Effects of high doses of cortisol on innate cellular immune response of seabream (Sparus aurata L.)

The effects of high doses of cortisol, biologically the most active corticosteroid in the circulating blood of teleost fish, on gilthead seabream (Sparus aurata L.) leucocytes were determined. Leucocytes isolated from the head-kidney (the principal haemopoietic organ of this fish species) were incubated with five different concentrations (ranging from 0 to 10(-1) mM) of cortisol for 30, 120, 240, 360, and 480 min and their effects on leucocyte viability and some of the main innate cellular immune responses were evaluated. The viability and the cytotoxic activity of leucocytes against tumor cells were not significantly affected by in vitro incubation with cortisol, at any of the assayed concentration or incubation times. With cortisol concentrations of 10(-9), 10(-7) or 10(-5) mM, the respiratory burst activity of head-kidney leucocytes were significantly depressed after 30 min of incubation. On the other hand, with cortisol concentrations of 10(-1) mM, the phagocytosis of yeast cells and the total peroxidase content of leucocytes were significantly depressed at incubation times longer than 240 min. To corroborate that the observed effects were due to the cortisol treatment, assays were developed using both cortisol and cycloheximide, which blocked the inhibitory effects of cortisol. The present results demonstrate that cortisol plays an important role in the down-regulation of phagocytic but not of cytotoxic activity in seabream leucocytes.     

A role for acidophilic granulocytes in the testis of the gilthead seabream (Sparus aurata L., Teleostei)

In mammals, a complex interaction between the immune and the reproductive systems has been described, in which testicular immune cells produce cytokines and growth factors which modulate gonad functions, while specific gonad cells influence the immune response in this organ. In this study we describe the presence of acidophilic granulocytes in the testis of the hermaphrodite teleost fish gilthead seabream (Sparus aurata L.) by using a specific monoclonal antibody. During the post-spawning stage of the testis, this cell type appears in the germinal compartment, accumulates interleukin (IL)-1beta and does not seem to be involved in the phagocytosis of degenerating cells. Moreover, in vitro, 11-ketotestosterone and 17beta-oestradiol, the principal fish sexual steroids, regulate the respiratory burst activity of acidophilic granulocytes obtained from the head-kidney (the bone marrow equivalent in fish) and the intracellular accumulation of IL-1beta by these cells. It is likely, therefore, that IL-1beta produced by testicular acidophilic granulocytes regulates important functions of the testis in fish.     

Growth hormone increases plasma levels of insulin-like growth factor (IGF-I) in a teleost, the gilthead seabream (sparus aurata)

A heterologous radioimmunoassay (RIA) was applied for the determination of immunoreactive (IR)--insulin-like growth factor (IGF-I) in a teleost, the gilthead seabream (Sparus aurata). Serial dilutions of the fish plasma gave a linear curve when added to constant amounts of 125I-labelled human IGF-I(53-70) and antiserum prepared against this fragment. The RIA was used to study the effect of GH on plasma levels of IR-IGF-I in S. aurata. A single injection of human recombinant GH (1 micrograms/g) resulted in a significant increase in IR-IGF-I at 29, 48 and 72 h, when compared with saline-injected fish. This novel observation suggests that in fish, as in mammals, circulating IGF-I levels are modulated by GH.     

Adhesion of leucocytes onto polytetrafluoroethylene (PTFE) vascular grafts and the effect of low molecular weight dextran (LMWD).

Platelets are known to interact with the surface of synthetic grafts. In the present study we have investigated another blood constituent, the white blood cell, to evaluate the contribution of leucocyte adhesion onto synthetic vascular graft surfaces. Furthermore the effect of low molecular weight dextran (LMWD) was evaluated in vivo. Polytetrafluoroethylene (PTFE) grafts were interpositioned in the femoral circulation in 12 healthy adult sheep. Autologous leucocytes were labelled with Indium-111-oxine and re-injected i.v. after purity and functional evaluation. Graft uptake of 111In-labelled leucocytes were studied continuously for 2.5 hours and two consecutive days using a gamma camera. Six animals received LMWD, 15 ml/h, i.v. infusion during the first day while the other six animals were infused with normal saline in the same amount and rate (controls). The labelled leucocytes showed normal phagocytosis of bacterias and the leucocyte purity was 74 +/- 6%. There was a rapid increase in graft activity initially. In control animals a continuous increase of graft activity was observed throughout the experiment. In the LMWD-treated animals graft activity remained on a steady level after the initial built up of activity and after 2.5 hours there was a highly significant difference between the groups, p less than 0.001. These differences were confirmed by in vitro activity measurement, autoradiography and histological examination of the grafts at the end of the experiment. It was concluded that leucocyte adhesion onto the surface of PTFE grafts occur during the early period after implantation and could therefore be a contributing factor in the thrombogenesis. Administration of LMWD seems to have a beneficial effect since less leucocyte adhesion occurred in those animals treated with LMWD.     

Human peripheral blood and pleural fluid eosinophils can be induced by immune complexes to release IgG immune complexes and aggregated IgE

The ability of IgG and IgE immune complexes and of phorbol myristate acetate (PMA), a soluble membrane activator, to stimulate hydrogen peroxide (H2O2) release and to induce oxygen radical-mediated cytotoxic activity by human peripheral blood (PBL) eosinophils and by PBL neutrophils was evaluated in normal volunteers and patients with hypereosinophilic malignant pleural effusions due to lung cancer. PMA stimulated a significant respiratory burst. Similar results were obtained with IgG IC stimulation, although the levels of H2O2 were lower. Agg IgE induced H2O2 release only by PBL and PE eosinophils and not by neutrophils. PMA stimulation resulted in detectable cytotoxic activity. IgG IC generated both PBL and PE eosinophil and PBL neutrophil cytotoxicity. Agg IgE induced significant cellular cytotoxicity in both PBL and PE eosinophils. This study suggests that eosinophil oxidative metabolic burst and cytotoxic activity stimulated by IgG and IgE immune complexes could represent a possible mechanism of parenchymal injury in eosinophilic disorders.     

Spleen in falciparum malaria: ultrastructural study

An ultrastructural study was undertaken of the spleen of 13 year-old-boy who died of falciparum malaria. The spleen revealed the following: both parasitized and non-parasitized erythrocytes are phagocytosed in large numbers by macrophages, littoral and reticular cells. Blood congestion and trapping of parasitized erythrocytes are commonly seen in splenic sinusoids and cords. Erythrocytes forming rosette structure around immuno-presenting cells is observed. The results of this study provide evidence that the mechanisms underlying splenic host defence in malaria include both immunological and non-immunological interaction with erythrocytes. Splenic trapping of parasitized erythrocytes is an important defence mechanism and the phagocytosis of erythrocytes probably accounts for anaemia.     

Effects of a natural parasitical infection (Lernanthropus kroyeri) on the immune system of European sea bass, Dicentrarchus labrax L

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.     

Osmoregulatory action of PRL,GH, and cortisol in the gilthead seabream (Sparus aurata L)

The osmoregulatory actions of ovine prolactin (oPRL), ovine growth hormone (oGH), and cortisol were tested in the euryhaline gilthead seabream Sparus aurata. Acclimated to sea water (SW, 40 ppt salinity, 1000 mOsm/kg H(2)O) or brackish water (BW, 5 ppt, salinity, 130 mOsm/kg H(2)O), injected every other day for one week (number of injections, 4) with saline (0.9% NaCl), oPRL (4 microg/g body weight), oGH (4 microg/g body weight) or cortisol (5 microg/g body weight), and transferred from SW to BW or from BW to SW 24h after the last injection. Fish were sampled before and 24h after transfer. Gill Na(+), K(+)-ATPase activity, plasma osmolality, plasma ions (sodium and chloride), plasma glucose, and muscle water moisture were examined. SW-adapted fish showed higher gill Na(+), K(+)-ATPase activity, plasma osmolality, and plasma ions levels than BW-adapted fish. Transfer from SW to BW decreased plasma osmolality and ions levels after 24h, while transfer from BW to SW increased these parameters, whereas gill Na(+),K(+)-ATPase activity was unaffected. oPRL treatment significantly decreased gill Na(+),K(+)-ATPase activity and increased plasma osmolality and ions in SW- and BW-adapted fish. This treatment minimizes loss of osmolality and ions in plasma after transfer to BW and increased these values after transfer to SW. No significant changes were observed in gill Na(+),K(+)-ATPase activity, plasma osmolality, and plasma ions in oGH-treated group with respect to saline group before or after transfer from SW to BW or from BW to SW. Treatment with cortisol induced, in SW-adapted fish, a significant increase of gill Na(+),K(+)-ATPase activity and decrease of plasma osmolality and plasma ions. In BW-adapted fish this treatment induced a significant increases in gill Na(+),K(+)-ATPase activity, plasma osmolality, and plasma ions. After transfer to SW cortisol-treated fish had higher plasma osmolality than the saline group. Our results support the osmoregulatory role of PRL in the adaptation to hypoosmotic environment in the gilthead seabream S. aurata. Further studies will be necessary to elucidate the osmoregulatory role of GH in this species. Cortisol results suggest a "dual osmoregulatory role" of this hormone in S. aurata.     

Cloning and Expression of Somatolactin, a Pituitary Hormone Related to Growth Hormone and Prolactin from Gilthead Seabream,Sparus aurata

A pituitary hormone, somatolactin (SL), belonging to the GH/PRL family, is produced in the intermediate lobe of the teleost pituitary. The function of this protein is uncertain. Clones coding for SL were isolated and sequenced from a gilthead seabream pituitary cDNA expression library. The nucleotide sequence of the larger cDNA isolated was 1.5 kb containing a 0.8-kb 3′-untranslated region and two potential polyadenylation signals (AATAAA). The mature polypeptide is composed of 207 amino acids, and a signal peptide of 24 residues was also found in the SL precursor. A potential N-glycosylation site Asn-Lys-Thr was identified in gilthead seabream SL. A comparison of the SL amino acid sequences of several fishes indicated that seven cysteine residues are characteristically present in all the SLs so far isolated. Six of those residues are present in homologous positions in SL and GHSparus aurataproteins. SL and GH fromS. auratashowed a 43% homology at the nucleotide level and 22% identity at the amino acid level. Expression of recombinant SL (rSL) inEscherichia coliand isolation from inclusion bodies led to a monomeric form of SL identical in electrophoretic mobility to one of the two forms of the native SL secreted from gilthead seabream pituitaries culturedin vitro.Further, a native glycosylated modified SL secretedin vitroas shown by N-glycosidase treatment was identified. Specific anti-SL antibodies that discriminate well against gilthead seabream GH and PRL in immunoblotting were also raised against rSL.     

Role of the oxidative metabolic burst in the antibody-dependent cellular cytotoxicity mediated by neutrophil polymorphonuclears

The purpose of the present study was to investigate the mechanisms by which neutrophil polymorphonuclears (PMN) mediate antibody-dependent cellular cytotoxicity (ADCC). Under experimental conditions which allow target cell phagocytosis, PMN were found to efficiently kill IgG-sensitized ox erythrocytes, as determined by the 51Cr release assay. Inhibition of the target cell ingestion by colchicine did not affect the PMN cytotoxic activity, suggesting that target cell phagocytosis does not represent an essential step in the PMN-mediated ADCC against erythrocytes. PMN from patients with Chronic Granulomatous Disease, who have defective oxidative metabolic burst, displayed an impaired ADCC activity, which was unaffected by changes in the phagocytic capacities induced by colchicine. The results indicate that, under the experimental conditions employed, both the intracellular and the extracellular target cell destruction by PMN involve oxygen-dependent mechanisms.     

Defective production of reactive oxygen intermediates (ROI) in a patient with recurrent amebic liver abscess

Previously, we reported the case of a man in the fourth decade of life afflicted with three independent episodes of amebic liver abscesses over a period of 4 years. Previous evidence has indicated that the cellular immune response is involved in protection against recurrent invasive amebic infection, and macrophage-mediated effector mechanisms appear important for host resistance to Entamoeba histolytica infection. The aim of the present work was to investigate locomotor activity and oxidative burst function of peripheral mononuclear cells of this individual after healing of the third amebic liver abscess. A locomotion assay using Boyden chemotaxis chambers and the respiratory burst evaluated by chemiluminescence were performed in both mononuclear phagocytes (MPs) and polymorphonuclear (PMN) leukocytes. Levels of salivary IgA and serum IgG anti-amebic antibodies were followed during 48 months after the second amebic liver abscess. Results obtained showed a deficiency in MP but not in PMN leukocyte respiratory burst. Respiratory burst is a major microbicidal mechanism in MP leukocytes; this also has been considered as a host resistance strategy against E. histolytica. It may be at least one risk factor in our patient that was responsible for recurrence of amebic liver abscess.     

Effect of photoperiod on the fish innate immune system: a link between fish pineal gland and the immune system

The pineal gland via its secretory product, melatonin, influences the light-dark rhythm in most vertebrates including fish. Apart from the information concerning this circadian rhythm, the interrelation of the melatonin with other physiological processes has not been considered in fish. Thus, we evaluated the changes in the humoral innate immune system of seabream (Sparus aurata L.) and sea bass (Dicentrarchus labrax L.) specimens exposed to a constant light-dark photoperiod (12 hr L:12 hr D). Serum was obtained from blood samples collected at 02:00, 08:00 hr (light-on), 14:00, 20:00 hr (light-off) and at 08:00 hr again. Among the humoral innate immune responses, complement, lysozyme and peroxidase activities were determined. Complement activity was higher during the day than during the night in both fish species. Seabream lysozyme activity reached its maximum at 20:00 and 02:00 hr but was hardly affected in sea bass. Finally, the peroxidase activity of seabream was significantly higher at 08:00 hr than during the rest of the cycle while, in sea bass, it showed little variation. The present results demonstrate that the humoral innate immune system has a circadian rhythm based on the light-dark cycle and that this cycle might be affected by the pineal gland.     

Correlation of cytotoxic activity in lungs to recovery of normal and gamma-irradiated cotton rats from respiratory syncytial virus infection

Cotton rats (Sigmodon hispidus) that were exposed to 300, 600, or 900 rads of gamma irradiation and inoculated intranasally 2 days later with respiratory syncytial virus (RSV) exhibited prolonged virus shedding and delayed humoral and cytotoxic immune responses compared with comparably inoculated nonirradiated control rats. In nonirradiated animals and in animals exposed to 300 and 600 rads, levels of virus declined and then disappeared from the lungs during the period in which cytotoxic activity was maximal in the lungs of these animals. In contrast, in the group of cotton rats exposed to 900 rads of irradiation, local cytotoxic activity remained low throughout the 11-day observation period, and virus was not eliminated from the lungs. Although virus-neutralizing antibodies in serum and lavage fluids from these animals may have been involved, correlation of antibody concentrations with virus clearance from lungs was not as evident. These data suggest that cytotoxic effector cells have a positive role in eliminating RSV from the lungs of unprimed cotton rats.     

Investigation of the in vitro Gender-Specific Partitioning of Mefloquine in Malarial Infected Red Blood Cells and Plasma

The investigation of gender-specific partitioning of the antimalarial drug mefloquine to cellular and fluid blood compartments was performed using blood collected from a female and male healthy subject that were infected with Plasmodium falciparum PCM2 clone and spiked with mefloquine (0.25, 1, and 5 μM). Mefloquine concentrations in red cells of both female and male subjects were significantly higher than plasma, which suggests an intensive uptake by red cells. This was supported by a high ratio of mefloquine concentrations in the parasitized and non-parasitized red cells of about 4-fold. Gender-specific partitioning of mefloquine in parasitized blood was seen only in plasma where significantly higher concentrations were observed in female compared with male plasma. Down-adjusting the therapeutic dose of mefloquine in female patients with malaria is not advisable because mefloquine concentrations in the target cellular compartment are similar in both genders.     

Effects of gonadotropin-releasing hormone agonist and dopamine antagonist on hypothalamus-pituitary-gonadal axis of pre-pubertal female red seabream (Pagrus major)

The effects of GnRH agonist (GnRHa) on the hypothalamus-pituitary-gonadal axis were studied in female pre-pubertal red seabream. Sexually immature 16-month-old fish were implanted intramuscularly with cholesterol pellets containing GnRHa or GnRHa in combination with domperidone, putative dopamine antagonist, and reared for 10-20 days. In both GnRHa and GnRHa+domperidone implanted groups, vitellogenesis was observed on Day 10 and ovulation was observed on Day 20, while ovarian development was not observed in the control fish throughout the experimental period. The levels of GnRH receptor mRNA were significantly higher in both GnRHa implanted groups than in the control. The expressions of all three gonadotropin subunit genes were up-regulated and serum luteinizing hormone levels were increased by the GnRHa implantation. Serum testosterone and estradiol-17beta levels were also increased on Day 10 and maintained high levels on Day 20. On the other hand, seabream (sb) GnRH mRNA levels in the brain were relatively low and unchanged in all experiment groups. The present study first shows that GnRH alone can induce precocious puberty in red seabream. These results indicate that the system of pituitary-gonadal axis has already been developed in 16-month-old fish and the commencement of sbGnRH secretion may be an important physiological event for the onset of puberty in the red seabream.     

Influence of skinfold sum and peak VO(2) on immune function in children

OBJECTIVE: To measure the relationship of skinfold sum and peak VO(2) power with immune function in children. DESIGN: Cross-sectional, with all children tested twice during a 2 month period for peak VO(2), sum of two skinfolds, and immune function, with data from the two measures averaged and then correlated (alpha level, < or = 0.01). Immune measures included leukocyte and lymphocyte subset counts, delayed-typed hypersensitivity (DTH), global IgG antibody response over 4 weeks to pneumococcal vaccination (pIgG), salivary IgA concentration (sIgA), PHA-stimulated lymphocyte proliferation (PHA-SLP), natural killer cell activity (NKCA), and granulocyte and monocyte phagocytosis and oxidative burst activity. SUBJECTS: Seventy-three children (n=42 males, n=31 females) ranging in age from 7 to 13 y (mean+/-s.d. age, 9.9+/-1.7 y). The mean skinfold sum was 28.9+/-17.1 mm, and peak VO(2) 45.8+/-8.1 ml/kg/min. RESULTS: Peak VO(2), skinfold sum, and immune measures did not differ significantly by age or gender. Therefore, correlations were made on combined indices for all subjects. Peak VO(2) and the skinfold sum were not significantly correlated with NKCA, oxidative burst activity, plgG or DTH. Peak VO(2) was negatively correlated with monocyte phagocytosis (r=-0.30, P=0.012) and positively correlated with PHA-SLP (6.25 microg/ml; r=0.35, P=0.004). The skinfold sum was positively correlated with the total leukocyte count (r=0.39, P<0.001), granulocyte count (r=0.36, P=0.002), monocyte count (r=0.38, P=0.001), monocyte phagocytosis (r=0.41, P<0.001), granulocyte phagocytosis (r=0.35, P=0.003), and sIgA (r=0.32, P=0.006), and negatively correlated with PHA-SLP (6.25 microg/ml; r=-0.39, P=0.001). CONCLUSIONS: Data from this study indicate that a high skinfold sum is related to elevated leukocyte subset counts and monocyte/granulocyte phagocytosis, and low PHA-SLP in children.     

Immune-enhancing role of vitamin C and zinc and effect on clinical conditions

Vitamin C concentrations in the plasma and leukocytes rapidly decline during infections and stress. Supplementation of vitamin C was found to improve components of the human immune system such as antimicrobial and natural killer cell activities, lymphocyte proliferation, chemotaxis, and delayed-type hypersensitivity. Vitamin C contributes to maintaining the redox integrity of cells and thereby protects them against reactive oxygen species generated during the respiratory burst and in the inflammatory response. Likewise, zinc undernutrition or deficiency was shown to impair cellular mediators of innate immunity such as phagocytosis, natural killer cell activity, and the generation of oxidative burst. Therefore, both nutrients play important roles in immune function and the modulation of host resistance to infectious agents, reducing the risk, severity, and duration of infectious diseases. This is of special importance in populations in which insufficient intake of these nutrients is prevalent. In the developing world, this is the case in low- and middle-income countries, but also in subpopulations in industrialized countries, e.g. in the elderly. A large number of randomized controlled intervention trials with intakes of up to 1 g of vitamin C and up to 30 mg of zinc are available. These trials document that adequate intakes of vitamin C and zinc ameliorate symptoms and shorten the duration of respiratory tract infections including the common cold. Furthermore, vitamin C and zinc reduce the incidence and improve the outcome of pneumonia, malaria, and diarrhea infections, especially in children in developing countries.