Production Of Reactive Oxygen Intermediates Research Articles

Characterization of the pro-inflammatory roles of the goldfish (Carassius auratus L.) M17 protein

The interleukin-6 family of cytokines possesses a diversity of roles with significant redundancy. The roles of these molecules have been relatively well characterized in mammals, with limited attention in other species. Progress has been made in the discovery of homologous molecules in fish. Here we report the characterization of pro-inflammatory properties of recombinant goldfish M17. Recombinant goldfish M17 enhanced phagocytosis, primed production of reactive oxygen intermediates, and was chemotactic to macrophages. Treatment of goldfish macrophages with LPS, heat-killed and live Aeromonas hydrophila resulted in higher M17 mRNA levels. Recombinant M17 (RgM17) induced dose-dependent production of IFNγ and IL-1β1 in goldfish macrophages. Furthermore, treatment of macrophages with rgM17 resulted in upregulation of transcription factors that were important in the differentiation of myeloid progenitors into monocytes/macrophages (Runx1 and GATA2). Our results indicate that goldfish M17 is an essential inflammatory cytokine for proliferation and differentiation of goldfish progenitor cells.

Selective modulation of monocyte and neutrophil responses with activated protein C in preterm infants

Background Inflammation is associated with many disorders of preterm infants including periventricular leukomalacia, chronic lung disease, and necrotizing enterocolitis. Activated protein c (APC) has shown positive immunomodulatory effects. Objectives We aimed to study neutrophil and monocyte function in response to lipopolysaccharide (LPS) and APC stimulation ex vivo in preterm infants <32 weeks gestation over the first week of life compared to neonatal and adult controls. Methods Peripheral blood was taken on day 1, 3, and 7 and stimulated with LPS in the absence or presence of APC. Expression of toll-like receptor 4 (TLR4) and CD11b and reactive oxygen intermediate (ROI) release from neutrophils and monocytes was examined by flow cytometry. Results LPS induced neutrophil ROI in adults and preterm infants and was significantly reduced by APC. Baseline and LPS-induced monocyte ROI production in preterm neonates was increased compared to adult and term controls. Neutrophil TLR4 baseline expression was higher in term controls compared to preterm infants. Conclusion Increased systemic ROI release in preterm infants may mediate tissue damage, ROI was reduced by APC. However, due to the high risk of hemorrhage further examination of APC mutant forms with anti-inflammatory but decreased anticoagulant properties is merited.

Molecular mechanisms of inflammation in the pathogenesis of respiratory disorders in patients with pulmonary tuberculosis

Aim. To assess external respiration (ER) and its relationship with the activity of enzymes involved in purine metabolism in patients with acute and chronic forms of pulmonary tuberculosis (TB).Materials and methods. In patients with acute and chronic TB, we assessed the activity of adenosine deaminase (ADA)-1, 2 in the blood serum (eADA), mononuclear cells, and neutrophils, the concentration of ecto-5’-nucleotidase (eNT5E) in the blood serum, the level of CD26 (dipeptidyl peptidase-4, DPPIV) in the blood serum and mononuclear cells, production of reactive oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI) in mononuclear cells and neutrophils, as well as parameters of ER.Results. Patients with TB were found to have an increase in the concentration of eNT5E and eADA-2 activity in the blood serum, stimulated production of ROI in neutrophils, a decrease in the concentration of DPPIV (CD26) in mononuclear cells, and a fall in the production of RNI in mononuclear cells and neutrophils. In patients with chronic TB, a decrease in the activity of ADA-1 in mononuclear cells and a fall in the concentration of DPPIV (CD26) in the blood serum were noted. In patients with acute TB, a decrease in the activity of eADA-1 in the blood serum and ADA-1 in neutrophils, reduced production of ROI in mononuclear cells, and an increase in spontaneous production of ROI in neutrophils were revealed. Correlations were found between the parameters of ER and the concentration of eNT5E in the blood serum, spontaneous production of ROI in mononuclear cells and production of RNI in neutrophils in chronic TB, as well as between eADA-2 in the blood serum, ADA-1 in neutrophils, DPPIV (CD26) activity in mononuclear cells, and ROI and RNI production in mononuclear cells and neutrophils.Conclusion. The data obtained make it possible to associate regulation of external respiration with parameters of purine metabolism, in particular with the concentration and activity of enzymes responsible for generation and metabolism of adenosine, that determine its level outside cells and inside mononuclear cells and neutrophils, with expression of cofactor molecules, as well as with the duration of activation of cells in innate immunity, neutrophils, and monocytes/ macrophages, determined largely by the potential of adenosine regulation.

Captopril Combined with Furosemide or Hydrochlorothiazide Affects Macrophage Functions in Mouse Contact Hypersensitivity Response.

Hypertension is a chronic disease associated with chronic inflammation involving activated macrophages. Antihypertensive drugs (for example, angiotensin-converting enzyme inhibitors—ACEIs) used in the treatment of hypertension have immunomodulatory properties. On the other hand, the immunological effect of diuretics and combined drugs (diuretics + ACEI) is unclear. Therefore, we examined the influence of diuretics and combination drugs (ACEI + diuretic) on cellular response (contact hypersensitivity), production of reactive oxygen intermediates (ROIs), and nitric oxide (NO), and the secretion of interleukin-12 (IL-12). CBA mice were administered i.p. captopril (5 mg/kg) with or without hydrochlorothiazide (10 mg/kg) or furosemide (5 mg/kg) for 8 days. On the third day, the mice were administered i.p. mineral oil, and macrophages were collected 5 days later. In the presented results, we show that diuretics administered alone or with captopril increase the generation of ROIs and reduce the formation of NO by macrophages. Moreover, tested drugs inhibit the secretion of IL-12. Diuretics and combined drugs reduce the activity of contact hypersensitivity (both activation and induction phases). Our research shows that the tested drugs modulate the cellular response by influencing the function of macrophages, which is important in assessing the safety of antihypertensive therapy.

Diet-induced obesity in mice impairs host defense against Klebsiella pneumonia in vivo and glucose transport and bactericidal functions in neutrophils in vitro.

Obesity impairs host defense against Klebsiella pneumoniae, but responsible mechanisms are incompletely understood. To determine the impact of diet-induced obesity on pulmonary host defense against K. pneumoniae, we fed 6-wk-old male C57BL/6j mice a normal diet (ND) or high-fat diet (HFD) (13% vs. 60% fat, respectively) for 16 wk. Mice were intratracheally infected with Klebsiella, assayed at 24 or 48 h for bacterial colony-forming units, lung cytokines, and leukocytes from alveolar spaces, lung parenchyma, and gonadal adipose tissue were assessed using flow cytometry. Neutrophils from uninfected mice were cultured with and without 2-deoxy-d-glucose (2-DG) and assessed for phagocytosis, killing, reactive oxygen intermediates (ROI), transport of 2-DG, and glucose transporter (GLUT1-4) transcripts, and protein expression of GLUT1 and GLUT3. HFD mice had higher lung and splenic bacterial burdens. In HFD mice, baseline lung homogenate concentrations of IL-1β, IL-6, IL-17, IFN-γ, CXCL2, and TNF-α were reduced relative to ND mice, but following infection were greater for IL-6, CCL2, CXCL2, and IL-1β (24 h only). Despite equivalent lung homogenate leukocytes, HFD mice had fewer intraalveolar neutrophils. HFD neutrophils exhibited decreased Klebsiella phagocytosis and killing and reduced ROI to heat-killed Klebsiella in vitro. 2-DG transport was lower in HFD neutrophils, with reduced GLUT1 and GLUT3 transcripts and protein (GLUT3 only). Blocking glycolysis with 2-DG impaired bacterial killing and ROI production in neutrophils from mice fed ND but not HFD. Diet-induced obesity impairs pulmonary Klebsiella clearance and augments blood dissemination by reducing neutrophil killing and ROI due to impaired glucose transport.

Association of recurrent upper respiratory tract infections with low production of oxygen intermediates in children

ObjectivesUpper respiratory tract infections in children generally have significant morbidity and mortality. There is little data available about functional immaturity of the immune system and the child's susceptibility to infections at the beginning of their lives, thus, justifying a more specific immunological analysis. MethodAnalysis of hemograms and innate and adaptive immune responses in 95 children between age 1 to 6 years with episodes of recurrent respiratory infections (test group n = 39) and without these episodes (control group n = 56) was carried out. The production of reactive oxygen intermediates by peripheral blood cells stimulated by phorbol myristate acetate was analyzed. Additionally, the number of B lymphocytes, auxiliary T lymphocytes, and cytotoxic cells was determined using flow cytometry. ResultsResults from both groups did not show statistically significant differences in red blood cells, total leukocytes count, and the differential neutrophils, eosinophils, basophils, lymphocytes, and monocytes count. The analysis of the number of B lymphocytes, auxiliary T lymphocytes (LTCD4), and cytotoxic cells (LTCD8) also did not show any difference between both groups. However, the production of radical oxygen intermediates was significantly reduced in the test group as compared to the control group (p < 0.05). ConclusionsThere was no difference in the analysis of hemograms, leukograms, or the number of lymphocytes, LTCD4, LTCD8, or LTCD19. The reduced production of oxygen intermediates in the affected group suggests that these children's microbicide capacity is compromised, which may be related to their recurrent respiratory infections.

ISOLYQUIRITIGENIN AFFECTS PHAGOCYTES FUNCTIONS AND INCREASES MICE SURVIVAL RATE IN STAPHYLOCOCCAL INFECTION

The results of studying the effect of isoliquiritigenin on animal survival in the model of staphylococcal infection and the function of human and animal phagocytes are presented in this article.The aim of the investigation was to study the effect of an isoliquiritigenin preliminary administration on the survival of animals against the background of staphylococcal infection, as well as on the function of phagocytes in mice and humans.Materials and methods. To assess the survival of Balb/C mice, a model of infection caused by Staphylococcus aureus J49 ATCC 25923 with the construction of Kaplan-Meier curves, was used. The effect on the phagocytes functions was studied by assessing the peptone-induced migration of phagocytes into the abdominal cavity of Balb/C mice, the absorption activity of phagocytes (neutrophils and monocytes) of human blood, as well as their production of reactive oxygen intermediates (ROIs) using а flow cytometry.Results. It was found out that a preliminary triple intraperitoneal administration of isoliquiritigenin (30 mg/kg) increases the survival rate of Balb/C mice in staphylococcal infection caused by Staphylococcus aureus J49 ATCC 25923. At the same time, isoliquiritigenin dose-dependently activates the production of reactive oxygen intermediates by human neutrophils and monocytes without statistically significantly suppressing a phagocytic activity of monocytes and neutrophils against fluoresceinisothiocyanate-labeled S. aureus J 49 ATCC 25923, as well as peptone-induced migration of phagocytes into the abdominal cavity of mice.Conclusion. Thus, a preliminary administration of isoliquiritigenin increases the survival rate of mice with staphylococcal infection and increases the production of reactive oxygen intermediates by phagocytes. The data obtained, can become the basis for further research of antibacterial and immunotropic effects of isoliquiritigenin in order to find new drugs for the treatment of staphylococcal infection.

Discoordination of the processes of activation and suppression of immunocompetent cells during mesotherapy with hyaluronic acid

Objective. The study aimed to assess the dynamics of the systemic immune response in patients during mesotherapy with hyaluronic acid (HA).Materials and methods. The observation group included 26 women who received the first-time mesocorrection with drugs based on hyaluronic acid (HA). Injections of low molecular weight HA were carried out according to standard methods in a course of 5 procedures with an interval of 14 days. The parameters of the innate and adaptive immune response were studied before and two weeks after the end of the course. The skin condition was assessed by dermatoscopy (Aramo Smart Lite 300, Southern Korea).Results. The clinical effect of the course of procedures was reflected in the subjective improvement in the appearance. The improvement was confirmed by hardware analysis, which recorded an increase in hydration, a smoothing of the skin relief, and a decrease in the depth of wrinkles. At the end of the course, redistribution of lymphocyte populations towards natural killer cells and B-lymphocytes was revealed with a decrease in the total number of T cells. The antibody production of immunoglobulins of classes M and G was increased, the serum content of Ig A and IgE was reduced, the number of both T-effectors and T-lymphocytes with immunosuppressive activity increased. Changes in the neutrophil system were characterized by the inhibition of the production of reactive oxygen intermediates; the dynamics of the expression of Toll-like receptors by monocytes was ambiguous.Conclusion. The results of the study confirmed the active involvement of the factors of innate and adaptive systemic response in the cosmetic effect, which manifested itself immediately after mesotherapy as systemic dysregulatory immune changes.

Evaluation of triazophos induced immunotoxicity of spleen and head kidney in fresh water teleost, Channa punctata

The utilization of pesticides has increased for destroying pests and protecting crops in the agriculture field. Triazophos is a commonly used organophosphorous insecticide that causes alterations in haematological and histological parameters in fish. The present study was designed to evaluate the effect of triazophos induced innate and cell mediated immunotoxicity in freshwater teleost, Channa punctata. Fishes were exposed to triazophos at concentrations 5 and 10% of LC50 value for 10 and 20 days. Splenic and head kidney macrophage phagocytosis, nitric oxide production and superoxide production were assayed to evaluate the innate immunity. Cell-mediated immunity was measured through splenic and head kidney lymphocyte proliferation in presence of T and B cell mitogens. Results of the present study revealed that macrophage phagocytosis was significantly reduced after in vivo triazophos treatment. Differential suppressive effect of triazophos was also observed where mitogen induced splenic and head kidney lymphocyte proliferations were reduced after 10 and 20 days treatment. Concentration dependent effect of triazophos was observed in in vivo studies where the production of reactive oxygen and nitrogen intermediates were suppressed. This study describes the first investigation of the effect of triazophos on immune functions and will help to determine appropriate ecotoxicity and immunotoxicity in freshwater teleosts.

Characterization of innate immune response to Brucella melitensis infection in goats with permissive or restrictive phenotype for Brucella intramacrophagic growth

Caprine brucellosis is a chronic, world-wide distributed disease which causes reproductive failure in goats and Brucella melitensis, its causative agent, bears a great zoonotic potential. There is evidence suggesting that some cattle and pigs have an innate ability to resist Brucella infection, but this has not yet been investigated in goats. In this study, we compared caprine macrophages that exhibit extreme restriction and permissiveness to B. melitensis’ intracellular growth in vitro. Monocyte derived macrophages (MDMs) from 110 female goats were cultured and challenged in vitro with B. melitensis 16 M. After initial screening, 18 donor goats were selected based on their macrophages ability to restrict or allow bacterial intracellular growth and some elements of humoral and cellular immunity were studied in depth. MDMs that were able to restrict the pathogen’s intracellular growth showed enhanced bacterial internalization, although there were no differences between groups in the production of reactive oxygen and nitrogen intermediates following 48 h treatment with heat-killed B. melitensis. Moreover, there were no differences between groups in the level of antibodies reacting with keyhole limpet hemocyanin (natural antibodies, NAbs) or with Brucella LPS antigens (cross-reacting antibodies, CrAbs), although a strong positive correlation between individual levels of IgM NAbs and IgM CrAbs was detected. Altogether, these results represent an initial step in understanding innate primary host response to B. melitensis, and deciphering which mechanisms may determine a successful outcome of the infection in goats.

The diterpene Manool extracted from Salvia tingitana lowers free radical production in retinal rod outer segments by inhibiting the extramitochondrial F1 Fo ATP synthase.

Uncontrolled oxidative stress production, especially in the outer retina is one of the causes of retinal degenerations. Mitochondria are considered the principal source of oxidative stress. However, a Reactive Oxygen Intermediates (ROI) production in the retinal photoreceptor layer seems to depend also on the expression of an extramitochondrial oxidative phosphorylation (OxPhos) machinery in the rod outer segments (OS). In fact, OS conduct aerobic metabolism, producing ATP through oxygen consumption, although it is devoid of mitochondria. As diterpenes display an antioxidant effect, we have evaluated the effect Manool, extracted from Salvia tingitana, on the extramitochondrial OxPhos and the ROI production in the retinal rod OS. Results confirm that the OxPhos machinery is ectopically expressed in the OS and that F1 Fo -ATP synthase is a target of Manool, which inhibited the OS ATP synthesis, binding the F1 moiety with high affinity, as analysed by molecular docking. Moreover, the overall slowdown of OxPhos metabolism reduced the ROI production elicited in the OS by light exposure, in vitro. In conclusion, data are consistent with the antioxidant properties of Salvia spp., suggesting its ability to lower oxidative stress production, a primary risk factor for degenerative retinal diseases. SIGNIFICANCE OF THE STUDY: Here we show that Manool, a diterpene extracted from Salvia tingitana has the potential to lower the free radical production by light-exposed rod outer segments in vitro, by specifically targeting the rod OS F1 Fo -ATP synthase belonging to the extramitochondrial OxPhos expressed on the disk membrane. The chosen experimental model allowed to show that the rod OS is a primary producer of oxidative stress linked to the pathogenesis of degenerative retinal diseases. Data are also consistent with the antioxidant and anti-inflammatory action of Salvia spp., suggesting a beneficial effect also in vivo.

ПРИМЕНЕНИЕ ФОТОМОДИФИКАЦИИ КРОВИ НИЗКОИНТЕНСИВНЫМ ОПТИЧЕСКИМ ИЗЛУЧЕНИЕМ В КОМПЛЕКСНОМ ЛЕЧЕНИИ ПАЦИЕНТОВ С НЕСТАБИЛЬНОЙ СТЕНОКАРДИЕЙ

The in vivo effect of low-intensity optical radiation (LIOR) on the blood of patients with unstable angina (UA) was studied. The integration of LIOR in the combined therapy of patients with UA proved to result in positive changes in the oxygen uptake system. Changes in the absorption spectra of the patients' blood samples, blood oxygenation characteristics, and the proportion of metabolic products were studied at all stages of light exposure. The absorption of LIOR by the blood was found to lead to its photomodification, which was manifested in changes of the partial pressure of blood gases, the content of oxyhemoglobin and the degree of saturation of hemoglobin with oxygen. LIOR affects the oxygen exchange in the body of patients with UA, changes the delivery and consumption of oxygen by tissues, initiates positive changes in the balance between the production of reactive oxygen intermediates, acting as physiologically active compounds, and their inhibition by antioxidant systems.

Changes in Somatic Cell Count, Composition and Cytokine Levels in Milk from Cows with Mastitis Due to Mixed Infections

Background: Mastitis is a mammary gland inflammation that is very common worldwide, mostly caused by bacteria, and causes enormous economic losses. Many microorganisms cause this disease. The most common causes of mastitis by these microorganisms are Staphylococcus aureus (S. aureus), Escherichia coli (E. coli) and Streptococcus agalactiae (S. agalactiae). The anti-inflammatory properties of transforming growth factor (TGF)-β include: 1) limiting interferon (IFN)-γ production; 2) increasing the expression of the ınterleukine (IL)-1 receptor antagonist; 3) inhibiting macrophage production of chemokines, pro-inflammatory cytokines, nitric oxide, and reactive oxygen intermediates; and 4) increasing macrophage clearance of bacterial debris and damaged parenchymal cells. It is stated that cytokines and milk composition change in case of mastitis. In this study, it was aimed to reveal the changes in milk TGF-β1 and Tumor necrosis factor (TNF)-α concentrations and milk composition in mixed infections caused by three pathogens causing mastitis.Materials, Methods &amp; Results: In this study, milk samples from 90 cows were divided into 5 groups. Tumor necrosis factor (TNF)-α and TGF-β1 concentrations and milk composition were determined in these milk samples. The California Mastitis Test (CMT) was applied to the cows included in the study and scoring was done. According to the CMT results of the milk samples taken, CMT(-) cows were included in group 1 (n = 22). Those with the CMT(+) were taken to the microbiology laboratory for analysis within 2 h. After the bacteria was determined, combination groupings were formed. Group 2 (n = 17) with S. aureus and E. coli, group 3 (n = 21), S. agalactiae with S. aureus, S. agalactiae and E. coli together, group 4 (n = 8) and CMT (+) but no bacterial growth formed group 5 (n = 22).Somatic cell counts were made in the milk samples taken from the cows belonging to the groupings. Somatic cell count was measured with the DeLaval Cell Counter® (Cell Counter DCC) device. Mineral matter, fat, protein, lactose, electrical conductivity and specific gravity were measured in milk samples using Lactoscan Milk Analyzer (Milkotronic/EUROPE). Milk samples were then stored at -80°C to measure TGF-β1 and TNF-α. Tumor necrosis factor-α and TGF-β1 concentrations in milk samples were measured using ELISA kits (Sunred Biological Technology).Discussion: Changes in milk TNF-α and TGF-β1 concentration and milk composition were determined in milk samples with mastitis caused by mixed infection. The TNF-α concentration of group 4 was higher than the other groups. On the other hand, the highest concentration of TGF-β1 was found in group 2. While the number of somatic cells in group 1 was lower than in groups 2, 3, and 4, there was no statistical difference between groups 1 and 5. The lowest milk fat ratio was found in group 1, and it was found to be statistically lower than groups 2, 3, and 4. While the rate of solid-non-fat of group 1 increased compared to groups 2 and 3, the highest protein ratio was found in groups 1 and 5. There was no difference between the 5 groups in terms of mineral matter ratios. While the specific gravity was highest in group 1, there was no statistical difference between the other 4 groups. Overall, it was concluded that there was an increase in TNF-α and TGF-β1 concentrations and a change in milk composition in samples with bacterial growth.Keywords: bovine mastitis, cytokine, milk composition, raw milk, transforming growth factor.

Menstrual blood-derived stromal cells modulate functional properties of mouse and human macrophages

Menstrual blood-derived stromal cells (MenSCs) are emerging as a strong candidate for cell-based therapies due to their immunomodulatory properties. However, their direct impact on innate immune populations remains elusive. Since macrophages play a key role in the onset and development of inflammation, understanding MenSCs implication in the functional properties of these cells is required to refine their clinical effects during the treatment of inflammatory disorders. In this study, we assessed the effects that MenSCs had on the recruitment of macrophages and other innate immune cells in two mouse models of acute inflammation, a thioglycollate (TGC)-elicited peritonitis model and a monobacterial sepsis model. We found that, in the TGC model, MenSCs injection reduced the percentage of macrophages recruited to the peritoneum and promoted the generation of peritoneal immune cell aggregates. In the sepsis model, MenSCs exacerbated infection by diminishing the recruitment of macrophages and neutrophils to the site of infection and inducing defective bacterial clearance. Additional in vitro studies confirmed that co-culture with MenSCs impaired macrophage bactericidal properties, affecting bacterial killing and the production of reactive oxygen intermediates. Our findings suggest that MenSCs modulate the macrophage population and that this modulation must be taken into consideration when it comes to future clinical applications.

Sclareol modulates free radical production in the retinal rod outer segment by inhibiting the ectopic f1fo-atp synthase

We have previously shown that the retinal rod outer segments (OS) produce reactive oxygen species in the function of illumination in vitro, establishing a relationship among the extra-mitochondrial oxidative phosphorylation and phototransduction. This source of oxidative stress in the OS can be modulated by polyphenols, acting as inhibitors of F1Fo-ATP synthase. The present study aimed at exploring whether sclareol, a diterpene, interacts with F1Fo-ATP synthase mitigating the light-induced free radical production in the rod OS.Characterization of bovine retinal sections was conducted by immunogold analysis. Reactive oxygen intermediates production, oxygen consumption, the activity of the four respiratory complexes and ATP synthesis were evaluated in purified bovine rod OS. Molecular docking analyses were also conducted.Sclareol reduced free radical production by light-exposed rod OS. Such antioxidant effect was associated with an inhibition of the respiratory complexes and oxygen consumption (OCR), in coupled conditions. Sclareol also inhibited the rod OS ATP synthetic ability. Since the inhibitor effect on respiratory complexes and OCR is not observed in uncoupled conditions, it is supposed that the modulating effect of sclareol on the ectopic oxidative phosphorylation in the rod OS targets specifically the F1Fo-ATP synthase. This hypothesis is confirmed by the in silico molecular docking analyses, which shows that sclareol binds the F1 moiety of ATP synthase with high affinity.In conclusion, a beneficial effect of sclareol can be envisaged as a modulator of oxidative stress in the photoreceptor, a risk factor for the degenerative retinal diseases, suggestive of its potential beneficial action also in vivo.

Relevance of Mitochondrial Dysfunction in the Reserpine-Induced Experimental Fibromyalgia Model

Fibromyalgia (FM) is one of the most common musculoskeletal pain conditions. Although the aetiology of FM is still unknown, mitochondrial dysfunction and the overproduction of reactive oxygen intermediates (ROI) are common characteristics in its pathogenesis. The reserpine experimental model can induce FM-related symptoms in rodents by depleting biogenic amines. However, it is unclear whether reserpine causes other pathophysiologic characteristics of FM. So far, no one has investigated the relevance of mitochondrial dysfunction in the reserpine-induced experimental FM model using protection- and insult-based mitochondrial modulators. Reserpine (1mg/kg) was subcutaneously injected once daily for three consecutive days in male Swiss mice. We carried out analyses of reserpine-induced FM-related symptoms, and their modulation by using mitochondrial insult on ATP synthesis (oligomycin; 1mg/kg, intraperitoneally) or mitochondrial protection (coenzyme Q10; 150mg/kg/5days, orally). We also evaluated the effect of reserpine on mitochondrial function using high-resolution respirometry and oxidative status. Reserpine caused nociception, loss in muscle strength, and anxiety- and depressive-like behaviours in mice that were consistent with clinical symptoms of FM, without inducing body weight and temperature alterations or motor impairment. Reserpine-induced FM-related symptoms were increased by oligomycin and reduced by coenzyme Q10 treatment. Reserpine caused mitochondrial dysfunction by negatively modulating the electron transport system and mitochondrial respiration (ATP synthesis) mainly in oxidative muscles and the spinal cord. These results support the role of mitochondria in mediating oxidative stress and FM symptoms in this model. In this way, reserpine-inducing mitochondrial dysfunction and increased production of ROI contribute to the development and maintenance of nociceptive, fatigue, and depressive-like behaviours.

Immunological Activities of Crude Polysaccharides Extracts of Climacodon pulcherrimus (Phanerochaetaceae) in Lipopolysaccharide-Induced Rat Macrophages and Neutrophils' Responses.

Mushroom contains a wide variety of components with high immune-modulating properties. Mushroom polysaccharides are known as immunocorrectors with wide areas of usage, which balance the immune system without resulting in the entire immune suppression or overreaction. To explore the potentially immunomodulatory role of Climacodon pulcherrimus, the present study was intended to evaluate the in vitro immunomodulatory activity of C. pulcherrimus polysaccharides (CPP) on rat neutrophils and macrophages. CPP was studied on lipopolysaccharide-induced neutrophils and macrophages' responses. The effect on phagocytosis, release of reactive oxygen intermediates (ROI), nitric oxide (NO), lysosomal enzyme release and neutrophil myeloperoxidase, and neutrophil adherence was assessed. The cytotoxicity of CPP against macrophages using 3-(4,5-dimethylthiazole-2-yl)-2, 5-diphenyltetrazolium bromide assay and neutral red inclusion assay was also assessed. ENPC (endopolysaccharides) markedly enhanced phagocytosis of macrophages and neutrophils, whereas the EXPC (exopolysaccharides) increased only that of neutrophils at 0.34 and 0.68 mg/mL. The production of NO evidently increased in ENPC- and EXPC-treated neutrophil and macrophage cultures. The production of ROI in a concentration-dependent manner increased in ENPC-treated (0.08-5.55 mg/mL) and EXPC-treated (0.17 and 0.34 mg/mL) macrophage cultures and ENPC-treated neutrophils, whereas it decreased in EXPC-treated neutrophils. The secretion of acid phosphatase increased in ENPC- and EXPC-treated macrophage and neutrophil cultures. Neutrophil myeloperoxidase release increased in EXPC-treated (5.55 mg/mL) cultures, whereas it decreased in ENPC-treated cultures. ENPC and EXPC increased the adhesion of neutrophils to microplate. These findings demonstrated that CPP displayed a dual mode of action, inhibiting or enhancing numerous phagocytic activities of macrophages and neutrophils.

IL-15 enhances the capacity of primary human macrophages to control Leishmania braziliensis infection by IL-32/vitamin D dependent and independent pathways.

How human macrophages can control the intracellular infection with Leishmania is not completely understood. IL-15 and IL-32 are cytokines produced by monocytes/macrophages that can induce antimicrobial mechanisms. Here, we evaluated the effects of recombinant human IL-15 (rhIL-15) on primary human macrophage infection and response to L. braziliensis. Priming with rhIL-15 reduced the phagocytosis of L. braziliensis and increased the killing of the parasites in monocyte-derived macrophages from healthy donors. rhIL-15 induced TNFα and IL-32 in uninfected cells. After infection, the high levels of rhIL-15-induced TNFα and IL-32 were maintained. In addition, there was an increase of NO and an inhibition of the parasite-induced IL-10 production. Inhibition of NO reversed the leishmanicidal effects of rhIL-15. Although rhIL-15 did not increase L. braziliensis-induced reactive oxygen intermediates (ROS) production, inhibition of ROS reversed the control of infection induced by rhIL-15. Treatment of the cells with rhIL-32γ increased microbicidal capacity of macrophages in the presence of high levels of vitamin D (25D3), but not in low concentrations of this vitamin. rhIL-15 together with rhIL-32 lead to the highest control of the L. braziliensis infection in high concentrations of vitamin D. In this condition, NO and ROS mediated rhIL-32γ effects on microbicidal activity. The data showed that priming of human macrophages with rhIL-15 or rhIL-32γ results in the control of L. braziliensis infection through induction of NO and ROS. In addition, rhIL-32γ appears to synergize with rhIL-15 for the control of L. braziliensis infection in a vitamin D-dependent manner.

Active fractions of mannoproteins derived from yeast cell wall stimulate innate and acquired immunity of adult and elderly dogs

Nutritional intervention in older dogs aims to increase lifespan and improve life quality as well as delay the development of diseases related to ageing. It is believed that active fractions of mannoproteins (AFMs) obtained through extraction and fractionation of yeast cell walls (Saccharomyces cerevisiae) may beneficially modulate the immune system. However, studies that have evaluated this component and the effects of ageing on the immune system of dogs are scarce. This study aimed to evaluate the immunological effects of AFMs in adult and elderly dogs. Three extruded iso-nutrient experimental diets were formulated: without addition of AFM (T0); with AFM at 400 mg/kg (T400); and with AFM at 800 mg/kg (T800). Thirty-six beagle dogs were used, and six experimental treatments, resulting in combinations of age (adult and elderly) and diet (T0, T400, and T800), were evaluated. On days zero, 14, and 28, blood samples were obtained for leucocyte phenotyping and phagocytosis assays. On days zero and 28, a lymphoproliferation test, quantification of reactive oxygen (H2O2) and nitrogen (NO) intermediate production, evaluation of faecal immunoglobulin A (IgA) content, and a delayed cutaneous hypersensitivity test (DCHT) were performed. Statistical analyses were performed with SAS software. Repeated measure variance analyses were performed, and means were compared by the Tukey test. Values of P ≤ 0.05 were considered significant, and values of P ≤ 0.10 were considered tendencies. Dogs fed T400 tended to have higher neutrophilic phagocytic activity than dogs fed T800 (P = 0.073). Regarding reactive oxygen intermediates, bacterial lipopolysaccharide (LPS)-stimulated neutrophils from animals that were fed T400 had a tendency to produce more H2O2 than those from animals fed the control diet (P = 0.093). Elderly dogs, when compared to adult dogs, had lower absolute T and B lymphocyte counts, lower auxiliary T lymphocyte counts, and higher cytotoxic T lymphocyte counts (P < 0.05). A significant effect of diet, age, and time with saline inoculation was noted for the DCHT. There was no effect of diet or age on faecal IgA content in dogs. This study suggests beneficial effects of mannoproteins on the specific and nonspecific immune responses in adult and elderly dogs.

Pediatric Intensive Care: Immunomodulation With Activated Protein C ex vivo.

Objective: Sepsis is major cause of morbidity and mortality in the Pediatric Intensive Care Unit (PICU). PICU patients may develop transient immune deficiency during sepsis. Activated Protein C (APC) has significant anti-inflammatory and cytoprotective effects. Clinical trials of APC in adult sepsis initially showed improved outcome but recent trials showed no benefit in adults or children. We aimed to assess the effects of APC treatment on innate immune responses in children.Design and Subjects: We compared neutrophil and monocyte responses to lipopolysaccharide (LPS) with and without APC treatment in PICU patients at the time of evaluation for sepsis compared with healthy adults and age-matched pediatric controls. We used flow cytometry to examine cell activation (CD11b expression), function [intracellular reactive oxygen intermediate (ROI) release] and LPS recognition [Toll like Receptor 4 (TLR4) expression].Results: PICU patients had significantly decreased protein c levels and LPS responses compared with adult and pediatric controls for all parameters. APC reduced LPS-induced neutrophil PICU TLR4 and adult ROI (p < 0.05). PICU non-survivors had increased LPS induced neutrophil and monocyte ROI production vs. survivors which was significantly reduced by APC.Conclusion: PICU patients demonstrate significantly reduced endotoxin reactivity which may predispose them to sepsis and alter effective antibacterial responses. APC reduces LPS-induced ROI production in adults and may have a role in treating severely compromised PICU patients especially given that newer APC forms are associated with decreased bleeding risk and enhanced anti-inflammatory effects.