulata, against C. tropicalis biofilms, by studying the cellular stress and antioxidant response in two experimental conditions: darkness and irradiation. The combination with Amphotericin B (AmB) was assayed to evaluate the synergic effect. Study design/MethodsBiofilms of clinical isolates and reference strain of Candida tropicalis were treated with AQs (AQ1 or AQ2) and/or AmB, and the biofilms depletion was studied by crystal violet and confocal scanning laser microscopy (CSLM). The oxidant metabolites production and the response of antioxidant defense system were also evaluated under dark and irradiation conditions, being the light a trigger for photo-activation of the AQs. The Reactive Oxygen Species (ROS) were detected by the reduction of Nitro Blue Tetrazolium test, and Reactive Nitrogen Intermediates (RNI) by the Griess assay. ROS accumulation was also detected inside biofilms by using 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) probe, which was visualized by CSLM. Superoxide dismutase (SOD) activity and the total antioxidant capacity of biofilms were measured by spectrophotometric methods.The minimun inhibitory concentration for sessile cells (SMIC) was determined for each AQs and AmB. The fractional inhibitory concentration index (FICI) was calculated for the combinations of each AQ with AmB by the checkerboard microdilution method. ResultsBiofilm reduction of both strains was more effective with AQ1 than with AQ2. The antifungal effect was mediated by an oxidative and nitrosative stress under irradiation, with a significant accumulation of endogenous ROS detected by CSLM and an increase in the SOD activity. Thus, the prooxidant-antioxidant balance was altered especially by AQ1. The best synergic combination with AmB was also obtained with AQ1 (80.5%) (FICI=0.74). ConclusionUnder irradiation, the oxidative stress was the predominant effect, altering the prooxidant-antioxidant balance, which may be the cause of the irreversible cell injury in the biofilm. Our results showed synergism of these natural AQs with AmB. Therefore, the photosensitizing AQ1 could be an alternative for the Candida infections treatment, which deserves further investigation.

Curcumin has protective and antioxidant properties on bull spermatozoa subjected to induced oxidative stress

Over the past decades, there has been an emphasis on assessment of the use of natural compounds in the prevention or repair of oxidative injury to spermatozoa. Curcumin (CUR) is a natural phenol with powerful antioxidant properties. The aim of the present study was to examine if CUR could reverse reactive oxygen species (ROS)-mediated alterations to the motility, viability and intracellular antioxidant profile of bull spermatozoa subjected to a prooxidant (i.e., ferrous ascorbate – FeAA). Spermatozoa were washed from recently collected semen samples, suspended in 2.9% sodium citrate and subjected to CUR treatment (5, 10, 25 and 50μmol/L) in the presence or absence of FeAA (150μmol/L FeSO4 and 750μmol/L ascorbic acid) during a 6h in vitro culture. Spermatozoa motility characteristics were assessed using the SpermVision computer-aided spermatozoa analysis (CASA) system. Cell viability was examined with the metabolic activity (MTT) assay, ROS generation was quantified using luminometry and the nitroblue-tetrazolium (NBT) test was used to quantify the intracellular superoxide formation. Cell lysates were prepared at the end of the culture to assess the intracellular activity of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) as well as the concentrations of glutathione (GSH) and malondialdehyde (MDA). Treatment with FeAA led to a reduced spermatozoa motility (P<0.001), viability (P<0.001) and decreased the antioxidant characteristics of the samples (P<0.001) but increased the ROS generation (P<0.001), superoxide production (P<0.001) and lipid peroxidation (P<0.001). The CUR treatment led to a preservation of spermatozoa motion (P<0.001), mitochondrial activity (P<0.001) and antioxidant characteristics (P<0.05 with SOD and GSH; P<0.01 with CAT and GPx), revealing the concentration range of 25–50μmol/L CUR to be the most effective for sustaining spermatozoa viability. Data from the present study suggest that CUR exhibits significant protective and ROS-scavenging characteristics which may prevent oxidative insults to spermatozoa and thus preserve the functional activity of male gametes.

Protective Effects of Quercetin on Selected Oxidative Biomarkers in Bovine Spermatozoa Subjected to Ferrous Ascorbate.

Quercetin (QUE) is a natural flavonol-type flavonoid with antibacterial, anti-inflammatory and anti-aggregatory properties. It is also a powerful reactive oxygen species (ROS) scavenger and chelating agent. The aim of this study was to assess the effectiveness of QUE to reverse ROS-mediated alterations to the motility, viability and intracellular antioxidant profile of bovine spermatozoa. Spermatozoa were washed out of fresh bovine semen, suspended in 2.9% sodium citrate and subjected to QUE treatment (7.5, 25, 50 and 100μmol/l) in the presence or absence of a pro-oxidant, that is ferrous ascorbate (FeAA; 150μmol/l FeSO4 and 750μmol/l ascorbic acid) during a 6-h invitro culture. Spermatozoa motion characteristics were assessed using the SpermVision computer-aided sperm analysis (CASA) system. Cell viability was examined with the metabolic activity (MTT) assay, ROS generation was quantified via luminometry, and the nitroblue tetrazolium (NBT) test was applied to quantify the intracellular superoxide formation. Cell lysates were prepared at the end of the invitro culture to investigate the intracellular activity of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) as well as the concentrations of glutathione (GSH) and malondialdehyde (MDA). FeAA treatment led to a reduced sperm motility (p<0.001), viability (p<0.001) and decreased the antioxidant parameters of the sperm samples (p<0.001) but increased the ROS generation (p<0.001), superoxide production (p<0.001) and lipid peroxidation (p<0.001). QUE administration resulted in a preservation of the spermatozoa vitality and antioxidant characteristics (p<0.01 with respect to the enzymatic antioxidants, p<0.001 in relation to GSH) with a concentration range of 50-100μmol/l QUE revealing to be the most effective. Our results suggest that QUE exhibits significant ROS-scavenging and metal-chelating properties which may prevent spermatozoa alterations caused by ROS, and preserve the functionality of male reproductive cells.

Respiratory Burst Process in Diabetic Children.

BackgroundIncreased rate of infections in diabetes mellitus (DM) is an accepted fact. Pathophysiologically, several tasks of the immune system could be involved including polymorphonuclear (PMN) functions.ObjectivesThe aim of this research was to evaluate the respiratory burst process of PMNs that is an essential part of phagocytosis, in children with DM.Patients and MethodsFifty two children with insulin dependent diabetes and 29 non-diabetic children were enrolled in this cross sectional study from 2010 to 2011. Nitroblue tetrazolium (NBT) test was done on PMNs taken from their heparinized blood. The resultant data was analyzed by SPSS version 16. P values were considered significant when it was under 0.05.ResultsMean NBTs were 72.1 ± 15.84 and 94.68 ± 5.31 in diabetics and non-diabetics, respectively (P < 0.001). Using Pearson correlation, there was no significant correlation between the NBT level and age, gender, duration of diabetes, daily insulin usage and blood HbA1C level.ConclusionsCompared to non-diabetics, respiratory burst process of polymorphonuclears is obviously decreased in diabetic children. This can explain one of the mechanisms involved in the increased rate of infections in DM.

Antioxidant and Immunomodulatory Activity of Hydroalcoholic Extract and its Fractions of Leaves of Ficus benghalensis Linn.

Background:Ficus benghalensis is a folk medicine indigenous plant of India. Several studies on this plant reported and focused on the biological profile of the plant.Objectives:This study is aimed to evaluate the antioxidant and immunomodulatory activity of F. benghalensis leaf extract using various in vitro screening methods of both parameters.Materials and Methods:Hydroalcoholic (FB1) extract and it's four fractions viz. n-hexane (FB2), n-butanol (FB3), chloroform (FB4), and water (FB5) of leaves of F. benghalensis investigated for their free radical scavenging activity using 1-1-diphneyl-2-picrylhydrazyl and 2, 2′-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) radicals. A dose-response curve was plotted and IC50 values were determined to assess antioxidant activity. Nitroblue tetrazolium test, phagocytosis of killed Candida albicans and candidacidal assay were carried out to assess the immunomodulatory activity. Positive non-lymphoid cell number, mean particle number of killed C. albicans, percent value of killed C. albicans by neutrophils were calculated and presented.Results:All extracts showed antioxidant and prominent immunomodulatory activity with compared to standard.Conclusions:Hydroalcoholic (FB1) extract and its four fractions viz. n-hexane (FB2), n-butanol (FB3), chloroform (FB4), and water (FB5) showed promising antioxidant and immunomodulatory activity.SUMMARY Hydroalcoholic extract and its fractions of F. benghalensis Linn exhibited different DPPH and ABTS scavenging activity in concentration dependent manner.The extract, fractions and reference antioxidants showed DPPH scavenging effect in the order of Vit-C > Quercetin > FB2 > FB1 > FB5 > FB4> FB3 and ABTS scavenging effect in the order of Vit-C > Quercetin > FB1> FB2 > FB5 > FB3> FB4.FB2 and FB3 showed promising immunomodulatory activity at all concentrations.

The influence of low-intensity laser radiation on the functional activity of neutrophils in the patients presenting with community-acquired pneumonia

The objective of the present study was to elucidate the influence of low-intensity laser radiation on the results of the nitroblue tetrazolium (NBT) test in the patients presenting with community-acquired pneumonia. A total of 100 patients with community-acquired pneumonia were available for the examination of whom 70 were treated with intravenous low-intensity laser irradiation of blood (ILIB) by means of the ILIB-405 technique during 7 days. The functional activity of neutrophils was estimated from their ability to reduce nitroblue tetrazolium in both spontaneous and stimulated NBT-tests. The analysis of the data obtained in the study has demonstrated the significant improvement of the results of the NBT tests in the group of patients receiving the ILIB treatments regardless of whether its content was originally elevated or reduced. The inclusion of intravenous low-intensity laser irradiation of blood in the combined treatment of the patients with community-acquired pneumonia appreciably promotes normalization of the bactericidal activity of neutrophils.

Comparative Efficiency and Impact on the Activity of Blood Neutrophils Isolated by Percoll, Ficoll and Spontaneous Sedimentation Methods

ABSTRACTStudies on the role of cells in physiological and pathological processes generally require isolation of some populations, such as neutrophils. In the literature, several methods used for isolating neutrophils are described; however, there is no consensus on the best technique to be used in cell functional studies. The present study compares the efficiency and impact on the chemotactic and phagocytic activity of neutrophils isolated from blood by three different methods: Percoll and Ficoll density centrifugation gradients and spontaneous sedimentation technique. The neutrophil chemotaxis, stimulated with lipopolysaccharide (LPS), autologous serum or homologous serum, was determined by using Boyden chambers. The phagocytic capacity was assessed by ingestion of zimosan particles, and digestion phase was analyzed by nitroblue tetrazolium test (NBT). The results obtained from neutrophil isolation by Percoll and Ficoll density gradients, as compared to spontaneous sedimentation technique, showed similar degrees of cell yields and higher purity; however, these methods affected neutrophil responsiveness, accompanied by elevated chemotaxis and reduced chemotactic capacity to respond to subsequent stimulation. Neutrophil isolation by spontaneous sedimentation, in contrast, did not affect cellular activity and resulted in cell preparation with high number of neutrophils. Although neutrophil phagocytosis results were similar between the different methods, digestion phase of phagocytosis was significantly enhanced after LPS-stimulation, only in the neutrophils isolated by spontaneous sedimentation technique. In conclusion, the present study shows that isolation of blood neutrophils by the spontaneous sedimentation technique is appropriate for the assessment of cellular activity, since it neither primes or activates the neutrophils nor does it affect their functional responsiveness.

ON THE ISSUE OF SEPSIS DIAGNOSTICS IN INFANTS

Introduction. Severe bacterial infections and bacterial sepsis remain one of the most topical problems in modern medicine. Stable high lethal outcome and continuous tendency to the growth of sickness rate are often caused by untimely making the diagnosis and administration of treatment respectively. In recent years C-reactive protein (CRP) and other proteins of acute inflammatory phase are used as biomarkers to diagnose sepsis. At the same time the problem of early diagnostics of purulent-septic diseases in infants cannot be considered completely solved. Objective. To improve diagnostics and treatment of generalized purulent-septic diseases in children, diagnostic and prognostic value of clinical-paraclinical signs in sick infants should be studied. Materials and methods. 19 infants hospitalized in the infectious department for infants of Chernivtsi Regional Pediatric Clinical Hospital (RPCH) for generalized infectious-inflammatory diseases were examined. The group of comparison included 29 infants with various somatic pathology (anemia, energy-protein insufficiency, rickets etc.). In addition to generally excepted methods all the infants underwent a comprehensive clinical-paraclinical examination including immunologic tests of I-II levels, detection of protein content of acute inflammatory phase. Results of the study. A high specificity and at the same time low sensitivity of the peripheral blood leukocytosis more than 10,5 g/L and stab neutrophil shifting of leukogram more than 12% to confirm sepsis of infants were found. The content of immunoglobulin G more than 10,0 g/L in the blood serum was characterized by a high specificity as well, although unreliable signs of clinical-epidemic risk. The content of C-reactive protein more than 10,0 mg/L in the blood serum of patients with sepsis was found in 72,0% of cases, the content of α-1-antitrypsin more than 3,7 g/L in the blood serum – in 71,4%, and concentration of ferritin over 54,0 ng/ml – in 80% of children. In the group of comparison the concentration of proteins in acute inflammatory phase was found in 35,7; 45,5 and 25,5% respectively (рφ 10 mg/ml and ferritin>54,0 ng/ml in the blood serum of infants was 4,7 and 12,0 respectively with 95% confidence interval (CI 1,3-17,1 and 0,2-181,0; χ2=10,9 and 10,0), which proved statistical reliability of the paraclinical changes indicated. None of the mentioned paraclinical signs taken separately is characterized by a high sensitivity and specificity in the diagnostics of sepsis in infants, and it requires a necessity of a comprehensive diagnostic approach. Considering the results obtained, we suggested to use a diagnostic pair of clinical-paraclinical signs to diagnose sepsis in infants: the results of spontaneous variant of nitro blue tetrazolium (NBT) test of neutrophil granulocytes in the blood increasing 17% of formazan-positive cells, and the content of C-reactive protein of the blood serum 10 mg/ml and more. There are the following indices of diagnostics value of the suggested complex of examinations: specificity – 64,0%, sensitivity – 92,0%, predicted value of a positive result – 75%, predicted value of a negative result – 88,0%. An attributive risk of septic process in infants with the indices mentioned was 62,0%, a relative risk – 5,7, and the correlation of chances was 2. Conclusions. 1.To diagnose sepsis in infants a comprehensive approach should be used, because none of separately taken clinical-paraclinical signs is characterized by synchronous high indices of diagnostic value and reliable risk of present generalized purulent-septic disease. 2.With the aim to find sepsis in infants a diagnostics complex of paraclinical signs is recommended to be used: the content of 17,0% and more of formazan-positive neutrophils in spontaneous NBT-test in combination with the values of C-reactive protein content in the blood serum of 10,0 mg and more.

A18: Tumor-associated neutrophils in ovarian cancer

Tumor-associated neutrophils (TANs) are the cell population that differs in morphofunctional characteristics from peripheral blood cells (Gregory and Houghton, 2011). The first study to identify the presence of TANs as an independent poor prognostic factor and to include TANs into a prognostic risk model was published in 2006 (Donskov , 2006). Specific signals during cancer progression have been shown to induce the emergence of a pro-tumor phenotype of neutrophils (PMN). Frinlender (2013) points out an anti-tumor phenotype of TANs. Of particular interest is the study of interaction of neutrophils with T cells because the latter are considered to be cytotoxic cells mediating antitumor immunity. The aim of the study was to assess the lymphoid cell infiltration (LCI) and the functional status of TANs in ovarian cancer. LCI was assessed by immunohistochemistry and the levels of mieloproxydase (MPO) and cationic proteins (CP) were evaluated by cytochemical methods in ovarian carcinoma surgical resection specimens. The results were presented as a mean cytochemical coefficient (MCC). The intensity of nitroblue tetrazolium (NBT) test was expressed as a percentage. Obtained results were analyzed by nonparametric statistical methods. The Kruskal–Wallis test was used to evaluate the differences between groups. We established that low intensity of infiltration in general and formation of marginal lymphoid cell ridge in some tumors are typical for malignant ovarian neoplasms. More intensive infiltration was found in the regions of the rapid growth of tumor cells, i.e. invasion zone. In some cases, a group of tumor cells was separated from the main part of the parenchyma and surrounded by lymphoid elements. Conditions are created under which active spread of cancer cells in the parenchyma is associated with intensive lymphoid cells infiltration in the stroma. Total count and density of lymphoid cells can significantly vary: from single lymphoid elements uniformly scattered in the tumor tissue to focal accumulations. In tumor tissue location of lymphoid cells is irregular, so most of the infiltration is in the stroma and among the cancer cells only solitary lymphocytes are found. Lymphoid cells which make up the infiltration include 12.3 ± 1.52% of macrophages, 22.1 ± 2.3% of plasma cells, 62.5 ± 1.1% of lymphocytes and 3.1 ± 0.9% of PMN. Total amount of cells was 224.1 ± 32.8 per 1 mm 2 of tumor. Lymphocytes in ovarian carcinomas do not form large clusters; they are disposed among other lymphoid cells in the stroma on the periphery of the tumor. Plasma cells have a classical morphological structure and often locate in the stroma and on the periphery of the tumor. Macrophages are an integral part of the tumor infiltrate. In the tumor they become stretched and angular. Macrophages usually have large dimensions, and they are located in the stroma as well as in the parenchyma among the cancer cells. Segmented leukocytes (presented in the vast majority by neutrophils) are constantly found in the infiltrate of the stroma and the parenchyma, most often they are found near or among massive necrosis. Analysis of TANs functional activity in comparison with the peripheral blood neutrophils has shown that MPO activity elevated and amounted 2.21 ± 0.43 MCC against 1.40 ± 0.07 MCC in the peripheral blood neutrophils, the level of CP also increased and amounted 1.51 ± 0.39 MCC against 0.83 ± 0.43 MCC. TANs activity in NBT-test was not significantly different from that of the peripheral blood neutrophils. TANs compose the minor part of primary ovarian tumor infiltrate. Indicators of aerobic and anaerobic bactericidal activity of TANs significantly exceed that of the peripheral blood neutrophils in patients with ovarian cancer. This work was conducted in the framework of implementing the state assignment of the Ministry of Education and Science of the Russian Federation No. 1163 – Russia.

Chronic Granulomatous Disease Complicated With Dissemination of BCG Vaccine

SESSION TITLE: Tuberculosis Global Case Reports SESSION TYPE: Global Case Report Poster PRESENTED ON: Tuesday, October 27, 2015 at 01:30 PM - 02:30 PM INTRODUCTION: Chronic granulomatous disease is caused by a deficiency of phagocyte microbicidal function, due to the inability to produce hydrogen peroxide and superoxide anion. The most common form is X-linked and presented with recurrent pulmonary infections. CASE PRESENTATION: A male patient a one year and nine months old, presented with febriles syndrome treated with multiple antibiotics such as clabulanic amoxicillin due to reoccurrent infection in the whole respiratory system; anemia, axillary adenopathy, without symptoms of recovery. For this reason he was referred to our hospital for diagnosis and treatment. At admission, his general condition was bad, with the following BM16.5 kg/m2, the patient is between 50-25 percentile growth. Presented: asthenia, decreased appetite, fever syndrome with records of 38 °, oxygen desaturation, with a 92 % to 21 % FiO2, respiratory rate of 36 per minute. Physical examination presented: right axillary lymphadenopathy, mobile, painless, hard elastic 1cm diameter, crepitant widespread rales and rhonchi in both lung fields and Other symptoms such as, increased rate of breathing, breathlessness and wheezing. Treatment was initiated with Clarithromycin and Amoxicillin clavulanic. TB empirical first-line treatment is added. He continued, febrile with records of 38-39 ° C, with antipyretics and physical means. Added access nonproductive cough and increased respiratory compromise adding retractions, and tachypnea. Background: Fully vaccinated. From month of life was treated for recurrent respiratory tract infections. Five months olds requiring hospitalization for acute pneumonia record. Laboratory results: anemia, increased erythrocyte sedimentation rate, hyperglycemia, and mild oxygen desaturation. Serological tests, toxoplasmosis, HIV, EBV, parvovirus, CMV, Mycoplasma, Bartonella, Brucella, Leptosipirosis, all negative. Dosage IgA and IgG slightly above its normal value, IgE 541.6 Ul/ml. Elevation of gamma globulin. DNA- antibody, ANCAs, and Rheumatoid Factor negative. Thorax CT: multiple images of lymph nodes in the mediastinum Observed in lung parenchyma patchy areas of airspace occupation distributed in both lungs and cavitated images in the lower lobes. Axillary lymphadenopathy. Upper abdomen no abnormalities. Normal Echocardiogram. Ocular Fundus, right eye net edges whitish lesions without macular involvement Faced with suspected diagnosis of TB specific studies are requested: Gastric Lavage, Lymph Node and Cerebrospinal Fluid: Negative smear and culture. Blood cultures for mycobacteria: Negatives. Tuberculin Skin Test: Negative. DISCUSSION: Were raised various differential diagnosis: Infectious diseases such as tuberculosis, toxoplasmosis, aspergillosis, staphylococci sepsis, tuberculosis being the entity that most compatible with the characteristics presented by the patient, although the bacillus was not found by the methods referred. Immunological diseases: Wiskott Aldrich immunodeficiency. Severe combined immunodeficiency disease is a group with deficiency of humoral and cellular immunity caused by various genetic mutations and associated enzyme deficiencies. The most common form is X-linked early onset in the first months of life, and is presented as severe mucocutaneous candidiasis, dissemination of BCG vaccine, persistent diarrhea, lung infections, meningitis, septicemia. Complement deficiency can start at different ages. C3 deficiencies present with sino-pulmonary infections by encapsulated bacteria, pneumococcus, Neisseria meningitidis, Haemophilus influenza. Autoimmune disease Systemic lupus erythematosus, polyangiitis, Wegener's granulomatosis. CONCLUSIONS: The patient remained febrile despite therapy instituted and showed no improvement in respiratory symptoms so it is referred to a specialist center for studies to detect diseases of the immune system. Resulting poor response in nitroblue tetrazolium (NBT) test, it was assumed as a chronic granulomatous disease complicated with dissemination of BCG (Bacille Calmette-Guerin) vaccine, given that the child was vaccinated at birth. Treatment was started with interferon gamma, TB drugs, Trimethoprim-sulfamethoxazole and Itraconazol, and showed significant improvement 40 days after starting treatment. Reference #1: Chronic granulomatous disease, Current Opinion in Immunology, Volume 15, October 2003, Pages 578-584, P G Heyworth, and col. DISCLOSURE: The following authors have nothing to disclose: Gabriela Manonelles, Vanina Martin No Product/Research Disclosure Information

Resveratrol offers protection to oxidative stress induced by ferrous ascorbate in bovine spermatozoa

Resveratrol (RES) is a natural polyphenol and phytoestrogen exhibiting cardioprotective, anticancer, antibacterial and vasorelaxing properties. It is also a powerful reactive oxygen species (ROS) scavenger and chelating agent. This study was designed to determine the efficiency of RES to reverse the ROS-mediated impairment of the motility, viability and intracellular antioxidant profile of bovine spermatozoa. Spermatozoa were washed out of fresh bovine semen, suspended in 2.9% sodium citrate and subjected to RES treatment (5, 10, 25 and 50 μmol L−1) in the presence or absence of a pro-oxidant, i.e., ferrous ascorbate (FeAA; 150 μmol L−1 FeSO4 and 750 μmol L−1 ascorbic acid) during a 6-h in vitro culture. Spermatozoa motion parameters were assessed using the SpermVision computer-aided sperm analysis (CASA) system. Cell viability was examined with the metabolic activity (MTT) assay, and the nitroblue-tetrazolium (NBT) test was applied to quantify the intracellular superoxide formation. Cell lysates were prepared at the end of the in vitro experiments in order to investigate the intracellular activity of superoxide dismutase (SOD), catalase (CAT), as well as the concentrations of glutathione (GSH) and malondialdehyde (MDA). FeAA treatment led to a reduced sperm motility (P < 0.001) and viability (P < 0.001), decreased the antioxidant parameters of the samples (P < 0.001 in case of SOD; P < 0.01 with respect to CAT; P < 0.05 in relation to GSH) but increased the superoxide production (P < 0.001) and lipid peroxidation (P < 0.001). RES supplementation resulted in a preservation of the spermatozoa vitality and antioxidant characteristics (P < 0.001 in case of SOD; P < 0.01 with respect to 25–50 μmol L−1 RES and P < 0.05 in relation to 10 μmol L−1 RES; P < 0.05 in case of GSH), with 50 μmol L−1 RES proving to be the most effective RES concentration. Our results suggest that RES possesses significant antioxidant properties that may prevent the deleterious effects caused by ROS to spermatozoa, and preserve the fertilization potential of male reproductive cells.

A child with chronic granulomatous disease

A four-year-old African-American female with a history of asthma, a Staphylococcus aureus skin abscess, and two prolonged pneumonia episodes presented with a one-week history of productive cough, fever, and night sweats. Chest computed tomography revealed right lung consolidation, cavitation within the right azygoesophageal recess, and an enlarged paratracheal lymph node, and biopsy of the lymph node showed granulomas. Lung tissue culture grew Burkholderia cepacia. A nitroblue tetrazolium test indicated a diagnosis of chronic granulomatous disease (CGD). CGD is a rare primary immunodeficiency in which there is a functional defect in one of the genes encoding nicotinamide adenine dinucleotide phosphate oxidase, which leads to impaired killing of catalase-producing bacteria and fungi within the phagolysosome. CGD presents most commonly as re- current and prolonged pulmonary infections, but may also include repeated episodes of lymphadenitis, cutaneous infections, hepatic abscesses, and osteomyelitis. Staphylococcus aureus is the most commonly isolated organism, but infection with certain other bacteria such as Burkholderia cepacia is highly suggestive of CGD.

Presence of a Juvenile Idiopathic Arthritis and Chronic Granulomatous Disease in a Child.

Dear Editor, Chronic Granulomatous Disease (CGD) is a result of deficient oxidative burst and impaired generation of reactive oxygen species due to mutations in genes encoding for NADPH oxidase complex (1). CGD usually presents with recurrent infections such as pneumonia, lymphadenitis osteomyelitis and hepatic or other abscesses (2) Rheumatologic disorders is a rare manifestation of first presentation of CGD. Juvenile idiopathic arthritis (JIA) is the most common chronic rheumatic disease among children. It collectively refers to a group of chronic arthritis of at least six weeks duration in children or adolescents under the age of 16 (3). JIA is a common problem in immuodefficiency diseases but is not common in CGD. However, there is a little reports of concordances of rheumatologic diseases and immunodeficiencies. In addition to increased susceptibility to infections in patients with CGD, a higher incidence of sterile inflammatory disorders in these patients has been noted (4). Our patient is a young boy who was born to the related parents whose their previous two children were dead because of myelomeningocele and anencephaly, respectively. The patient was 2 years old when he suffered from pelvic pain .Considering clinical findings in addition to 4 + RF, anti-CCP: 200 and negative HLA B5, B8 and B27, he was diagnosed as poly articular JIA. He was on medication (prednisolone and metotraxate) for years without remission but he had any complications. When he was 6 years old he experienced several episodes of sinusitis, cervical lymphadenitis and pneumonia. His lymphadenitis was managed surgically because it did not respond to non invasive therapies. In his admission to hospital because of pneumonia several work ups were performed as a result of unsuccessful antibiotic therapy. Bronchoscopy revealed nothing. Complete bold count was normal (WBC = 9300 with 63% PMN). CD markers and all serum immunoglobulins level were in normal range. Nitroblue Tetrazolium (NBT) test in patients was 0 but in father and mother’s patient was 87 and 98, respectively (normal range: 52 - 88 μg). In addition, activity dihydrorhodamine (DHR) Flow Cytometric test reduced in patient (DHR = 8) and in father and mother’s patient was 27.5. The patient underwent CGD treatment based on the laboratory data, and clinical improvement was soon appeared. Now he is on JIA and CGD treatment with poor control of his joint involvements and he gradually developed ulnar and plantar deviation which has made him using braces. There are some reports of concordances of rheumatologic diseases and immunodeficiencies. There are reports of CGD accompanying Kawasaki disease (5) DiGeorge and JIA (6), CGD in a girl with SLE and CGD in a patient with antiphospholipid antibody syndrome (7). Lee and Yap were among the first researchers who reported a patient with CGD and JIA. Despite our patient, they reported a girl who developed JIA after CGD (8). De Ravin and colleagues also reported different cases of CGD which gradually developed different rheumatologic disorders among them there is a patient with JIA who had been previously diagnosed as CGD (7). The difference between these reports and ours was the priority of immunodeficiency in their cases and rheumatologic disorder in our case. Some authority believe CGD might itself be an associated risk factor for developing immune-based inflammatory disorders (4). However, there are authors that believe JIA acts as a predisposing factor for autoimmunity (7). Collectively, we believe that immunodeficiency and rheumatologic disorders are the two ends of a spectrum in which a patient might demonstrate different manifestations of each. So, for each immunodeficient or rheumatologic patient every sign and symptoms should be precisely considered because it might be a presentation of a new disorder.

Dose- and Time-Dependent In Vitro Effects of Divalent and Trivalent Iron on the Activity of Bovine Spermatozoa.

This in vitro study was designed to assess the impact of divalent (Fe(2+)) or trivalent (Fe(3+)) iron on the activity and oxidative balance of bovine spermatozoa at specific time intervals (0, 2, 8, 16, and 24h) during an in vitro culture. Forty-five semen samples were collected from adult breeding bulls and diluted in physiological saline solution supplemented with different concentrations (0, 1, 5, 10, 50, 100, 200, 500, 1000μmol/L) of FeCl2 or FeCl3. Spermatozoa motion parameters were assessed using the SpermVision™ computer-aided sperm analysis (CASA) system. Cell viability was examined with the metabolic activity 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and the nitroblue-tetrazolium (NBT) test was applied to quantify the intracellular superoxide formation. Both divalent and trivalent iron exhibited a dose- and time-dependent impact on the spermatozoa physiology and oxidative balance. Concentrations ≥50μmol/L FeCl2 and ≥100μmol/L FeCl3 led to a significant decrease of spermatozoa motility (P < 0.05) and mitochondrial activity (P < 0.001 with respect to 200-1000μmol/L FeCl2/FeCl3; P < 0.01 in case of 100μmol/L FeCl2/FeCl3), accompanied by a significant superoxide overproduction (P < 0.001 in terms of 200-1000μmol/L FeCl2 and 500-1000μmol/L FeCl3; P < 0.01 with respect to 100μmol/L FeCl2 and 100-200μmol/L FeCl3). On the other hand, concentrations below 10μmol/L FeCl2 and 50μmol/L FeCl3 proved to stimulate the spermatozoa activity, as shown by a significant preservation of the motility and viability characteristics (P < 0.001 in case of the motility parameters; P < 0.01 with respect to the spermatozoa viability), alongside a significant decline of the superoxide generation (P < 0.05). In a direct comparison, divalent iron has been shown to be more toxic than trivalent iron. Results from this in vitro study show that high concentrations of both forms of iron are toxic, while their low concentrations may have spermatozoa activity-promoting properties. In vitro concentrations of divalent or trivalent iron that could be regarded as critical are 50μmol/L FeCl2 and 100μmol/L FeCl3 when iron ceases to be an essential micronutrient in order to become a toxic risk factor.

LĖTINĖ GRANULIOMINĖ LIGA: LITERATŪROS APŽVALGA IR KLINIKINIŲ ATVEJŲ APRAŠYMAS

Chronic granulomatous disease (CGD) is an inherited X-linked or, less frequently, with somatic mutations linked disorder of phagocytic cells which presents in early childhood as recurrent granulomatous lesions and atypical bacterial or fungal infections of skin, lungs, gastrointestinal tract, liver, spleen or lymph nodes. Some patients remain undiagnosed until adulthood or rarely diagnosis is made post mortem. Haematopoietic stem cell transplantation (HSCT) can cure CGD, thus timely diagnosis is essential for saving life of such patients. We present two complicated clinical cases of children with CGD, diagnosed in Lithuania for the first time. After the history of recurrent epizodes of severe infections, reviewed family history, repeated spontaneous and stimulated nitroblue tetrazolium tests (NBT), CGD diagnosis was confirmed for both patients. Allogeneic HSCT is planned in both cases expecting curative effect. All children with recurrent atypical infections should be checked for immunodeficiency. Multidisciplinary approach is essential for making rapid and correct diagnosis based on life and family history, clinical manifestation, immunologic tests and genetic counseling. CGD is curable by HSCT, thus patients should be referred to a HSCT center without delay

Performance of Cage‐Raised, Overwintered Hybrid Striped Bass Fed Fish Meal‐ or Soybean‐Based Diets

AbstractWe conducted a 26‐week study to evaluate the performance of cage‐raised hybrid Striped Bass (female White Bass Morone chrysops × male Striped Bass M. saxatilis) fed fish meal (FM) and soybean (SOY; soybean meal + soy protein concentrate) based diets. A FM reference diet was formulated to contain crude protein (CP) at 450 g/kg, lipid at 120 g/kg, and an estimated digestible energy level of 13 MJ/kg. Two test diets (SOY and SOY+GBA) were formulated to replace 70% of the CP in the reference diet with SBM in the absence or presence of GroBiotic‐A (GBA), a yeast‐based prebiotic, at 20 g/kg. The other 12% of dietary FM in the reference diet was replaced with SPC in the test diets, further reducing FM inclusion to 70 g/kg. Each diet was fed to quadruplicate groups of 40 advanced juvenile hybrid Striped Bass (initial weight ∼ 54 g/fish) stocked into each of twelve 1‐m3 floating cages, which were equally divided into four 0.05‐ha, rubber‐lined ponds according to a randomized complete block design. Caged fish in each pond were fed one of the randomly assigned diets to apparent satiation once daily. After 26 weeks of feeding, results showed that (1) the overall growth performance, survival, and feed efficiency of the fish were unaffected by diet; (2) the intraperitoneal fat ratio was significantly lower in fish that received the SOY‐based diets; and (3) plasma glucose and lysozyme activity were significantly lower in fish that were fed the SOY‐based diets, whereas plasma osmolality, hematocrit, neutrophil oxidative radical production (nitroblue tetrazolium test), and extracellular superoxide anion production of head‐kidney macrophages were unaffected by diet. We conclude that low‐FM, SOY‐based diets can support adequate performance and normal physiological responses of hybrid Striped Bass during overwintering.

CURCUMIN IN MALE FERTILITY: EFFECTS ON SPERMATOZOA VITALITY AND OXIDATIVE BALANCE

The aim of this study was to assess the dose- and time-dependent effects of curcumin on bovine spermatozoa during short-term (0h, 2h, 6h) and long-term (12h, 24h) in vitro culture periods. Semen samples were collected from 20 adult breeding bulls, and diluted in physiological saline solution containing 0.5% DMSO together with 0, 1, 5, 10, 50 and 100 μM/L of curcumin. Spermatozoa motion parameters were determined using the SpermVisionTM and CASA (Computer Assisted Semen Analyzer) system. Cell viability was measured using the metabolic activity MTT assay, and the nitroblue-tetrazolium (NBT) test was used to assess the intracellular superoxide formation. The CASA analysis revealed that concentrations of 50 μM/L and 10 μM/L of curcumin were able to significantly prevent the decrease of motility and progressive motility (P

IN VITRO SUPPLEMENTATION OF RESVERATROL TO BOVINE SPERMATOZOA: EFFECTS ON MOTILITY, VIABILITY AND SUPEROXIDE PRODUCTION

The aim of this study was to assess the dose- and time dependent in vitro effects of resveratrol (RES), a natural polyphenol and phytoalexin with potential antiviral, anti-inflammatory and antioxidant properties on bovine spermatozoa during five different time periods (0h, 2h, 6h, 12h and 24h). Semen samples were collected from 20 adult breeding bulls, and diluted in physiological saline solution containing 0.5% DMSO together with 0, 1, 5, 10, 50, 100 and 200 μM/L RES. Spermatozoa motility was examined using the Sperm VisionTM and CASA (Computer Assisted Semen Analyzer) system. Cell viability was measured using the metabolic activity MTT assay, the nitroblue-tetrazolium (NBT) test was used to assess the intracellular superoxide formation. The initial CASA analysis showed no significant changes in the spermatozoa motion parameters, however a motion-promoting effect of 10 and 50 μM/L RES became significant after 2h (P