Luís Cláudio Fernandes

Differential effects of short-chain fatty acids on proliferation and production of pro- and anti-inflammatory cytokines by cultured lymphocytes

Short-chain fatty acids (SCFA) are produced by fermentation of water-soluble fiber by anaerobic bacteria in the large bowel. Fiber-rich diets decrease the risk of developing inflammatory bowel disease (IBD) and butyrate enemas are effective as a therapy in some patients. Crohns disease, one form of IBD, appears to involve an exagerated T helper-1 (Th1) lymphocyte phenotype, characterised by production of interleukin (IL)-2 and interferon (IFN)-gamma, that drives the inflammation. To examine whether SCFA influence pro- and anti-inflammatory cytokine production, rat mesenteric lymph node lymphocytes were cultured in the presence of acetate (10 mM), butyrate (1.5 mM) or propionate (2 mM) and the production of cytokines in response to concanavalin A determined. Butyrate, but not acetate or propionate, inhibited lymphocyte proliferation and IL-2 production. Acetate and propionate were able to partly prevent the inhibitory effect of butyrate on IL-2 production. Acetate and propionate increased IFN-gamma production, whereas butyrate inhibited it. Acetate and propionate in combination were able to prevent the inhibitory effect of butyrate on IFN-gamma production. IL-4 was not detected in any cultures. Acetate and propionate increased IL-10 production, which was not affected by butyrate. It is concluded that butyrate significantly inhibits Th1-type responses and that this might explain the therapeutic effect of butyrate in IBD patients. Acetate and propionate have less marked modulatory actions, and in some cases have effects that oppose those of butyrate. A combination of the three SCFA causes a shift in the T helper lymphocyte phenotype towards a more anti-inflammatory phenotype and this might explain the protective effects of fiber.

Pulmonary inflammation due to exercise-induced pulmonary haemorrhage in Thoroughbred colts during race training

This study investigated the putative roles of inflammation and platelet-activating factor (PAF) in exercise-induced pulmonary haemorrhage (EIPH). Two-year-old Thoroughbred colts (n=37) were exercised on a racetrack for 5months before commencement of the study. Each colt was then exercised at 15-16m/s over 800-1000m and broncho-alveolar lavage fluid (BALF) was collected 24h later. The colts were subsequently divided into two groups on the basis of BALF analysis; an EIPH-positive group (presence of haemosiderophages, n=23) and an EIPH-negative group (absence of haemosiderophages, n=14). BALF from the EIPH-positive group had a significantly higher protein concentration (0.39±0.28 vs. 0.19±0.12mg/mL, P=0.031), higher PAF bioactivity (0.18±0.12 vs. 0.043±0.05 340:380nm ratio, P=0.042) and a higher lipid hydroperoxide concentration compared to the EIPH-negative group. There was also a lower nitrite concentration and reduced production of superoxide anion and hydrogen peroxide by alveolar macrophages in the EIPH-positive group. There was evidence of pulmonary inflammation and a decreased innate immune response of alveolar macrophages in EIPH-positive colts compared with the EIPH-negative group.

Time motion analysis of football (soccer) referees during official matches in relation to the type of fluid consumed

The aim of the present study was to determine the effect of volume and composition of fluid replacement on the physical performance of male football referees. Ten referees were evaluated during three official matches. In one match the participants were asked to consume mineral water ad libitum, and in the others they consumed a pre-determined volume of mineral water or a carbohydrate electrolyte solution (6.4% carbohydrate and 22 mM Na+) equivalent to 1% of their baseline body mass (half before the match and half during the interval). Total water loss, sweat rate and match physiological performance were measured. When rehydrated ad libitum (pre-match and at half time) participants lost 1.97 ± 0.18% of their pre-match body mass (2.14 ± 0.19 L). This parameter was significantly reduced when they consumed a pre-determined volume of fluid. Sweat rate was significantly reduced when the referees ingested a pre-determined volume of a carbohydrate electrolyte solution, 0.72 ± 0.12 vs 1.16 ± 0.11 L/h ad libitum. The high percentage (74.1%) of movements at low speed (walking, jogging) observed when they ingested fluid ad libitum was significantly reduced to 71% with mineral water and to 69.9% with carbohydrate solution. An increase in percent movement expended in backward running was observed when they consumed a pre-determined volume of carbohydrate solution, 7.7 ± 0.5 vs 5.5 ± 0.5% ad libitum. The improved hydration status achieved with the carbohydrate electrolyte solution reduced the length of time spent in activities at low-speed movements and increased the time spent in activities demanding high-energy expenditure.

Platelet‐Activating Factor and Evidence of Oxidative Stress in the Bronchoalveolar Fluid of Thoroughbred Colts during Race Training

BACKGROUND Inflammatory airway disease (IAD) is prevalent in young racehorses during training, being the 2nd most commonly diagnosed ailment interrupting training of 2-year-old Thoroughbred racehorses. HYPOTHESIS That stabling and exercise cause oxidative stress, release of platelet-activating factor (PAF) and inflammation in airways of Thoroughbred colts. ANIMALS Colts in breeding farms (NC, n = 45), stabled for 30 days (EC, n = 40), and race trained (EX, n = 34). METHODS Cytological profile and parameters of bronchoalveolar lavage fluid (BALF) related to oxidative stress, bioactivity of the proinflammatory mediator PAF, catalase activity, and alveolar macrophage function. RESULTS Percentages of neutrophils and eosinophils in the BALF of the EX group were higher (5.4 +/- 6.4% versus 0.9 +/- 1.2%) than the upper limits for normal horses (3-5%). BALF from the EX group (45.6 +/- 2.8 cells/microL of BALF) also displayed significantly (P = .017) higher total nucleated cell count. PAF bioactivity and the total protein concentration in the BALF were higher in the EX group (0.0683 +/- 0.076 versus 0.0056 +/- 0.007 340 : 380 nm ratio P = .0039, 0.36 +/- 0.30 versus 0.14 +/- 0.15 mg of proteins/mL of BALF P < .001). Concentration of BALF hydroperoxides was higher in the EC group (104.7 +/- 80.0 versus 35.2 +/- 28.0 nmol/mg of proteins, P = .013) and catalase activity was higher in the EX group (0.24 +/- 0.16 versus 0.06 +/- 0.02 micromol H2O2/min/mg of proteins, P = .0021). Alveolar macrophage phagocytosis (P = .048) as well as production of superoxide anion (P = .0014) and hydrogen peroxide (P = .0011) were significantly lower in EX group. CONCLUSIONS AND CLINICAL IMPORTANCE Further studies should be performed to elucidate the role of PAF in the pathophysiology of IAD. Its presence in bronchoalveolar fluid of young athletic horses makes it a potential therapeutic target to be investigated.

The effects of peroxovanadate and peroxovanadyl on glucose metabolism in vivo and identification of signal transduction proteins involved in the mechanism of action in isolated soleus muscle

The insulin-like effects of peroxovanate (POV) and peroxovanadyl (PSV) on rates of lactate formation and glycogen synthesis were measured in isolated incubated soleus muscle preparations. In another experiment rats were made insulin deficient by streptozotocin injection and treated with POV and PSV (0.25 mM) administered in the drinking water and in the course of 7 days glycemia were determined. Also, signal transduction proteins ERK 1 and ERK 2 involved in the insulin signaling were measured in soleus muscle of diabetic rats treated with POV and PSV. Peroxides of vanadate and vanadyl significantly stimulated glucose utilization in soleus muscle preparations in vitro. The stimulation of glycogen synthesis and lactate formation by POV and PSV was similar to insulin stimuli. Rats treated with POV or PSV presented reduction of glycemia, food and fluid intake with amelioration of the diabetic state during the short period of treatment (7 days). POV and PSV modulated ERK1/2 phosphorilation and the insulin administration in these rats caused an addictive effect on phosphorilation state of these proteins.

Tumor growth reduction is regulated at the gene level in Walker 256 tumor-bearing rats supplemented with fish oil rich in EPA and DHA

We investigated the effect of fish oil (FO) supplementation on tumor growth, cyclooxygenase 2 (COX-2), peroxisome proliferator-activated receptor gamma (PPARγ), and RelA gene and protein expression in Walker 256 tumor-bearing rats. Male Wistar rats (70 days old) were fed with regular chow (group W) or chow supplemented with 1 g/kg body weight FO daily (group WFO) until they reached 100 days of age. Both groups were then inoculated with a suspension of Walker 256 ascitic tumor cells (3×107 cells/mL). After 14 days the rats were killed, total RNA was isolated from the tumor tissue, and relative mRNA expression was measured using the 2-ΔΔCT method. FO significantly decreased tumor growth (W=13.18±1.58 vs WFO=5.40±0.88 g, P<0.05). FO supplementation also resulted in a significant decrease in COX-2 (W=100.1±1.62 vs WFO=59.39±5.53, P<0.001) and PPARγ (W=100.4±1.04 vs WFO=88.22±1.46, P<0.05) protein expression. Relative mRNA expression was W=1.06±0.022 vs WFO=0.31±0.04 (P<0.001) for COX-2, W=1.08±0.02 vs WFO=0.52±0.08 (P<0.001) for PPARγ, and W=1.04±0.02 vs WFO=0.82±0.04 (P<0.05) for RelA. FO reduced tumor growth by attenuating inflammatory gene expression associated with carcinogenesis.

Agaricus brasiliensis mycelium supplementation in Sarcoma 180tumour-bearing mice reverses the immune response induced by the tumour

Abstract The biological properties of Agaricus brasiliensis mycelium, including their immuno-stimulating and anti-tumour activities, have previously been investigated. The aim of this study was to evaluate the effects of long-term intake of A. brasiliensis mycelium cultivated on wheat grains by solid-state culture on tumour growth and immune response in Sarcoma 180 tumour-bearing mice. Mice received dietary supplementation with 100% or 50% Agaricus mycelium cultivated on wheat grains for 14 weeks and were inoculated with Sarcoma 180 tumours. We found that A. brasiliensis had anti-tumour activity, and that dietary supplementation with a 50% concentration (A50S) prevented loss of body weight, inhibited tumour growth, induced the increase of CD4+ T cells and CD25+ CD4+ T subsets in peripheral organs and downregulated Tumor necrosis factor alpha production in plasma. Our results confirm that dietary supplementation with Agaricus may offer immuno-modulatory activity and protection against tumour growth.