Respiratory Burst

CARRASCO, M., M. DEL RIO, A. HERNANZ AND M. DE LA FUENTE. Inhibition of human neutrophil functions by sulfated and nonsulfated cholecystokinin octapeptides. PEPTIDES 18(3) [415][416][417][418][419][420][421] 1997.-The effects of CCK-8s and desulfated CCK-8 at concentrations ranging from 10 014 to 10 06 M were studied in vitro on several functions of human peripheral neutrophils: adherence to substrate, mobility (spontaneous and directed by a chemical gradient or chemotaxis), ingestion of inert particles (latex beads) or cells ( Candida albicans), and production of superoxide anion measured by the nitroblue tetrazolium reduction test. The effect of CCK-8s on intracellular levels of cAMP was investigated as well as the implication of calcium in the action of CCK-8s on phagocytic function using stimulants and inhibitors of both intracellular and extracellular calcium channels. The two peptides, at concentrations from 10 012 to 10 08 M , inhibited significantly both mobility and ingestion capacities and increased adherence to substrate. A dose-response relationship was observed with a maximum inhibition of neutrophil functions at 10 010 M. CCK-8s and desulfated CCK-8 induced in these cells a significant, but transient, increase of cAMP levels at 60 s. Moreover, CCK-8s was found to inhibit completely the stimulation of latex bead phagocytosis in neutrophils produced by the calcium ionophore A23187. These results suggest that CCK-8 is a negative modulator of several neutrophil functions and that the inhibition of these activities could be carried out through an increase of the intracellular cAMP levels and a decrease of the extracellular calcium input. ᭧ 1997 Elsevier Science Inc.

An 8 weeks growth experiment was conducted to determine the effects of dietary vitamin C on the survival, growth, tissue ascorbic acid contents and immunity of large yellow croaker (Pseudosciaena crocea) with initial weight of 17.82 ± 0.68 g. Seven practical diets were formulated to contain 0. 1, 12.2, 23.8, 47.6, 89.7, 188.5 and 489.0 mg ascorbic acid equivalent kg − 1 diet, supplied as L-ascorbyl-2-polyphosphate (LAPP). Each diet was fed to triplicate groups of fish in seawater floating cages (1.5 × 1.5 × 2.0 m), and each cage was stocked with 120 fish. Fish were fed twice daily (05:00 and 17:00) to apparent satiation for 8 weeks. The water temperature fluctuated from 19.5 to 25.5°C, the salinity from 25 to 28‰ and dissolved oxygen content was approximately 7 mg l − 1 during the experimental period. Results showed that the specific growth rate (SGR) (from 1.80 to 1.96% d − 1 ) had an increasing trend with the increase of dietary vitamin C, but no significant difference was observed among dietary treatments. No gross deficiency signs were observed in any of the experimental fish. Survival rate, however, significantly increased with increasing dietary vitamin C (P b 0.05). The vitamin C contents in liver and muscle correlated positively with the vitamin C in diets. The vitamin C requirement was estimated to be 28.2 mg kg − 1 based on survival, and 87.0 mg kg − 1 on liver content of vitamin C. The activities of serum lysozyme and alternative complement pathway (ACP), phagocytosis percentage (PP) and respiratory burst activity of head kidney significantly increased with increasing dietary vitamin C. The challenge experiment with Vibrio harveyi showed that fish fed the diets with supplementation of vitamin C had significantly lower cumulative mortality compared to the control group (66.7%), and the cumulative mortality (16.7%) in fish with 489.0 mg kg − 1 ascorbic acid was significantly lower than that (41.7%) in fish with 23.8 mg kg − 1 ascorbic acid. These results suggested that vitamin C significantly influenced the immune response and disease resistance of large yellow croaker.

Francisella noatunensis subsp. noatunensis, causing granulomatosis in cod, has been shown to reside within cod immune cells, mainly within monocytes and macrophages. In the present study, we analysed the ability of the bacterium to replicate within adherent cells isolated from head kidney by in vitro infection of leucocytes. Two different technical approaches for flow cytometry analyses were performed for detection of intracellular bacteria. The presence of the wild type was assessed after identification by intracellular binding of specific antibodies to the pathogen. The other way was to use green fluorescent protein (GFP) transformed bacterium for infection studies allowing direct measurements of fluorescence from infected cells. By both methods we found an increase in fluorescence in infected cells, verifying bacterial replication, both after 4 and 28 h post infection in leucocytes isolated from head kidney (HKL). The GFP transformed bacterium was similar to the wild type in growth and infectivity pattern, showing that it can be a valuable tool for further studies of infection routes and pathology. Further, F. noatunensis subsp. noatunensis was found to inhibit respiratory burst activity, a potent pathogen killing mechanism, in cod leucocytes, but not in such cells from salmon. Our findings may indicate that inhibition of respiratory burst during Francisella infection is a key to its intracellular existence. This strategy seems to be conserved through evolution as it is also observed during infections in higher vertebrates caused by bacteria within the Francisella genus. The results presented here, showing the intracellular existence of Francisella, its replication within leucocytes and the inhibitory effect on respiratory burst, strongly support that these factors contribute to disease and pathology in infected cod. The intracellular replication shown in the present study might contribute to explain the problems of obtaining protective vaccines against Francisella and effective antibiotic treatment of infected fish.

Botrytis cinerea is responsible for the gray mold disease on more than 200 host plants. This necrotrophic ascomycete displays the capacity to kill host cells through the production of toxins, reactive oxygen species and the induction of a plantproduced oxidative burst. Thanks to an arsenal of degrading enzymes, B. cinerea is then able to feed on different plant tissues. Recent molecular approaches, for example on characterizing components of signal transduction pathways, show that this fungus shares conserved virulence factors with other phytopathogens, but also highlight some Botrytis-specific features. The discovery of some first strain-specific virulence factors, together with population data, even suggests a possible host adaptation of the strains. The availability of the genome sequence now stimulates the development of high-throughput functional analysis to decipher the mechanisms involved in the large host range of this species.

The susceptibility, physiological and immune responses of the Pacific white shrimp Penaeus vannamei, under challenge with Vibrio alginolyticus were investigated for a 72-h period. The survival of shrimp challenged with V. alginolyticus was 86.7, 75.6, 57.8, 54.4, 48.9 and 44.4% after 12, 24, 36, 48, 60 and 72 h, respectively. No significant differences in immune parameters were observed among the control shrimp injected with saline during 72 h. However, the total hemocyte count (THC), phenoloxidase (PO), superoxide dimutase (SOD) activities, and respiratory bursts were decreased with V. alginolyticus challenged with V. alginolyticus after 12 h. The physiological parameters of hemolymph glucose, lactate, and lipid contents increased, and peaked at 36 h after infection. The challenge with V. alginolyticus augmented the magnitudes of the hyperglycemic, hyperlacticemic, and hyperlipidemic responses, following the same trends of the changes in the control shrimp. The observations of this study show that the Pacific white shrimp P. vannamei immune parameters of THC, phenoloxidase activity and respiratory burst, and physiological parameters of glucose, lactate, and lipids were changed due to infection by V. alginolyticus. In addition, hemolymph glucose and lipid were only significantly correlated with SOD activities, while the hemolymph lactate was found to be highly correlated with all the immune parameters examined, except the THC.

Ribose, a critical building block for nucleotides, plays an important role in energy metabolism, transcription, translation, and second messenger systems. This 5-carbon sugar, synthesized from glucose via the pentose phosphate pathway, has a rate-limiting step at glucose-6-phosphate dehydrogenase. Therefore, we hypothesized that when cells are required to proliferate or differentiate, as in an immune response, the requirement for D-ribose may be greater than what could be supplied by the synthetic pathway. We hypothesized that providing an exogenous source of D-ribose during cell differentiation will enhance the process of differentiation. We used a retinoic acid-induced HL-60 cell differentiation culture as a model of neutrophil maturation. The addition of 10 to 25 mmol/L D-ribose was shown to reduce cell proliferation and move the cell population toward apoptosis in a dose-dependent manner. The expression of a cell surface marker representing maturity (CD11b) significantly increased and a cell surface marker indicative of immaturity (CD117) significantly decreased. Functionally, the cells had a greater oxidative burst function dependent on time and dose. The mechanism by which ribose enhances HL-60 cell differentiation is not known; however, as adenosine triphosphate levels did not change, adenosine triphosphate is not thought to be involved. We conclude that in this cell culture model, ribose supplementation enhanced cellular differentiation and function. Thus, ribose might be conditionally essential during time of higher need as in an immune response.

Genomic DNA of 191 strains of the family Pseudomonadaceae, including 187 strains of the genus Xanthomonas, was cleaved by EcoRI endonuclease. After hybridization of Southern transfer blots with 2-acetylamino-fluorene-labelled Escherichia coli 16+23S rRNA probe, 27 different patterns were obtained. The strains are clearly distinguishable at the genus, species, and pathovar levels. The variability of the rRNA gene restriction patterns was determined for four pathovars ofXanthomonas campestris species. The 16 strains ofX. campestris pv. begoniae analyzed gave only one pattern. The variability of rRNA gene restriction patterns of X. campestris pv. manihotis strains could be related to ecotypes. In contrast, the variability of patterns observed for X. campestris pv. malvacearum was not correlated with pathogenicity or with the geographical origins of the strains. The highest degree of variability of DNA fingerprints was observed within X. campestris pv. dieffenbachiae, which is pathogenic to several hosts of the Araceae family. In this case, variability was related to both host plant and pathogenicity.

Fish have many non-specific and specific, humoral and cellular mechanisms to resist bacterial diseases. Non-specific humoral factors include growth inhibiting substances, e.g. transferrin and antiproteases; lysins, e.g. lysozyme, C-reactive protein (CRP), bactericidal peptides and most importantly, complement which has lytic, proinflammatory, chemotactic and opsonic activities thus making a link with non-specific phagocyte responses. These are primarily executed by neutrophils and macrophages. The phagocytes contain many hydrolytic enzymes and when stimulated by bacteria produce reactive oxygen species (ROS), especially the hydroxyl radical, via generation of superoxide anions and nitric oxide. ROS are highly bactericidal. In addition, the neutrophils possess large quantities of myeloperoxidase (MPO) which generates bactericidal hypohalite ions from halide ions and H 2 O 2 . Macrophages can receive MPO from neutrophils and this may enhance their bactericidal activity. Antibody constitutes a specific humoral defence by inhibiting bacterial adherence or invasion of non-phagocytic host cells and neutralising bacterial toxins. Antibody can act as an opsonin and by activating complement by the classical pathway may lyse the bacterial cell, activate inflammation, phagocyte influx and further enhance phagocytosis. Macrophage bactericidal activity can be markedly increased by cytokines released by T lymphocytes on contact with their specific antigen. Pathogenic bacteria have evolved many ways of subverting these defence mechanisms of fish. These include the in vivo production of high a$nity iron uptake systems (e.g. siderophores and their surface receptors, in Aeromonas salmonicida, Vibrio anguillarum). Some bacteria e.g. V. anguillarum strains of serotype 01, while being resistant to the alternative pathway of complement activation in normal serum, are killed in immune serum. In other bacteria, surface layers, capsules and elongated lipopolysaccharide (LPS) 0-antigens protect against classical complement-mediated killing (e.g. A. salmonicida, Photobacterium piscicida, V. anguillarum serotype 02 respectively). While some important pathogens (e.g. P. piscicida) are readily killed by macrophages, others (A. salmonicida) possess specialised superoxide dismutase and catalase to resist ROS and are toxic themselves to macrophages. Renibacterium salmoninarum uses antibody, complement and other serum components to activate the respiratory burst of macrophages on immediate contact thus exhausting this macrophage bactericidal mechanism before the bacterium is phagocytosed. Subsequently, R, salmoninarum is taken up by the macrophage where it multiplies as an intracellular pathogen. However, these two highly adapted pathogens, A. salmonicida and R. salmoninarum are both killed by activated macrophages indicating that the whole concerted e#orts of specific and non-specific, humoral and cellmediated immune responses are required for immunity against these two important pathogens. 1999 Academic Press 291

The inception of ecological immunology has led to an increase in the number of studies investigating the impact of environmental stressors on host immune defence mechanisms. This in turn has led to an increased understanding of the importance of invertebrate groups for immunological research.

Resveratrol-3,5,4 -trihydroxystilbene-possesses antioxidant activities in vitro. It dose-dependently inhibited the generation of peroxyl, hydroxyl, peroxides, and lipid peroxidation products in cell free systems. Oxidative burst of whole human blood stimulated with PMA, fMLP, OpZ, and A23187 was inhibited in a concentration-dependent way, indicating suppression of both receptor and nonreceptor activated chemiluminescence by resveratrol. Results from isolated human neutrophils revealed that resveratrol was active extracellularly as well as intracellularly in inhibiting the generation of reactive oxygen species. Liberation of ATP and analysis of apoptosis showed that in the concentration of 100 M, resveratrol did not change the viability and integrity of isolated neutrophils. Western blot analysis documented that resveratrol in concentrations of 10 and 100 M significantly decreased PMAinduced phosphorylation of PKC / II. Dose-dependent inhibition of nitrite production and iNOS protein expression in RAW 264.7 cells indicated possible interference of resveratrol with reactive nitrogen radical generation in professional phagocytes. The results suggest that resveratrol represents an effective naturally occurring substance with potent pharmacological effect on oxidative burst of human neutrophils and nitric oxide production by macrophages. It should be further investigated for its pharmacological activity against oxidative stress in ischaemia reperfusion, inflammation, and other pathological conditions, particularly neoplasia.

The presence of a residual amount of unstable proteins in wines is a concern for winemakers because these polymers can precipitate from solution during storage causing appearance of sediments and hazes. Such precipitates are commonly the result of denaturation and subsequent aggregation of heat-unstable wine proteins. It has been demonstrated that the vast majority of the wine proteins derive from grapes and that proteins responsible for haze formation are Pathogenesis-Related (PR) proteins, in particular thaumatin-like (TL) proteins and chitinases. Moreover, these proteins are highly resistant to acidic pHs, proteolysis and fermentation conditions. However, they can became insoluble during wine storage and thus originate perceptible turbidity in the bottles. Despite the efforts made in the recent past, the white wine protein instability is still a main problem during white wine making and bentonite treatments are even now indispensable to stabilise white wines.

a b s t r a c t A feeding trial was conducted for 60 days to study the immunomodulatory role of three different immunostimulants yeast extract (YE), brewer's yeast (BY) and spirulina (SP) in Labeo rohita fingerlings. Four hundred and fifty fingerlings (avg. wt 3.35 ± 0.15 g) were randomly distributed in ten treatments and fed with either of ten iso-nitrogenous and iso-caloric semi-purified diets, prepared with three incremental levels (1%, 2% and 4%) of different immunostimulants except the control. Growth parameters did not vary significantly (p > 0.05) among the experimental groups. Haematology and serum parameters was performed before Aeromonas hydrophila challenge whereas respiratory burst activity was analysed following challenge. The respiratory burst activity, total leucocyte count, serum total protein and globulin was significantly higher (p < 0.05) in YE 1% supplemented group. The survival (%) after challenging with A. hydrophila was also highest in the YE fed groups. The results indicate that among the different sources and levels of immunostimulants, YE at lower inclusion level is more effective in promoting the immune status of L. rohita fingerlings.

Hydroxytyrosol (HT) (also known as dihydroxyphenylethanol (DPE)) is a polyphenol extracted from virgin olive oil. HT is known to exert an antioxidant effect but the mechanism of action and the identity of the reactive oxygen molecule(s) targeted are not known. In this study, we show that HT inhibits luminol-amplified chemiluminescence of human neutrophils stimulated with N-formyl-methionyl-leucylphenylalanine (fMLP), phorbol myristate acetate (PMA) and opsonized zymosan. This effect was dose-dependent and occurred immediately after the addition of HT. However, HT had no effect on lucigenin-amplified chemiluminescence, suggesting that it does not inhibit NADPH oxidase activation or scavenge superoxide anions. Furthermore, HT inhibited H 2 O 2 -dependent-dichlorofuoroscein (DCFH) fluorescence of activated neutrophils, as measured by flow cytometry. Finally, HT inhibited luminol-amplified chemiluminescence in a cell-free system consisting of H 2 O 2 and HRPO. These results suggest that HT, a polyphenol derived from olive oil, could exert its antioxidant effect by scavenging hydrogen peroxide but not superoxide anion released during the respiratory burst. #

The present study was conducted to evaluate the effect of various feeding patterns of emodin on growth, non-specific immune response, and disease resistance to Aeromonas hydrophila in juvenile Wuchang bream. Healthy Megalobrama amblycephala (initial weight: 3.47 AE 0.032) were grown in a circulating water system for 8 weeks. Five groups were studied: one control group was fed with a basal diet for eight weeks (Pattern 1, P1), and three treatment groups were fed with a trial diet of 30 mg emodin kg À1 at oneweek (Pattern 2, P2), two-week (Pattern 3, P3), four-week (Pattern 4, P4) intervals. The final treatment group maintained the trial diet for the entire eight-week study duration (Pattern 5, P5). Results indicated that different feeding patterns of emodin significantly influenced the weight gain rate of Wuchang bream (P < 0.05). Fish in the P4 treatment group had significantly higher rates of weight gain (WG) than those in other treatment groups. There were no significant differences in survival rates or feed conversion ratios (FCR) between treatment groups and the control group. White blood cell count (WBC), respiratory burst activity, superoxide dismutase (SOD) activity, myeloperoxidase (MPO) activity and tumor necrosis factor-a (TNF-a) activity were shown to increase at first and then decrease from P3 condition to P5 condition. Fish under P4 treatment showed the most significant improvement of all tested parameters compared to control. Significantly higher levels (P < 0.05) of plasma aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activity were observed in P2 and P4 treatment groups when compared with the control group, while no significant differences were observed in the AST and ALT activity of fish in P2, P3, P4 and P5 treatment groups. In a bacterial challenge experiment with A. hydrophila, fish under P4 and P5 treatment showed lower cumulative mortality than the control group. The results of this study suggest that an initial 4-week feeding interval is recommended for the economic and practical culture of M. amblycephala.

Farming of the flat oyster Ostrea edulis in Europe is severely constrained by the protozoan Bonamia ostreae. The introduction of the resistant species Crassostrea gigas has been a relief for the farmers, while the pilot programmes to select O. edulis strains resistant to bonamiosis performed in various countries can be seen as a promising strategy to minimise the effects of bonamiosis. However, the physiological bases of this differential susceptibility remain unknown. A search for an explanation of the intra and interspecific differences in oyster susceptibility to bonamiosis was accomplished by comparing some immune parameters among various O. edulis stocks and C. gigas. On December 2003, naïve and Bonamia-relatively resistant flat oysters from Ireland, Galician flat oysters and Pacific oysters C. gigas were deployed in a Galician area affected by bonamiosis; haemolymph samples were taken in February and May 2004. A new oyster deployment at the same place was carried out on June 2004 and haemolymph sampling was performed on April 2005. On November 2004, new sets of Irish flat oysters and C. gigas were deployed in Ireland and haemolymph sampling was performed in June 2005. Various haemocytic parameters were measured: total and differential haemocyte count, phagocytic ability, respiratory burst (superoxide anion [ ] and hydrogen peroxide [H 2 O 2 ]) and nitric oxide [NO] production. The comparison of the parameters was carried out at 3 levels: (1) between O. edulis and C. gigas, (2) among O. edulis stocks with different susceptibility to bonamiosis, and between Bonamia-infected and non infected O. edulis. In addition, haemocyte-B. ostreaein vitro encounters were performed to analyse interspecific differences in the haemocytic respiratory burst, using flow cytometry. Significant differences associated with total and differential haemocyte count, and respiratory burst between O. edulis and C. gigas were detected, which could be linked to differences in susceptibility to bonamiosis between both species. Additionally, significant changes in total and differential haemocyte count, and respiratory burst of O. edulis associated with B. ostreae infection were found. However, no consistent difference in any haemocyte parameter between the O. edulis stocks involved in the study was recorded.

The white shrimp Litopenaeus vannamei were challenged with tryptic soy broth (TSB)-grown Vibrio alginolyticus at a dose of 1!10 6 colony-forming units (cfu) shrimp ÿ1 , and then placed in water containing concentrations of nitrite-N at 0 (control), 1.12, 5.15, 11.06 and 21.40 mg l ÿ1 . Mortality of shrimp in 5.15, 11.06 and 21.40 mg l ÿ1 was significantly higher than those in the control solution after 48e168 h. L. vannamei that had been exposed to control, 0.98, 4.94, 9.87 and 19.99 mg l ÿ1 nitrite-N for 96 h were examined for THC (total haemocyte count), phenoloxidase activity, and respiratory burst (release of superoxide anion). The THC and phenoloxidase activity decreased when the shrimp were exposed to 4.94, 9.87 and 19.99 mg l ÿ1 nitrite-N, whereas, the respiratory burst increased significantly at 9.87 and 19.99 mg l ÿ1 nitrite-N after 96 h. It is therefore suggested that nitrite in water caused a depression in the immune ability of L. vannamei and an increased susceptibility to V. alginolyticus infection, together with an increase of superoxide anion production, possibly to cytotoxic levels for the host.

The pharmacological profile of the lignan 7-hydroxymatairesinol (HMR/lignan™, HMR) includes chemopreventive effects, antioxidant properties, and mild proestrogenic activity. The present study was devised to investigate the effects of HMR on THP-1 cells, an established model of human monocytes, and on human polymorphonuclear leukocytes (PMNs). In THP-1 cells, HMR concentration-dependently reduced LPSstimulated tumor necrosis factor (TNF)-α secretion in the supernatant. HMR at low, sub-μM concentrations also reduced TNF-α mRNA, which was however enhanced by supra-μM concentrations of HMR. In human PMNs, HMR concentration-dependently reduced ROS production induced by either N-formyl-Met-Leu-Phe, phorbol myristate acetate or angiotensin II, as well as interleukin-8 production induced by either N-formyl-Met-Leu-Phe or angiotensin II. Results indicate that HMR is an effective inhibitor of both monocytic THP-1 cells and of human PMNs and warrant further studies to assess their relevance for the prevention and treatment of several conditions characterized by chronic systemic inflammation.

Neutrophils represent the front-line defence cells in protecting organisms against infection and play an irreplaceable role in the proper performance of the immune system. As early as within the first minutes of stimulation, neutrophilic NADPH oxidase is activated, and cells release large quantities of highly toxic reactive oxygen species (ROS). These oxidants can be highly toxic not only for infectious agents but also for neighboring host tissues. Since flavonoids exhibit antioxidant and anti-inflammatory effects, they are subjects of interest for pharmacological modulation of ROS production. The present paper summarizes contemporary knowledge on the effects of various flavonoids on the respiratory burst of mammalian neutrophils. It can be summarized that the inhibitory effects of flavonoids on the respiratory burst of phagocytes are mediated via inhibition of enzymes involved in cell signaling as well as via modulation of redox status. However, the effects of flavonoids are even more complex, and several sites of action, depending upon the flavonoid structure and way of application, are included.

Both aqueous and methanolic fractions derived from the Tibetan preparation PADMA-28 (a mixture of 22 plants) used as an anti-atherosclerotic agent, and which is non-cytolytic to a variety of mammalian cells,were found to strongly inhibit (1) the killing of epithelial cells in culture induced by 'cocktails' comprising oxidants, membrane perforating agents and proteinases; (2) the generation of luminol-dependent chemiluminescence in human neutrophils stimulated by opsonized bacteria; (3) the peroxidation of intralipid (a preparation rich in phopholipids) induced in the presence of copper; and (4) the activity of neutrophil elastase. It is proposed that PADMA-28 might prove beneficial for the prevention of cell damage induced by synergism among pro-inflammatory agonists which is central in the initiation of tissue destruction in inflammatory and infectious conditions.

Pyrethroids are widely used insecticides of low acute toxicity in mammals but the consequences of long-term exposure are of concern. Their insecticidal action is related to neurotoxicity and, in addition, there are indications of mammalian immunotoxicity. In this work the effect of 60 days permethrin (150 mg kg -1 body weight/day) exposure on two types of leukocytes (monocytes and lymphocytes) in adolescent rats was investigated. In particular, the monocyte respiratory burst response was first investigated, followed by studies on the degree and type of lymphocyte DNA damage induced by permethrin at this stage of life. Permethrin treatment reduces the monocyte respiratory burst response to phorbol myristate acetate, thereby decreasing superoxide anion (65%) and hydrogen peroxide (37%) production. Moreover an increase [correction made here after initial online publication] in monocyte plasma membrane fluidity in the hydrophilic-hydrophobic interface of the lipid bilayer was measured. Data obtained from the comet assay show that permethrin induces lymphocyte DNA lesions at both formamido pyrimidine glycosylase (Fpg) and endonuclease III (Endo III) sites in adolescent rats. Our results indicate the key role of permethrin in oxidative stress whose consequences lead to biochemical and functional changes. The reduced phagocyte respiratory burst induced by permethrin treatment and the type of DNA damage measured could represent new relevant aspects of pyrethroid toxicity which should be considered for human health.

The total haemocyte count (THC), differential haemocyte count (DHC), phenoloxidase activity, respiratory bursts (release of superoxide anion), superoxide dismutase activity, phagocytic activity and clearance efficiency to the pathogen Photobacterium damsela were measured when tiger shrimp Penaeus monodon (13.5 AE 1.5 g) were individually injected with saline or dopamine at 10 À8 , 10 À7 , or 10 À6 mol shrimp À1 . Results showed that a transient period of immunosuppression occurred between 2 and 8 h after injection of dopamine for all immune parameters except circulating haemocytes, and all immune parameters had returned to control values within 8e16 h after receiving dopamine. The injection of dopamine also significantly increased the mortality of P. monodon challenged with the pathogen Pho. damsela. These results suggest that stress-inducing dopamine suppresses the immune system, which in turn promotes the susceptibility of P. monodon to Pho. damsela.

White shrimp Litopenaeus vannamei (Boone) held in 35& seawater were challenged with Vibrio alginolyticus at a dose of 3 ! 10 5 colony-forming units (cfu) shrimp ÿ1 , and then placed in water containing concentrations of Cu 2+ at 0 (control), 1, 5, 10 and 20 mg l ÿ1 . Mortality of shrimp in 5, 10 and 20 mg l ÿ1 Cu 2+ was significantly higher than those in 1 mg l ÿ1 Cu 2+ and the control solution after 24e96 h. In another experiment, L. vannamei which had been exposed to control, 1, 5, 10 and 20 mg l ÿ1 Cu 2+ for 24, 48 and 96 h were examined for THC (total haemocyte count), phenoloxidase activity, respiratory burst (release of superoxide anion), phagocytic activity and clearance efficiency to V. alginolyticus. Copper concentrations at 1 mg l ÿ1 or greater for 24 h resulted in decreased THC, phenoloxidase activity, phagocytic activity and clearance efficiency, whereas copper concentration at 20 mg l ÿ1 caused significant increase in respiratory burst of L. vannamei. In conclusion, concentration of Cu 2+ at 1 mg l ÿ1 or greater increased the susceptibility of L. vannamei to V. alginolyticus infection by a depression in immune ability. The release of superoxide anion by L. vannamei exposed to 20 mg l ÿ1 Cu 2+ was considered to be cytotoxic to the host.

The total haemocyte count (THC), phenoloxidase activity, respiratory burst and phagocytic activity and clearance efficiency to Vibrio alginolyticus were examined when the white shrimp Litopenaeus vannamei were injected with different types (type I to type V) of carrageenan at 6 µg g − 1 shrimp. Results showed that the THC, phenoloxidase activity, respiratory burst, and phagocytic activity against V. alginolyticus were significantly higher in shrimp that received any type of carrageenan after 24 h. Results also showed that the THC and PO activity maintained significantly higher in shrimp that received type IV carrageenan as compared to those that received other types of carrageenan after 96 h. In a separate experiment, L. vannamei which had been injected with different types of carrageenan after 3 h were challenged with V. alginolyticus at 7.34 × 10 6 cfu shrimp − 1 , and placed in seawater. The survival of shrimp that received type IV carrageenan was significantly higher than that of shrimp that received type I, II, III and V carrageenan, and shrimp that received saline as well as the control shrimp after 120 h. It was concluded that L. vannamei that received type IV carrageenan (λ-carrageenan) exhibited higher immune ability of L. vannamei as well as resistance against V. alginolyticus infection.

It is well documented that the polysaccharide glucomannan (GM), an abundant constituent of the fungal cell wall, in the form of particulate induces strong activation of phagocytes, however, the effects of soluble GM are not known. Activation of phagocyte anti-microbial mechanisms is a crucial part of the innate host defense against invading pathogens. However, under uncontrolled inflammatory conditions they contribute to damage of surrounding tissues. Thus, to prevent these deleterious effects, the activation of phagocytes is a tightly regulated process. Therefore, in this study we analyzed the effect of soluble GM on some neutrophil functions such as reactive oxygen species production, degranulation, and receptor mobilization at the plasma membrane. Soluble GM at the tested concentrations did not stimulate oxidative burst of phagocytes directly but significantly potentiated oxidative burst in response to opsonized zymosan particles. GM induced significant phosphorylation of p47phox subunit of NADPH oxidase on Ser345. This priming effect of GM was accompanied by time and concentration dependent degranulation characterized by increased surface expression of receptors stored in neutrophil granules (CD10, CD11b, CD14, CD35, and CD66b). Degranulation was further confirmed by increase of elastase activity in media. Thus, it could be suggested that soluble GM induces priming of phagocytes connected with their degranulation, the increase of surface receptor expression, and potentiation of oxidative burst response to opsonized particles through the activation of NADPH oxidase.

After transmission by infected mosquitoes, malaria sporozoites rapidly travel to the liver. To infect hepatocytes, sporozoites traverse Kupffer cells, but surprisingly, the parasites are not killed by these resident macrophages of the liver. Here we show that Plasmodium sporozoites and recombinant circumsporozoite protein (CSP) suppress the respiratory burst in Kupffer cells. Sporozoites and CSP increased the intracellular concentration of cyclic adenosyl mono-phosphate (cAMP) and inositol 1,4,5-triphosphate in Kupffer cells, but not in hepatocytes or liver endothelia. Preincubation with cAMP analogues or inhibition of phosphodiesterase also inhibited the respiratory burst. By contrast, adenylyl cyclase inhibition abrogated the suppressive effect of sporozoites. Selective protein kinase A (PKA) inhibitors failed to reverse the CSP-mediated blockage and stimulation of the exchange protein directly activated by cAMP (EPAC), but not PKA inhibited the respiratory burst. Both blockage of the low-density lipoprotein receptor-related protein (LRP-1) with receptor-associated protein and elimination of cell surface proteoglycans inhibited the cAMP increase in Kupffer cells. We propose that by binding of CSP to LRP-1 and cell surface proteoglycans, malaria sporozoites induce a cAMP/EPAC-dependent, but PKA-independent signal transduction pathway that suppresses defence mechanisms in Kupffer cells. This allows the sporozoites to safely pass through these professional phagocytes and to develop inside neighbouring hepatocytes.

Neutrophils have a crucial function in the defense against bacteria and fungi. Indeed, during chronic, severe neutropenia and in case of severe neutrophil dysfunctions, the patients may suffer recurrent and sometimes life-threatening infections. This article describes the clinical symptoms, the theory behind the antimicrobial systems of neutrophils, the methods to diagnose the various aberrations, and the possibilities for treating these patients. A few of the most common causes of neutropenia and neutrophil dysfunctions are described in detail, including recent genetic information regarding the cause of these diseases. q

Rodríguez AB, Nogales G, Ortega E, Barriga C. Melatonin controls superoxide anion level: Modulation of superoxide dismutase activity in ring dove heterophils. J. Pineal Res. 1998; 24:9–14. © Munksgaard, CopenhagenRodríguez AB, Nogales G, Ortega E, Barriga C. Melatonin controls superoxide anion level: Modulation of superoxide dismutase activity in ring dove heterophils. J. Pineal Res. 1998; 24:9–14. © Munksgaard, CopenhagenAbstractThe pineal gland and the immune system in mammals and birds are functionally connected, with some recent findings showing that melatonin, the major secretory product of the pineal gland in vertebrates, is a free-radical scavenger and general antioxidant. In birds, heterophils, with their ability to ingest and kill different antigens, play a central role in the host defense mechanism. This in vitro study investigated the possible antioxidant action of melatonin in heterophils isolated from adult male and female ring dove)Streptopelia risoria). The superoxide anion levels (O2−) and the superoxide dismutase (SOD) activity were evaluated as indicators of the metabolic burst of heterophils after ingestion of inert particles (latex beads) at the basal time (0 min) and 15, 30,45, and 60 min of incubation with 100 μM melatonin. The heterophil O2− levels, measured by the nitroblue tetrazolium reduction test, were lower after incubation in the presence of melatonin at all the times studied, a time-dependent effect. Latex beads produced a significant increase in SOD activity, whereas there was a significant decrease in the presence of melatonin. In addition, when heterophils were incubated in the presence of both latex beads and melatonin, the indole appeared to annul the enhancement of SOD activity (U-525nm) produced by latex beads. In conclusion, our findings show that the neurohormone melatonin could be a useful antioxidant.The pineal gland and the immune system in mammals and birds are functionally connected, with some recent findings showing that melatonin, the major secretory product of the pineal gland in vertebrates, is a free-radical scavenger and general antioxidant. In birds, heterophils, with their ability to ingest and kill different antigens, play a central role in the host defense mechanism. This in vitro study investigated the possible antioxidant action of melatonin in heterophils isolated from adult male and female ring dove)Streptopelia risoria). The superoxide anion levels (O2−) and the superoxide dismutase (SOD) activity were evaluated as indicators of the metabolic burst of heterophils after ingestion of inert particles (latex beads) at the basal time (0 min) and 15, 30,45, and 60 min of incubation with 100 μM melatonin. The heterophil O2− levels, measured by the nitroblue tetrazolium reduction test, were lower after incubation in the presence of melatonin at all the times studied, a time-dependent effect. Latex beads produced a significant increase in SOD activity, whereas there was a significant decrease in the presence of melatonin. In addition, when heterophils were incubated in the presence of both latex beads and melatonin, the indole appeared to annul the enhancement of SOD activity (U-525nm) produced by latex beads. In conclusion, our findings show that the neurohormone melatonin could be a useful antioxidant.

The immune response of European sea bass, Dicentrarchus labrax, to a natural infection by the copepod parasite Lernanthropus kroyeri was evaluated for the first time in vivo. The results clearly demonstrated the triggering of the fish immune system by the parasite. Lysozyme activity and alternative complement pathway were involved in the early action against the parasitical infection, whilst classical complement and respiratory burst (RB) activity took over in the later stages of infection. It was hypothesized that the levels of alternative and classical complement and RB stimulation indexes may determine the resistance capacity of the fish to the parasite. It is not clear how parasites can survive despite the strong immunological arsenal deployed by the fish. The continual increase of prevalence and severity of parasite infection suggested that the parasite’s mechanism of evasion of the immune system was extremely successful. The contrasting decrease in the negative effects of parasites on the fish health (such as gills anaemia) suggested that an equilibrium between the parasites and their hosts was reached in chronic infection. These dynamic interactions between parasites and fish hosts were probably the main determinant of host specificity.

Molecular mechanisms of the crosstalk between mitochondria and NADPH oxidase through reactive oxygen species -studies in white blood cells and in animal models (doi: 10.1089/ars.2012.4953) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. 2 2 Word count main text: 8,484 words (excluding references and figure legends) Number of references: 69 Number of figures (color: 2 online, 0 hardcopy): 9

The reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is part of the microbicidal arsenal used by human polymorphonuclear neutrophils (PMNs) to eradicate invading pathogens. The production of a superoxide anion (O 2 -) into the phagolysosome is the precursor for the generation of more potent products, such as hydrogen peroxide and hypochlorite. However, this production of O 2 is dependent on translocation of the oxidase subunits, including gp91 phox , p22 phox , p47 phox , p67 phox , p40 phox , and Rac2 from the cytosol or specific granules to the plasma membrane. In response to an external stimuli, PMNs change from a resting, nonadhesive state to a primed, adherent phenotype, which allows for margination from the vasculature into the tissue and chemotaxis to the site of infection upon activation. Depending on the stimuli, primed PMNs display altered structural organization of the NADPH oxidase, in that there is phosphorylation of the oxidase subunits and/or translocation from the cytosol to the plasma or granular membrane, but there is not the complete assembly required for O 2 generation. Activation of PMNs is the complete assembly of the membrane-linked and cytosolic NADPH oxidase components on a PMN membrane, the plasma or granular membrane. This review will discuss the individual components associated with the NADPH oxidase complex and the function of each of these units in each physiologic stage of the PMN: rested, primed, and activated. J. Leukoc. Biol. 78: 1025-1042; 2005.

Oxidized low-density lipoproteins (oxLDL) play a crucial role in atherogenesis mainly via their capacity to bind and to activate macrophages. However, the role of the protein LDL moiety in this process is not yet established. In this study, human LDL were exposed to hypochlorous acid (HOCl), a selective protein oxidant, or copper sulfate (CuSO 4 ), a major lipid oxidant, and tested for their capacity to activate the NADPHoxidase of human THP-1-and U937-derived macrophages as measured by lucigenin chemiluminescence (CL). Compared to native LDL which had no effect, HOCl-oxLDL triggered potent CL responses in both U937 and THP-1 cells but only when these were fully differentiated into macrophages by phorbol myristate acetate. In contrast, Cu-oxLDL only triggered a moderate CL response of U937 cells and had little effect on THP-1 cells. While delipidation did not affect HOCl-oxLDL-induced CL response it abolished that induced by Cu-oxLDL. Interestingly, U937 cells showed higher CL responses to both types of oxLDL than THP-1 cells, a finding which could be related to their higher expression of the scavenger receptor CD36. Taken together these results strongly support the role of the protein moiety in oxLDL-induced macrophage activation.

TGF-␤1 (TGF) has been implicated in the pathogenesis of several chronic infections and is thought to promote microbial persistence by interfering with macrophage function. In rats with experimental pulmonary cryptococcosis, increased lung levels of TGF were present at 12 mo of infection. Within the lung, expression of TGF localized to epithelioid cells and foamy macrophages in areas of inflammation. Increased TGF expression was also observed in the lungs of experimentally infected mice and a patient with pulmonary cryptococcosis. TGF reduced Ab and serum-mediated phagocytosis of Cryptococcus neoformans by rat alveolar macrophages (AM) and peripheral blood monocytes, and this was associated with decreased chemokine production and oxidative burst. Interestingly, TGF-treated rat AM limited both intracellular and extracellular growth of C. neoformans. Control of C. neoformans growth by TGF-treated rat AM was due to increased secretion of lysozyme, a protein with potent antifungal activity. The effects of TGF on the course of infection were dependent on the timing of TGF administration relative to the time of infection. TGF treatment of chronically infected rats resulted in reduced lung fungal burden, while treatment early in the course of infection resulted in increased fungal burden. In summary, our studies suggest a dual role for TGF in persistent fungal pneumonia whereby it contributes to the local control of infection by enhancing macrophage antifungal efficacy through increased lysozyme secretion, while limiting inflammation by inhibiting macrophage/monocyte phagocytosis and reducing associated chemokine production and oxidative burst.

A feeding trial was conducted for 60 days to determine the effect of dietary tannin on growth and health status of Labeo rohita fingerlings. One hundred and eighty fingerlings (average weight 3.2 g) were randomly distributed into six treatment groups with three replicates each. Six isonitrogenous (crude protein 339.8-351.1 g/kg feed) and isocaloric (16.51-16.65 MJ DE/kg) practical diets were prepared with graded levels of tannin, viz. control, T 1 (2.5 g/kg), T 2 (5 g/kg), T 3 (10 g/kg), T 4 (15 g/kg) and T 5 (20 g/kg). Addition of tannin up to 20 g/kg did not affect the weight gain (%), specific growth rate, feed conversion ratio, protein efficiency ratio, and apparent net protein utilization of fingerings significantly (P>0.05). The hepato somatic index value of control group was lowest than the other experimental groups. The muscle cholesterol level and haemoglobin count in the different treatments was significantly increased compared with the control. The immunological parameters like total leucocyte count and respiratory burst activity was significantly increased in tannin-fed groups. Based on the results of the present study, it is concluded that short-term feeding for 60 days of hydrolysable tannin has no adverse effect on growth A u t h o r ' s p e r s o n a l c o p y A.K. Prusty et al. / Animal Feed Science and Technology 136 (2007) 96-108 97 of L. rohita fingerlings. Hence feeding of hydrolysable tannin on a long-term basis needs further study.

The opportunistic human pathogen Pseudomonas aeruginosa is the predominant micro-organism of chronic lung infections in cystic fibrosis (CF) patients. P. aeruginosa colonizes the CF lungs by forming biofilm structures in the alveoli. In the biofilm mode of growth the bacteria are highly tolerant to otherwise lethal doses of antibiotics and are protected from bactericidal activity of polymorphonuclear leukocytes (PMNs). P. aeruginosa controls the expression of many of its virulence factors by means of a cell-cell communication system termed quorum sensing (QS). In the present report it is demonstrated that biofilm bacteria in which QS is blocked either by mutation or by administration of QS inhibitory drugs are sensitive to treatment with tobramycin and H 2 O 2 , and are readily phagocytosed by PMNs, in contrast to bacteria with functional QS systems. In contrast to the wild-type, QS-deficient biofilms led to an immediate respiratory-burst activation of the PMNs in vitro. In vivo QS-deficient mutants provoked a higher degree of inflammation. It is suggested that quorum signals and QS-inhibitory drugs play direct and opposite roles in this process. Consequently, the faster and highly efficient clearance of QS-deficient bacteria in vivo is probably a two-sided phenomenon: down regulation of virulence and activation of the innate immune system. These data also suggest that a combination of the action of PMNs and QS inhibitors along with conventional antibiotics would eliminate the biofilm-forming bacteria before a chronic infection is established.

The total haemocyte count (THC), differential haemocyte count (DHC), phenoloxidase activity, respiratory burst (release of superoxide anion), superoxide dismutase activity, phagocytic activity and clearance efficiency to the pathogen Vibrio alginolyticus were measured when the white shrimp Litopenaeus vannamei (9.4e11.3 g) were injected individually with sodium alginate at 10, 20 or 50 mg g ÿ1 . No significant differences in THC, DHC and superoxide dismutase activity were observed among the shrimp injected with saline and those injected with sodium alginate at 10, 20 or 50 mg g ÿ1 . However, L. vannamei injected with sodium alginate at 20 mg g ÿ1 increased its phenoloxidase activity and respiratory burst after 2 days and one day, respectively. L. vannamei injected with sodium alginate at 50 mg g ÿ1 maintained a higher phagocytic activity and clearance efficiency to V. alginolyticus after 4 days. In another experiment, L. vannamei which had been injected with sodium alginate, were challenged with V. alginolyticus at 2!10 5 colonyforming units (CFU) shrimp ÿ1 and then placed in seawater of 34&. The survival of shrimp that received sodium alginate at either dose was significantly higher than that of control shrimp at the termination of the experiment (6 days after the challenge). It is therefore concluded that L. vannamei received sodium alginate at 10 mg g ÿ1 or more and increased its immune ability and resistance from V. alginolyticus infection. Fish & Shellfish Immunology 17 (2004) 41e51 www.elsevier.com/locate/fsi V. damsela [2], V. harveyi [3], V. parahaemolyticus [4], and V. vulnificus [5], and viruses like monodon baculovirus (MBV) [6], white spot syndrome virus (WSSV) [7], infectious hypodermal and hematopoietic necrosis virus (IHHNV) [8], yellow head virus (YHV) [9] and Taura syndrome virus (TSV) [10] in Taiwan. Consequently, the total farmed production of penaeid shrimp declined from 82,598 ton in 1987 to 8878 ton in 2001.

The d-mannose binding lectin ArtinM from Artocarpus integrifolia, previously known as KM+ and artocarpin, is considered a stimulant of Th1-type immunity, which is able to confer resistance to some intracellular pathogens. In addition, ArtinM induces neutrophil migration by haptotaxis through simultaneous interactions of its carbohydrate recognition domains (CRDs) with glycans expressed on the extracellular matrix and the neutrophil surface. In the present study, we have expanded the characterization of ArtinM as a neutrophil activator. Exposure of neutrophils to ArtinM for 15 min resulted in tyrosine phosphorylation of intracellular proteins, a process that was selectively inhibited by d-mannose or mannotriose. Shortly after stimulation, neutrophils secreted high levels of LTB 4 and underwent shedding of L-selectin from their surface. Exposure to ArtinM enhanced neutrophil functions, such as respiratory burst and zymozan and Listeria monocytogenes phagocytosis. In addition, ArtinM-stimulated neutrophils displayed increased CXCL-8 secretion and TLR2 gene transcription. These results demonstrate that Art-inM is able to induce potent neutrophil activation, a feature that should be strongly considered in the assessment of the lectin capacity to confer resistance against infections.

The purpose of this study was to analyze the potential of various types of biodegradable microspheres (MS) (i) to activate in vitro cell line-derived macrophages (RAW 264.7, Mf), and primary peritoneal and bone marrow-derived mouse Mf, to prolong the release and presentation of microencapsulated synthetic malaria antigens by Mf after uptake of antigen-loaded MS, and (ii) to stimulate an immune response in mice against a microencapsulated synthetic malaria antigen. The MS were made of various types of poly(lactide-co-glycolide) (PLGA) or chitosan cross-linked with tripolyphosphate. PLGA, but not chitosan MS, were efficiently ingested by Mf. Upon exposure to the various MS types, Mf increased only the production of reactive oxygen intermediates (ROI), while the production of nitric oxide (NO), tumor necrosis factor alpha (TNF-a), and the expression of cyclooxigenase-2 (COX-2), inducible NO synthase (iNOS), the cell surface markers MHC class I and II, and CD 86 remained unaffected. In vitro release of the microencapsulated antigen from PLGA50:50 MS followed a pulsatile pattern and extended over 14 weeks. This prolonged antigen release was also mirrored in the significantly prolonged antigen presentation over more than 7 days by Mf after uptake of antigen-loaded PLGA MS. Finally, antigen-loaded PLGA MS induced a solid immune response in mice after a single s.c.-injection, which was only slightly inferior to the antibody titers measured with the control formulation with Montanide ISA720. These results suggest that MS are well tolerated by Mf. The prolonged antigen 0168-3659/$ -see front matter D Journal of Controlled Release 109 www.elsevier.com/locate/jconrel presentation by Mf, as measured in vitro, along with the capacity to induce a strong immune response in animals emphasize that biodegradable MS are a very promising delivery system for both preventive and immunotherapeutic vaccines. D