Higher Respiratory Burst Activity Research Articles

Influences of Bacillus subtilis and fructooligosaccharide on growth performances, immune responses, and disease resistance of Nile tilapia, Oreochromis niloticus.

The present study investigated the effects of Bacillus subtilis and fructooligosaccharide (FOS) on growth performances, immunity improvement, and disease resistance of Nile tilapia (Oreochromis niloticus). The fish (24.5 ± 1.6 g) were fed a basal diet (G1), diets supplemented with 1 g/kg (G2), 3 g/kg (G3) and 5 g/kg (G4) of FOS as well as diets supplemented with 1 × 109 CFU/g (G5), 3 × 109 CFU/g (G6) and 5 × 109 CFU/g (G7) of B. subtilis for 56 days. After the feeding trial, the complement C3, IL-1β, TNF-α, IFN-γ, hsp70 gene expression in the liver was then analyzed by a quantitative Real-time PCR. Then, fish were infected with Streptococcus agalactiae, and the survival rate was recorded. The results showed that FOS and B. subtilis had no significant effect (P > 0.05) on growth performances and survival rate. Lysozyme activity was significantly greater in the G4, G5, G6, and G7 groups. Also, all fish fed FOS and B. subtilis showed significantly (P < 0.05) higher respiratory burst activity than other groups. The expressions of complement C3, IL-1β, TNF-α, IFN-γ, and hsp-70 in the liver were significantly higher for fish fed 5 g/kg of FOS as well as for fish that received any concentration level of B. subtilis (P < 0.05) used in the study. After the S. agalactiae challenge test, the survival rate of fish-fed diets supplemented with FOS and B. subtilis was slightly higher than for the control group. The results indicated that FOS and B. subtilis could stimulate immune responses and immune-related genes in tilapia. However, further investigation of other prebiotics or herbs in combination with B. subtilis is encouraged at molecular levels and screening for beneficial metabolites that may increasingly improve digestive enzymes, growth performances, and health benefits in tilapia. In addition, on-farm experiments are needed.

Different dietary protein levels for Podocnemis unifilis subadult farming: hematological and biochemical assessment

ABSTRACT This study evaluated the physiological responses of subadult yellow-spotted Amazon river turtle, Podocnemis unifilis, to incremental crude protein levels (29%, 32%, 35%, and 39% CP) as extruded commercial feeds. The hematological and plasma biochemical parameters of P. unifilis were analyzed at 60, 120, and 180 days of the feeding trial. The increase in total thrombocyte, leukocyte, lymphocyte, heterophil, and eosinophil numbers, besides high respiratory burst activity showed an improvement in animal immune defense response to incremental protein in diets, acting together to maintain the integrity of the tissues against antigens and infectious agents. Elevated subadult P. unifilis plasma total cholesterol and triglyceride levels observed after increasing the feeding time (until 180 days) with incremented protein levels in artificial diets during captivity are a nutritional warning, related to the condition of being less physically active. Based on these results, periodic physiological evaluations are particularly important to ensure the healthy and adequate nutritional conditions of captive-bred animals, such as maintaining lower stocking densities of animals to avoid stress and, if possible, providing them with fresh food in addition to extruded artificial feed.

Dietary supplementations of methionine improve growth performances, innate immunity, digestive enzymes, and antioxidant activities of rohu (Labeo rohita).

The dietary composition of fish constitutes numerous immune and growth-promoting substance; during the current study, methionine was amended in the diet of Labeo rohita (L. rohita), in order to appraise the diverse modulatory effects of methionine supplementation. Fish were grouped into three on the basis of methionine as methionine supplemented group 1 (MSG1), methionine supplemented group 2 (MSG2), and CTRL (control group). We assessed the effects of methionine at the end. Captivating results were obtained from the methionine supplementing groups. The results depict significantly high (p < 0.05) innate immunity parameters, including myeloperoxidase, IgG concentrations, lysozyme, respiratory burst, and total bacteriolytic activity of complement in MSG2. Furthermore, ameliorate growth performances were also noticed such as high weight gain, FCE%, and body length in MSG2, respectively. We also measuredhigh level of antioxidant digestive enzymes, gut length, and survival rate in MSG2 compared to the rest of the groups. The results were further validated by measuring the stress parameters, such as aspartate aminotransferase (AST), glucose, and hematocrit (HCT%), and the concentration remains low in MSG2. Lastly, after 90 days, fish were challenged with Aeromonas hydrophila, the highest survival was observed in the MSG2 and MSG1 compared to CTRL. These results ultimately give the baseline to the credible effects of methionine on L. rohita.

Dietary carotenoprotien extracted from shrimp shell waste augments growth, feed utilization, physio-metabolic responses and colouration in Oscar, Astronotus ocellatus (Agassiz, 1831)

Carotenoprotein, a complex of carotenoids and protein, extracted from shrimp shell waste can be an effective source of carotenoid as well as antioxidant in fish diets. In the present study, the nutritional potential of carotenoprotein extracted from shell wastes of Parapeneopsis stylifera on growth, physiometabolic changes and coloration in high-value ornamental fish, oscar (Astronotus ocellatus) was assessed in a 120 days feeding trial. Four iso‑nitrogenous (30–31% CP) and isolipidic (11.34–11.55% CL) diets were formulated with graded levels of carotenoprotein inclusion like 0%, 5%, 10% and 15% and designated as Control, T1, T2 and T3 respectively. At the end of the feeding trial, growth indices such as final body weight, weight gain, and specific growth rate indicated that fish fed with 10 and 15% levels of carotenoprotein incorporated diet showed higher growth compared to control and T1. Oxidative stress enzymes like SOD and Catalase were lower in 15% and 10% carotenoprotein incorporated diets respectively in both gills and liver. Stress indicators like serum glucose and cortisol activity were found to be higher in control than in diets with 10% (T2) as well as 15% (T3), suggesting the stress-reducing ability of the carotenoprotein in fish feed. Relatively higher respiratory burst activity in fishes of T3 group indicated higher immunity status of the fish. Quantitative colour values in terms of redness, chroma in the fin region as well as carotenoid content in scale, skin and muscle of fish were higher in T2 (10% carotenoprotein incorporated diet) as compared to T3 (15% carotenoprotein incorporated diet). Overall, this study clearly showed that dietary incorporation of as little as 10% carotenoprotein is advisable to ornamental fish farmers for enhanced growth, colouration, as well as antioxidant activity in this popular high value ornamental fish for maximizing their profit.

Dietary butyric acid improved growth, digestive enzyme activities and humoral immune parameters in Barramundi (Lates calcarifer)

A six-week feeding trial was conducted to determine the effect of butyric acid (BA) inclusion in diet on performance of Barramundi (Lates calcarifer). Fish (12.0 ± 0.2 g) were fed with four experimental diets contained 0.0 (control), 2.5, 5.0 and 10.0 g BA/kg in triplicate. Fish fed on 5 and 10 g BA/kg diets had higher growth and total alkaline protease and lipase activities than the other treatments. Fillet protein content in BA-supplemented groups was higher than the control. Liver catalase activity was highest in 2.5 g BA/kg group compared with the other groups. The values of liver superoxide dismutase activity and serum total protein concentration in 5 and 10 g BA/kg treatments were higher than those in the control (p < .05). Moreover, the highest and the lowest levels of serum lysozyme activity were noticed in the 10 g BA/kg and the control groups, respectively (p < .05). Also, the highest respiratory burst activity was observed in fish fed 5 g BA/kg feed. Supplementing diet with 2.5 or 5 g BA/kg resulted in higher serum haemolytic activity in fish compared with the other groups. The number of red and white blood cells increased in fish fed BA-supplemented diets compared with the control (p < .05). In conclusion, the results of this study showed inclusion of BA at 5 g/kg diet in L. calcarifer juveniles improved growth and upgraded the general health condition by enhancing fish antioxidant enzyme activities and haemato-immunological responses.

Dietary supplementation of thiamine and pyridoxine-loaded vanillic acid-grafted chitosan microspheres enhances growth performance, metabolic and immune responses in experimental rats

In the present investigation, the effect of dietary supplementation of thiamine and pyridoxine loaded vanillic acid-grafted chitosan microspheres (TPVGC) on growth, metabolic and immune responses in Wistar strain albino rats was studied. Eight experimental groups, namely four groups each for male and female rats were fed with 0, 0.4, 0.8 and 1.6% of TPVGC in the diet. At the end of 45days feeding trials, both male and female rats supplemented with TPVGC had higher weight gain% and specific growth rate than the control groups. Significantly (p<0.05) lower blood glucose level and higher respiratory burst activity were recorded in the treatment groups than the control groups of both male and female rats. Activity of metabolic enzymes (aspartate amino transferase, alanine aminotransferase, alkaline phosphatase and acid phosphatase) and antioxidant enzymes (superoxide dismutase, catalase and glutathione S-transferase) were significantly higher (p<0.05) in the control groups and a decreasing trend in the same was observed with a gradual increase in the inclusion level of TPVGC in the diet of the treatment groups. However, a reverse trend was observed for acetylcholine esterase. It was inferred that dietary supplementation of thiamine and pyridoxine loaded vanillic acid-grafted chitosan enhanced the growth performance, metabolic and immune responses in the animal-model.

Immuno-stimulating impacts of dietary enrichment of vitamin C and E combinations in labeo Rohita Fingerlings

A 30 day feeding trial was conducted to determine the effect of dietary enrichment of vitamin C (VC) and vitamin E (VE) on growth and health status of Labeo rohita fingerlings. Two hundred and forty fingerlings (average weight 3.53 ± 0.04 g) were randomly distributed into four treatment groups with three replicates each. Four iso-nitrogenous (crude protein 40.09–40.21g 100g−1 feed) and iso-caloric (396.73–399.9 Kcal DE 100g −1) purified diets were prepared with different combinations of VC and VE, viz. control (VC @ 1g kg−1 + VE @0.1g kg−1), T1 (VC @2g kg−1 + VE @ 0.1g kg−1), T2 (VC @1g kg−1 + VE @0.2g kg−1), T3 (VC @ 2g kg−1 + VE @0.2g kg−1). In the present study, higher vitamin supplementation in the diets had no significant effect on the percentage weight gain, specific growth rate and FCR values, in the absence of any stressors. Although, VC and VE supplemented diets at different concentrations showed no significant effect on the catalase activity, significant differences were observed in the SOD values. The VC fed at high level along VE showed significantly higher respiratory burst activity as compared to control irrespective of vitamin E inclusion level. Significant differences observed in the lysozyme activity and survival rate, before and after Aeromonas hydrophila infection, signifies the immune-protective and immune-stimulating effects of VC and VE in Labeo rohita fingerlings.

Flow cytometric measurement of respiratory burst activity and surface expression of neutrophils for septic patient prognosis

The hyperinflammation that begins with sepsis is essential for eradicating infection but also causes hypoperfusion and organ failure. To understand the innate immune status of septic patients, the functional and phenotypic changes of neutrophils during sepsis and their clinical implication were studied. Seventy-four patients who were admitted to intensive care unit due to severe sepsis or septic shock were enrolled. Surface antigens of neutrophils (CD64, CD10, and CD16) were detected by flow cytometry. Respiratory burst activity (RBA) was measured by flow cytometry using 2',7'-dichlorofluorescein diacetate and phorbol-12-myristate-13-acetate. The parameters were serially examined at Days 1 and 8 in septic shock patients. High CD64 and low CD10 and CD16 on Day 1 was associated with sepsis severity (P = 0.003, 0.017, and 0.007, respectively). On Day 1, RBA and CD64 were higher in survivors than in nonsurvivors of septic shock patients (P = 0.012 and 0.027, respectively), and on Day 8, CD10 and CD16 were higher in survivors than in nonsurvivors (P = 0.019 and 0.036, respectively). High RBA and high CD64 on Day 1 showed low 28-day mortality in univariate analysis (P = 0.018 and 0.034, respectively). In multivariate analysis, RBA maintained statistical significance (P = 0.042) but CD64 revealed only a tendency (P = 0.064). Neutrophil surface antigen (CD64, CD10, and CD16) could reflect sepsis severity. High CD64 expression and high RBA at early phase of sepsis might be associated with better prognosis, whereas high expression of CD10 and CD16 at late phase of sepsis might be associated with better prognosis. © 2015 International Clinical Cytometry Society.

Excretory products of the cestode, Schistocephalus solidus, modulate in vitro responses of leukocytes from its specific host, the three-spined stickleback (Gasterosteus aculeatus)

Helminth parasites have evolved remarkable strategies to manipulate the immune system of their hosts. During infections of three-spined stickleback (Gasterosteus aculeatus) with the cestode Schistocephalus solidus prominent immunological changes occur, presumably due to manipulative activity of the parasite. We hypothesise that excretory/secretory products of the parasite are involved in the manipulation of the stickleback's immune system and that this may depend on the individual parasite and its origin. We therefore produced S. solidus conditioned cell culture media (SSCM) with parasites from different origins (Norway, Spain and Germany) and exposed head kidney leukocytes (HKL) from un-infected sticklebacks in cell cultures to SSCM. After in vitro culture, HKL were subjected to differential cell counts (granulocytes/lymphocytes) by means of flow cytometry. Leukocyte sub-populations were analysed for cell viability and changes in cell morphology. The respiratory burst activity was measured with a luminescence assay. Exposure of HKL to SSCM induced an up-regulation of respiratory burst activity after already 1 h, which was still elevated at 24 h, but which was in some cases significantly down-regulated after 96 h. Respiratory burst was positively correlated with the number of live granulocytes in the culture, suggesting that the respiratory burst activity was changed by SSCM effects on granulocyte viability. After 1 h and 24 h of HKL culture, no lymphocyte responses to SSCM were detectable, but after 96 h lymphocyte viability was significantly decreased with SSCM from Spanish S. solidus. In these cultures, residual lymphocytes increased in size, suggesting that cell death and activation might have occurred in parallel. The highest respiratory burst activity was induced by SSCM from Spanish parasites, in particular when they were grown in sympatric sticklebacks. The in vitro HKL responses to SSCM depended on the individual parasite and its population of origin, suggesting that in vivo, S. solidus excretory products are regulated individually, possibly to balance the interplay of each individual host–parasite pair.

Phagocytosis and Respiratory Burst Activity in Lumpsucker (Cyclopterus lumpus L.) Leucocytes Analysed by Flow Cytometry

In the present study, we have isolated leucocytes from peripheral blood, head kidney and spleen from lumpsucker (Cyclopterus lumpus L.), and performed functional studies like phagocytosis and respiratory burst, as well as morphological and cytochemical analyses. Different leucocytes were identified, such as lymphocytes, monocytes/macrophages and polymorphonuclear cells with bean shaped or bilobed nuclei. In addition, cells with similar morphology as described for dendritic cells in trout were abundant among the isolated leucocytes. Flow cytometry was successfully used for measuring phagocytosis and respiratory burst activity. The phagocytic capacity and ability were very high, and cells with different morphology in all three leucocyte preparations phagocytised beads rapidly. Due to lack of available cell markers, the identity of the phagocytic cells could not be determined. The potent non-specific phagocytosis was in accordance with a high number of cells positive for myeloperoxidase, an enzyme involved in oxygen-dependent killing mechanism present in phagocytic cells. Further, high respiratory burst activity was present in the leucocytes samples, verifying a potent oxygen- dependent degradation. At present, the specific antibody immune response could not be measured, as immunoglobulin or B-cells have not yet been isolated. Therefore, analyses of the specific immune response in this fish species await further clarification. The present study presents the first analyses of lumpsucker immunity and also the first within the order Scopaeniformes.

Inhibition ability of probiotic, &lt;i&gt;Lactococcus lactis&lt;/i&gt;, against &lt;i&gt;A. hydrophila&lt;/i&gt; and study of its immunostimulatory effect in tilapia (&lt;i&gt;Oreochromis niloticus&lt;/i&gt;)

The present study was designed to investigate the inhibition ability of probiotic, Lactococcus lactis RQ516, against A. hydrophila in vitro and its immunostimulatory effect in tilapia, Oreochromis niloticus as growth promoter. Six tanks were used (T-1 and control treated with and without RQ516, respectively) and three replicates per treatment. The inhibition ability assay in vitro showed that the minimum diameter of the inhibition zone (P < 0.05) was 7.43 ± 0.47 mm at 6 h and the final diameter at 24 h was 14.77 ± 1.17 mm. After 40 days, probiotic treatment as water additives significantly improved the final weight and daily weight gain (DWG) (P < 0.05). A significant increase (P < 0.05) in the total protein and globulin concentration of tilapia blood serum could be found in T-1 compared with the control. These immune responses were generally more pronounced with probiotic-treated tilapia. As for immune responses, the higher respiratory burst activity (RBA), lysozyme content (LC), myeloperoxidase (MPO) and superoxide dismutase (SOD) activities were observed in T-1 (P < 0.05) than the control. It indicated that probiotic Lactococcus lactis RQ516 was beneficial for tilapia in terms of increasing final weight, DWG and the concentrations of serum protein and globulin and enhancing immune responses. International Journal of Engineering, Science and Technology, Vol. 2, No. 7, 2010, pp. 73-80

Passive immune-protection of small abalone against Vibrio alginolyticus infection by anti- Vibrio IgY-encapsulated feed

Small abalone ( Haliotis diversicolor supertexta) is a high value-added shellfish. It however has been suffering Vibrio alginolyticus infections, which cause mass death of small abalone and thus great economic losses, particularly in artificial aquaculture. In this study, we attempted to treat small abalone with anti- Vibrio IgY to elicit a passive immunity directly against V. alginolyticus infections. Anti- Vibrio IgY was alginate encapsulated in egg powders as feed, which may avoid antibody inactivation in the gastrointestinal tract of small abalone. The feed was tested for the stability of anti- Vibrio IgY in a gastrointestinal mimic environment. The result showed anti- Vibrio IgY retained activity as high as 90% after 4 h exposure to pancreatic enzymes. Addition of 0, 5 or 10% anti- Vibrio IgY-encapsulated egg powders into a basal diet to form abalone diet formulae. Small abalones fed with the anti- Vibrio IgY formulae showed a relatively high respiratory burst activity than those without anti- Vibrio IgY treatments. The survival rates of small abalones fed with 5 or 10% anti- Vibrio IgY egg powders were in the range of 65–70% 14 days post- V. alginolyticus challenge (1 × 10 6 c.f.u.), which was significantly higher than 0% of those fed without anti- Vibrio IgY. The anti- Vibrio IgY-encapsulated formulae were thus concluded to be an effective means to prevent small abalone from V. alginolyticus infection, and may be practical in use in abalone aquaculture.

Enhanced innate immune parameters in Labeo rohita (Ham.) following oral administration of Bacillus subtilis

The present study assessed the use of Bacillus subtilis in fish as a probiotic. The bacterium was administered orally at three different doses 0.5 × 10 7 (T 2), 1 × 10 7 (T 3), 1.5 × 10 7 (T 4) cfu/g feed to Labeo rohita for two weeks. The positive control group (T 1) and negative control group (T 5,) were fed feed without B. subtilis for the same period. On the 15th day blood and serum were sampled to determine respiratory burst activity (NBT assay), differential leukocyte counts (DLC) and serum bactericidal activity. Fishes were challenged intraperitoneally with Aeromonas hydrophila O:18 after two weeks in the treatment groups (T 2, T 3 and T 4) and also in the positive control group(T 1), while the negative control group (T 5) was challenged with phosphate buffered saline (PBS, pH 7.2) only. The respiratory burst activity and DLC were assessed on the 3rd day post-challenge. B. subtilis treated fish showed significantly higher ( P < 0.05) respiratory burst activity and bactericidal activity during the pre-challenge compared with the control groups. The highest respiratory burst activity (0.37 ± 0.03) and serum bactericidal activity were recorded in the group (T 4) fed feed containing B. subtilis at 1.5 × 10 7 cfu/g feed. Granulocyte numbers were significantly higher ( P < 0.05) in treatment groups in comparison to the control in both the pre- and post-challenge periods. The result suggests that B. subtilis can enhance certain innate immune responses in rohu.

Comparison of respiratory burst activity of inflammatory neutrophils in ayu ( Plecoglossus altivelis) and carp ( Cyprinus carpio)

Neutrophils of ayu ( Plecoglossus altivelis) were previously shown to have unusually high respiratory burst activity (RBA). To understand this unique character of ayu neutrophils, the RBAs of resting and inflammatory neutrophils of ayu and carp ( Cyprinus carpio) were compared. Inflammation was induced in the peritoneal cavity by injecting killed-bacteria. The RBA of peritoneal-exudate (inflammatory) neutrophils was measured after stimulation with phorbol myristate acetate (PMA). Resting neutrophils were obtained from kidney and blood of non-injected fish. In carp, the RBA of inflammatory neutrophils was much higher than that of resting neutrophils. On the other hand, in ayu no significant difference was observed. The RBA of neutrophils was already high in the kidney stock. The process of inflammation did not further enhance RBA. In addition to PMA, other stimulants (zymosan, opsonized-zymosan, and zymosan-treated serum) were used to measure RBA. Even with these stimulants, the RBA of inflammatory neutrophils was always higher than that of kidney neutrophils in carp. On the other hand in ayu, the RBA of kidney neutrophils was already high in the kidney stock, and no significant difference was observed between peritoneal and kidney neutrophils in ayu. These results indicate ayu neutrophils have spontaneously activated characteristics with the respect to the ROS generation in the kidney hematopoietic-stock.

Redox regulation of thyroid hormone-induced Kupffer cell-dependent IκB-α phosphorylation in relation to inducible nitric oxide synthase expression

Thyroid hormone-induced calorigenesis promotes oxidative stress in the liver with higher respiratory burst activity in Kupffer cells, which could increase the expression of redox-sensitive genes. Our aim was to test the hypothesis that L-3,3′,5-triiodothyronine (T3) triggers inducible nitric oxide synthase (iNOS) expression in rat liver by upstream mechanisms involving the inhibitor of κB (IκB) kinase activation. T3 administration (daily doses of 0.1 mg/kg for three consecutive days) induced a calorigenic response, with maximal increases in the content of hepatic thiobarbituric acid reactants or protein carbonyls and NOS activity at 48–72 h after treatment, compared to control values. In this time interval, the serum levels of tumor necrosis factor-α (TNF-α; ELISA) are enhanced, concomitantly with higher liver IκB-α phosphorylation (Western blot analysis), NF-κB DNA binding (electrophoretic mobility shift assay), and iNOS mRNA expression (reverse transcription-polymerase chain reaction). These changes and the increase in hepatic NOS activity are abolished by the administration of either α-tocopherol (100 mg/kg) or the Kupffer cell inactivator gadolinium chloride (10 mg/kg) prior to T3. It is concluded that T3-induced oxidative stress triggers the redox upregulation of liver iNOS expression through a cascade initiated by TNF-α produced by Kupffer cells and involving IκB-α phosphorylation and NF-κB activation, a response that may represent a defense mechanism by protecting the liver from cytokine-mediated lethality and ROS toxicity.

Dietary l-ascorbic acid affects growth, nonspecific immune responses and disease resistance in juvenile grouper, Epinephelus malabaricus

An 8-week feeding trial was conducted to estimate the adequate dietary l-ascorbic acid (AA) requirements of juvenile grouper and its effect on nonspecific immune responses and disease resistance when challenged with Vibrio carchariae. AA supplemented at 0, 20, 50, 80, 150, 250, 400 and 800 mg/kg diet in the basal diet providing of 0, 3, 14, 27, 46, 76, 135 and 288 AA mg/kg diet, respectively. Each diet was fed to triplicate groups of grouper with initial body weight of 7.58±0.05 g. Fish fed diets supplemented with ≥46 mg AA/kg had significantly ( P<0.05) greater weight gain (WG) and feed efficiency (FE) than fish fed diets with ≦14 mg AA/kg. After the final weighing, 10 fish were randomly taken from each aquarium and injected with V. carchariae. Mortality was recorded daily for 7 days and nonspecific immune parameters were monitored in the remaining fish. Higher cumulative mortality was in fish fed the unsupplemented diet or the 3-mg AA/kg diet than other dietary groups. Fish fed the 288-mg AA/kg diet had higher respiratory burst activity (superoxide O 2 −) of head kidney leukocytes and alternative complement activity than fish fed the unsupplemented diet and diets with ≦76 mg AA /kg, respectively. Lysozyme activity was higher in fish fed diets with ≥76 mg AA than fish fed diets with ≦27 mg AA/kg. These data indicated that the adequate dietary AA concentration for optimum growth of juvenile grouper is 45.3 mg AA/kg diet, and six times the optimum dietary AA level was needed to enhance the nonspecific immune responses and maintain survival of the fish infected with bacteria.

Superoxide release and cellular gluthatione peroxidase activity in leukocytes from children with persistent asthma

Asthma is an inflammatory condition characterized by the involvement of several mediators, including reactive oxygen species. The aim of the present study was to investigate the superoxide release and cellular glutathione peroxidase (cGPx) activity in peripheral blood granulocytes and monocytes from children and adolescents with atopic asthma. Forty-four patients were selected and classified as having intermittent or persistent asthma (mild, moderate or severe). The spontaneous or phorbol myristate acetate (PMA, 30 nM)-induced superoxide release by granulocytes and monocytes was determined at 0, 5, 15, and 25 min. cGPx activity was assayed spectrophotometrically. The spontaneous superoxide release by granulocytes from patients with mild (N = 15), moderate (N = 12) or severe (N = 6) asthma was higher at 25 min compared to healthy individuals (N = 28, P < 0.05, Duncan test). The PMA-induced superoxide release by granulocytes from patients with moderate (N = 12) or severe (N = 6) asthma was higher at 15 and 25 min compared to healthy individuals (N = 28, P < 0.05 in both times of incubation, Duncan test). The spontaneous or PMA-induced superoxide release by monocytes from asthmatic patients was similar to healthy individuals (P > 0.05 in all times of incubation, Duncan test). cGPx activity of granulocytes and monocytes from patients with persistent asthma (N = 20) was also similar to healthy individuals (N = 10, P > 0.05, Kruskal-Wallis test). We conclude that, under specific circumstances, granulocytes from children with persistent asthma present a higher respiratory burst activity compared to healthy individuals. These findings indicate a risk of oxidative stress, phagocyte auto-oxidation, and the subsequent release of intracellular toxic oxidants and enzymes, leading to additional inflammation and lung damage in asthmatic children.

Construction and functional activities of chimeric mouse-human immunoglobulin G and immunoglobulin M antibodies against the Neisseria meningitidis PorA P1.7 and P1.16 epitopes.

We studied the in vitro protective activities of human immunoglobulin G1 (IgG1), IgG3, and IgM antibodies against group B meningococci by constructing sets of chimeric mouse-human antibodies (chIgG1, chIgG3, and chIgM, respectively) with identical binding regions against the P1.7 and P1.16 epitopes on PorA. This was done by cloning the V genes of three mouse hybridoma antibodies and subsequently transfecting vectors containing the homologous heavy- and light-chain genes into NSO cells. Cell clones secreting intact human chIgG1, chIgG3, or chIgM antibodies originating from three parent mouse antibodies were isolated. The functional affinities appeared to be similar for all human isotypes and surprisingly also for the pentameric chIgM antibody. chIgG1 exhibited greater serum bactericidal activity (SBA) than chIgG3, while chIgG3 was more efficient in inducing a respiratory burst (RB) associated with opsonophagocytosis than chIgG1 was. On the other hand, chIgM exhibited SBA similar to that of chIgG1, but it exhibited much higher RB activity than chIgG3 and chIgG1 exhibited. The antibodies against the P1.16 epitope were more efficient in terms of SBA than the antibodies against the P1.7 epitope were; thus, 10- to 40-fold-lower concentrations of antibodies against P1.16 than of antibodies against P1.7 were needed to induce SBA. On the other hand, antibodies against these epitopes were equally effective in inducing RB. Our results revealed differences in the functional activities of human chIgG1, chIgG3, and chIgM antibodies against meningococci, which might influence their protective effects against meningococcal disease.

Flow cytometric analysis of the neutrophil respiratory burst of ayu, Plecoglossus altivelis: comparison with other fresh water fish

Neutrophils of vertebrates undergo respiratory burst activity (RBA) as a defense mechanism against bacterial infections. We report here that ayu ( Plecoglossus altivelis) have unusually high RBAs even when they are in a healthy condition. Kidney and blood leukocytes were obtained from ayu, rainbow trout ( Oncorhynchus mykiss), carp ( Cyprinus carpio), eel ( Anguilla japonica), and pond smelt ( Hypomesus nipponensis). Neutrophil RBA was measured by flow cytometry using dihydrorhodamine after stimulation with phorbol myristate acetate. The amount of RBA of neutrophils from both blood and kidney was significantly higher in ayu than in the other species (e.g. the fluorescence intensity of ayu blood neutrophils was about 3–7 times higher than that from trout and carp, and that of ayu kidney neutrophils was 2–19 times higher than that of rainbow trout, carp, eel, and pond smelt). This unique character of ayu neutrophils was invariable even at different ages, locations, and sex-maturation stages.

Polytrauma induces increased expression of pyruvate kinase in neutrophils

Polytrauma (PT) leads to systemic activation of polymorphonuclear neutrophils (PMNs). Organ damage commonly found in these patients is ascribed to respiratory bursts of activated PMNs. With the use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, PMN extracts from PT patients were found to contain a clear protein band not seen in control PMNs from healthy volunteers. This band was identified by amino acid sequencing and Western blotting as pyruvate kinase (PK). Enzymatic assays revealed a 600-fold increase in PK activity in PMNs of PT patients, with the highest levels occurring between the fifth and seventh posttraumatic day. In lymphocytes, no such increase was detectable. As PK is a major regulatory enzyme in glycolysis, glucose-dependent lactate production in PMNs from PT patients was assayed. These cells showed a higher glycolytic lactate production than controls. It was additionally demonstrated that acute activation of respiratory burst activity depends mainly on breakdown of glucose to lactate via the pentose-phosphate pathway and glycolysis. In PMNs from PT patients, this glucose-dependent respiratory burst activity was more than twofold higher than in controls. The increase in expression and activity of PK in PMNs from PT patients may contribute to the high glucose-dependent respiratory burst activity seen in these cells.