Detection of Reactive Oxygen Species in Human Neutrophils Under Various Conditions of Exposure to Galectin.

Reactive oxygen species (ROS) have been extensively studied in biology in the past years. This class of molecules can be derived from endogenous sources (e.g., phagocytic cells as neutrophils, eosinophils, monocytes, macrophages, and organelles as mitochondria and peroxisomes) and participate in physiological and pathological conditions. The beneficial and harmful effects of ROS depend on redox regulation, which establishes the balance between their production and the activity of antioxidant systems to prevent oxidative stress in vivo. Neutrophils are the immune effectors most well depicted with an intense oxidative burst in response to tissue inflammation. Several proteins and members of the galectin family are involved in this fine modulation of ROS production by neutrophils. Interestingly, studies have indicated that Galectin-1 (Gal-1) can up- or downregulate ROS production by neutrophils even when exposed to N-formyl-Met-Leu-Phe (fMLP) or Phorbol Myristate Acetate (PMA), both of which are potent neutrophil stimulants that trigger high levels of ROS production. Similarly, Galectin-3 (Gal-3) induces ROS in neutrophils from a sterile or nonsterile inflammatory environment, possibly creating a negative loop that could control ROS production. Besides, superoxide production is also induced by Galectin-8 (Gal-8) and Galectin-9 (Gal-9) in neutrophils but in a different manner. We describe herein the luminol and lucigenin-dependent chemiluminescence technique by using a luminometer as a method of assessment to measure ROS production by human neutrophils isolated and exposed to purified human recombinant Gal-1. The protocol described herein could be applied for the investigation of the role of other galectins in the modulation of ROS production by neutrophils.

Role of neuroendocrine modulation and biochemistry in the sepsis in Piaractus mesopotamicus.

Sepsis is a systemic process with multifactorial pathophysiology that affects most animal species. It is responsible for high rates of morbidity and mortality. This work aimed to study the biochemical and neuroendocrine changes of the sepsis process in Piaractus mesopotamicus after Aeromonas hydrophila inoculation analyzing changes in blood leukocyte and differences in neuroendocrine-biochemical modulation using RNA-seq. Fish showed hypercortisolemia, inhibition of glucose absorption, followed by hypocortisolemia and then hyperglycemia. Thyroid hormones (T3 and T4) showed immediate decrease in serum and T4 increased 6 h post-inoculation (HPI). Sepsis-induced hormonal alterations triggered changes in the metabolic pathways increasing protein and lipid catabolism, use of transient anaerobic glycolysis and liver injury. A reference transcriptome was constructed based on blood leukocytes from P. mesopotamicus. The assembly resulted in total 266,272 contigs with a N50 of 2786 bp. There was a reorganization of plasma membrane of leukocytes at the beginning of the septic process with increased expression of neuroendocrine receptors and with continuous flow of neurotransmitters, hormones and solutes with compensatory regulation at 6 HPI. Three and nine HPI seemed to be critical, the expression of a number of transcription factors was increased, including the modulatory DEGs related to glucocorticoid and thyroid hormones induced and suppressed (FDR < 0.05). Neuroendocrine modulation can regulate leukocytes and biochemical parameters of peripheral blood, being important sources for the study of the pathophysiology of sepsis. These finding highlights the importance of further studies focusing on biochemical-neuroendocrine changes in blood leukocytes and systemic sepsis.

Modulation of stress and innate immune response by corticosteroids in pacu (Piaractus mesopotamicus).

Understanding how stress and corticosteroid modulates the innate immune response is one of the keys to improving productivity and reducing losses in intensive aquaculture. Thus, we investigated the effects of dietary corticosteroids (7 days; long-term exposure) and transport (4 h; short-term stress) on stress and innate immune response in pacu. For this end, fish were fed with diets containing dexamethasone (100 mg kg-1) or hydrocortisone (200 mg kg-1), followed by transport, and then were intraperitoneally inoculated with heat-killed Aeromonas hydrophila or PBS (sham-inoculation). Fish were sampled after a 7-day feeding period, immediately post-transport and 24 h post-transport and inoculation. The dietary treatment of corticosteroids decreased resting cortisol levels by inhibiting the production of cortisol on the hypothalamus pituitary interrenal-axis. Further, both corticosteroids reduced hematocrit, red blood cells, haemoglobin and hemolytic activity of the complement, while they increased glucose levels and serum lysozyme concentrations. The transport increased cortisol and glucose levels and reduced the humoral immune defenses such as serum lysozyme concentration and hemolytic activity of the complement system. Interestingly, the hemolytic activity of the complement system increased sharply in fish fed with corticosteroids immediately post-transport, when they had their HPI-axis partially suppressed by the corticosteroids. This finding suggests a stimulatory effect of the catecholamines released during the transport on the activity of the complement system. Our results are highly valuable to understanding the stress and innate immune responses to long-term exposure to corticosteroids and short-term stress in fish and may provide insights into how corticosteroids modulate the innate immune system.

Hematological and immune changes in Piaractus mesopotamicus in the sepsis induced by Aeromonas hydrophila.

The pathogenesis of sepsis involves complex systems and multiple interrelationships between the host and pathogen producing high mortality rates in various animal species. In this study, hematological disturbances, innate immunity and survival during the septic process in Piaractus mesopotamicus inoculated with Aeromonas hydrophila were studied. For this aim, fish blood samples were taken from control and infected groups 1, 3, 6, and 9 h post-inoculation (HPI). Leukogram showed reduction in the number of leukocytes and thrombocytes, followed by cessation of leukocyte chemotaxis 6 HPI and severe morphological changes in leukocytes and erythrocytes. At 3 HPI production of reactive oxygen species increased and at 6 HPI decreased. There was no change in serum lysozyme concentration and lytic activity of the complement system, despite the progressive increase in serum lytic activity and bacterial agglutination. Finally, the changes in clinical signs due to aeromonosis and increasing septicemia resulted in a reduction in survival to 57.14% after 36 HPI. It was possible concluded that these hematological and immune are crucial event in the worsening of sepsis in P. mesopotamicus, and these findings are utility for diagnosing and understanding the pathophysiology sepsis in pacu induced by A. hydrophila.

Epigenetic alterations are associated with monocyte immune dysfunctions in HIV-1 infection.

Monocytes are key cells in the immune dysregulation observed during human immunodeficiency virus (HIV) infection. The events that take place specifically in monocytes may contribute to the systemic immune dysfunction characterized by excessive immune activation in infected individuals, which directly correlates with pathogenesis and progression of the disease. Here, we investigated the immune dysfunction in monocytes from untreated and treated HIV + patients and associated these findings with epigenetic changes. Monocytes from HIV patients showed dysfunctional ability of phagocytosis and killing, and exhibited dysregulated cytokines and reactive oxygen species production after M. tuberculosis challenge in vitro. In addition, we showed that the expression of enzymes responsible for epigenetic changes was altered during HIV infection and was more prominent in patients that had high levels of soluble CD163 (sCD163), a newly identified plasmatic HIV progression biomarker. Among the enzymes, histone acetyltransferase 1 (HAT1) was the best epigenetic biomarker correlated with HIV - sCD163 high patients. In conclusion, we confirmed that HIV impairs effector functions of monocytes and these alterations are associated with epigenetic changes that once identified could be used as targets in therapies aiming the reduction of the systemic activation state found in HIV patients.

Aloe vera bathing improved physical and humoral protection in breeding stock after induced spawning in matrinxã (Brycon amazonicus).

In this study, we show that induced spawning causes stress, an intense loss of epithelia and immunosuppression, decreasing physical and humoral protection in fish, effects that were prevented or improved in fish bathed with Aloe vera. A. vera has several medicinal properties, including wound healing and immunostimulatory effects, which we observed in this study. Fish bathed with A. vera had a higher number of epidermal goblet cells and, in general, an improved wound healing rate compared with the control after induced spawning. These effects might be related to (1) the stimulation of leukocyte activity, represented here by the increased leukocyte respiratory activity triggered by A. vera (leukocytes are recognized as playing an important role in wound repair); (2) the antimicrobial properties of A. vera, which decrease wound infection and accelerate the healing process; and (3) several mechanisms that explain the healing effect of A. vera (increased collagen synthesis, rate of epithelialization, and anti-inflammatory and moisturizing effects). Our results also suggest that caution is necessary during the induced spawning process, especially during stripping, and A. vera bathing is recommended after intensive aquaculture operations.

Comparative study of the effects of combined oral contraceptives in hemostatic variables: an observational preliminary study.

Thrombotic risk is associated with the estrogen dose and type of progestin in combined oral contraceptives. Studies published since 1990 showed that third-generation progestins have larger risk to contribute to thrombosis development than the second-generation. However, there are conflicts in the literature regarding the thrombotic risk associated to the drospirenone progestin. So, this study aimed to evaluate the effects of 3 formulations of contraceptives containing ethinylestradiol (EE) (20 and 30 µg) combined with drospirenone versus levonorgestrel combined with EE (30 µg) in hemostatic parameters. This cross-sectional study included 70 healthy women between 18 and 30 years, BMI 19 to 30 kg/m², not pregnant, non-smokers, and users or non-users (control) of contraceptives for a minimum period of 6 months. The following parameters were assessed: prothrombin time (PT), Factor VII, activated partial thromboplastin time (aPTT), Factor XII, fibrinogen, Factor 1 + 2, Protein C, Protein S, antithrombin, D-dimers, and plasminogen activator inhibitor-1. Significant alterations were found in PT, aPTT, fibrinogen, D-dimers, and protein S, all favoring a state of hypercoagulation for contraceptive containing DRSP/20EE. Both contraceptives containing DRSP/30EE and LNG/30EE promoted changes that favor the hypercoagulability in the coagulant variable PT and in the anticoagulant variables Protein S and Protein C, respectively. We suggest that the progestin drospirenone can contribute to an inadequate balance among procoagulant, anticoagulant, and fibrinolytic factors, since that the contraceptive containing the lowest dose of estrogen and drospirenone (DRSP/20EE) caused a higher number of hemostatic changes.

Acute aerocystitis in Nile tilapia bred in net cages and supplemented with chromium carbochelate and Saccharomyces cerevisiae.

Oreochromis niloticus bred in net cages were supplemented with cell wall of Saccharomyces cerevisiae (Sc) (0.3%) or chromium carbochelate (Cr) (18 mg/kg of feed) or in association (Sc + Cr), for 90 days. After this period, acute inflammation was induced in the swim bladder by inoculation of 3 × 10(8) CFU of inactivated Streptococcus agalactiae, and another group received 0.65% saline solution (control). Twelve, 24, and 48 h after stimulation, the inflammation was evaluated through total and differential counting of accumulated cells, and through leukocyte respiratory burst in the blood, cortisolemia, glycemia and serum lysozyme concentration. The results showed that there were greater total numbers of cells in the exudate of fish inoculated with inactivated bacterium than in those injected with saline solution, with predominance of lymphocytes, thrombocytes, macrophages and granulocytes. Tilapia supplemented with Cr presented increased total numbers of cells with significant accumulation of lymphocytes and reductions in cortisolemia and glycemia, but the different treatments did not have any influence on leukocyte respiratory burst or serum lysozyme concentration. Tilapia supplemented with Sc and the Cr + Sc association did not present significant changes to the variables evaluated, despite higher accumulation of lymphocytes in the inflammatory exudate from fish treated with Sc. The results indicate that tilapia bred in net cages and supplemented with Cr presented higher total accumulation of cells at the inflammatory focus, thus indicating an increase in the inflammatory response induced by the bacterium, probably due to the reduction in cortisolemia and higher glucose consumption. Thus, supplementation with Cr had beneficial action, which facilitated development of acute inflammation induced by the bacterium, but did not affect neither leukocyte respiratory burst in the blood nor serum lysozyme concentration.

Galectin-3 plays a modulatory role in the life span and activation of murine neutrophils during early Toxoplasma gondii infection.

Galectins are beta-galactoside-binding lectins involved in several biological processes and galectin-3 (Gal-3) is related to modulation of immune and inflammatory responses. This study aimed to evaluate the role of Gal-3 in the life span and biological functions of murine neutrophils during in vitro infection by virulent Toxoplasma gondii RH strain. Inflammatory peritoneal neutrophils (Nphi) from C57BL/6 wild-type (WT) and Gal-3 knockout (KO) mice were cultured in the presence or absence of parasites and analyzed for phosphatidylserine (PS) exposure and cell death using Annexin-V and propidium iodide staining, and cell viability by MTT assay. Cell toxicities determined by lactate dehydrogenase (LDH), degranulation by lysozyme release, and cytokine production were measured in Nphi culture supernatants. Phorbol myristate acetate (PMA)- or zymosan-dependent reactive oxygen species (ROS) were measured in Nphi cultures. Our results demonstrated that Gal-3 is involved in the increase of the viable Nphi number and the decrease of PS exposure and cell death following T. gondii infection. We also observed that Gal-3 downmodulates T. gondii-induced Nphi toxicity as well as Nphi degranulation regardless of infection. Furthermore, Gal-3 expression by Nphi was associated with increased levels of IL-10 in the beginning and decreased levels of TNF-alpha later on, regardless of parasite infection, as well as with decreased levels of IL-6 and increased IL-12 levels, following early parasite infection. Our results also showed that Gal-3 suppresses PMA- but not zymosan-induced ROS generation in Nphi following T. gondii infection. In conclusion, Gal-3 plays an important modulatory role by interfering in Nphi life span and activation during early T. gondii infection.

Superoxide anion production by neutrophils is associated with prevalent clinical manifestations in systemic lupus erythematosus.

To determine the relation between neutrophil function and the clinical characteristics of systemic lupus erythematosus (SLE), the superoxide anion (O2-) production by neutrophils, mediated by FcgammaR and FcgammaR/CR cooperation, was studied in 64 SLE patients classified according to their prevalent clinical manifestations. Three clinically distinct patterns were designated: (1) manifestations associated with the occurrence of cytotoxic antibodies (SLE-I group); (2) manifestations associated with circulating immune complexes (IC; SLE-II group), and (3) manifestations associated with IC and cytotoxic antibodies (SLE-III group). O2- production was evaluated by a lucigenin-dependent chemiluminescent assay in neutrophils stimulated with IC-IgG opsonized or not with complement. No difference in O2- production was observed when neutrophil responses from healthy controls were compared to the unclassified patients. However, when the SLE patient groups were considered, the following differences were observed: (1) SLE-I neutrophils showed lower O2- production mediated by the IgG receptor (FcgammaR) with the cooperation of complement receptors (FcgammaR/CR) than observed in the SLE-II, SLE-III, and healthy groups; (2) neutrophils from the SLE-II group showed a decreased [Formula: see text] production mediated by FcgammaR/CR compared to the SLE-III group, (3) SLE-III neutrophils produced more O(2)(-) than neutrophils from the SLE-II and control groups, and (4) CR showed inefficiency in mediating the O2- production by neutrophils from the SLE-I group. Comparative experiments on the kinetics of chemiluminescence (CL; Tmax and CLmax) disclosed differences only for the SLE-I group. Taken together, these results suggest that differences in oxidative metabolism of neutrophils mediated by FcgammaR/CR may reflect an acquired characteristic of disease associated with distinct clinical manifestations.

Receptores para imunoglobulina G (FcyR) / Receptors for immunoglobulin G (FcyR)

Os receptores para IgG (Fc'gama'R) fazem uma importante ligação entre as respostas imunes humoral e celular. Estes receptores medeiam várias respostas biológicas tais como: fagocitose, endocitose, captura e clearance de imunocomplexos, citotoxicidade e liberação de mediadores inflamatórios. Os Fc'gama'R humanos pertencem à superfamília das imunoglobulinas e três classes principais destes receptores são descritas, as quais apresentam várias isoformas. Estas moléculas diferem quanto à afinidade e especificidade para os isotipos de IgG, distribuição celular, sinalização intracelular e pesos moleculares. Além disso, o polimorfismo genético é responsável por variações entre os indivíduos. A diversidade estrutural e funcional dos Fc'gama'R faz destas moléculas importantes alvos para a imunoterapia. A ativação e desativação de células efetoras via Fc'gama'R pode ser explorada para o desenvolvimento de novas terapias para o câncer, doenças infecciosas e desordens auto-imunes. Esta revisão descreve detalhes estruturais e funcionais, relevância clínica e alguns usos terapêuticos dos Fc'gama'R