Stabilization of serum antibody responses triggered by initial mucosal contact with the antigen independently of oral tolerance induction

Initial contacts with a T-dependent antigen by mucosal routes may result in oral tolerance, defined as the inhibition of specific antibody formation after subsequent parenteral immunizations with the same antigen. We describe here an additional and permanent consequence of these initial contacts, namely, the blockade of secondary-type responsiveness to subsequent parenteral contacts with the antigen. When repeatedly boosted ip with small doses (3 µg) of ovalbumin (OVA) (or lysozyme), primed B6D2F1 mice showed progressively higher antibody responses. In contrast, mice primed after a single oral exposure to the antigen, although repeatedly boosted, maintained their secondary antibody titers on a level which was inversely proportional to the dose of antigen in the oral pretreatment. This phenomenon also occurred in situations in which oral tolerance was not induced. For example, senile 70-week-old B6D2F1 mice pretreated with a single gavage of 20 mg OVA did not become tolerant, i.e., they formed the same secondary levels of anti-OVA antibodies as non-pretreated mice. However, after 4 weekly challenges with 3 µg OVA ip, orally pretreated mice maintained the same anti-OVA serum levels, whereas the levels of control mice increased sequentially. This "stabilizing" effect of mucosal exposure was dose dependent, occurred with different proteins and was triggered by single or multiple oral or nasal exposures to the antigen.

Chronic postnatal administration of methylmalonic acid provokes a decrease of myelin content and ganglioside N-acetylneuraminic acid concentration in cerebrum of young rats

Levels of methylmalonic acid (MMA) comparable to those of human methylmalonic acidemia were achieved in blood (2-2.5 mmol/l) and brain (1.35 µmol/g) of rats by administering buffered MMA, pH 7.4, subcutaneously twice a day from the 5th to the 28th day of life. MMA doses ranged from 0.76 to 1.67 µmol/g as a function of animal age. Control rats were treated with saline in the same volumes. The animals were sacrificed by decapitation on the 28th day of age. Blood was taken and the brain was rapidly removed. Medulla, pons, the olfactory lobes and cerebellum were discarded and the rest of the brain ("cerebrum") was isolated. Body and "cerebrum" weight were measured, as well as the cholesterol and triglyceride concentrations in blood and the content of myelin, total lipids, and the concentrations of the lipid fractions (cholesterol, glycerolipids, phospholipids and ganglioside N-acetylneuraminic acid (ganglioside-NANA)) in the "cerebrum". Chronic MMA administration had no effect on body or "cerebrum" weight, suggesting that the metabolites per se neither affect the appetite of the rats nor cause malnutrition. In contrast, MMA caused a significant reduction of plasma triglycerides, but not of plasma cholesterol levels. A significant diminution of myelin content and of ganglioside-NANA concentration was also observed in the "cerebrum". We propose that the reduction of myelin content and ganglioside-NANA caused by MMA may be related to the delayed myelination/cerebral atrophy and neurological dysfunction found in methylmalonic acidemic children.

Actions of Crotalus durissus terrificus venom and crotoxin on the isolated rat kidney

Many studies have reported the occurrence of lethal acute renal failure after snakebites. The aim of the present investigation was to determine alterations in renal function produced by Crotalus durissus terrificus venom and crotoxin as well as the histological alterations induced by these venoms. Isolated kidneys from Wistar rats weighing 240 to 280 g were perfused with Krebs-Henseleit solution containing 6 g% of previously dialyzed bovine serum albumin. The effects of Crotalus durissus terrificus venom and crotoxin were studied on glomerular filtration rate (GFR), urinary flow (UF), perfusion pressure (PP) and percentage sodium tubular transport (%TNa+). The infusion of Crotalus durissus terrificus venom (10 µg/ml) and crotoxin (10 µg/ml) increased GFR (control80 = 0.78 ± 0.07, venom80 = 1.1 ± 0.07, crotoxin80 = 2.0 ± 0.05 ml g-1 min-1, P<0.05) and UF (control80 = 0.20 ± 0.02, venom80 = 0.32 ± 0.03, crotoxin80 = 0.70 ± 0.05 ml g-1 min-1, P<0.05), and decreased %TNa+ (control100 = 75.0 ± 2.3, venom100 = 62.9 ± 1.0, crotoxin80 = 69.0 ± 1.0 ml g-1 min-1, P<0.05). The infusion of crude venom tended to reduce PP, although the effect was not significant, whereas with crotoxin PP remained stable during the 100 min of perfusion. The kidneys perfused with crude venom and crotoxin showed abundant protein material in the urinary space and tubules. We conclude that Crotalus durissus terrificus venom and crotoxin, its major component, cause acute nephrotoxicity in the isolated rat kidney. The current experiments demonstrate a direct effect of venom and crotoxin on the perfused isolated kidney.

Indirect effects of oral tolerance to ovalbumin interfere with the immune responses triggered by Schistosoma mansoni eggs

The objective of the present study was to investigate whether the injection of a tolerated protein (indirect effects) affects the formation of granulomas around Schistosoma mansoni eggs trapped in the lungs after intravenous (iv) injection into normal (noninfected) C57BL/6 mice (6 animals per group). To induce oral tolerance to chicken egg ovalbumin a 1/5 dilution of egg white in water was offered ad libitum in a drinking bottle for 3 days. Control mice received water. After 7 days, control and experimental animals were injected iv with 2,000 S. mansoni eggs through a tail vein. In some mice of both groups the iv injection of eggs was immediately followed by intraperitoneal (ip) immunization with 10 µg of dinitrophenylated conjugates of ovalbumin (DNP-Ova) emulsified in complete Freund's adjuvant (CFA) or only CFA; 18 days later, mice were bled and killed by ether inhalation. The lungs were fixed in formalin and embedded in paraffin. Serial sections of 5 µm were stained with Giemsa, Gomori's silver reticulin and Sirius red (pH 10.2). Granuloma diameters were measured in histological sections previously stained with Gomori's reticulin. Anti-DNP and anti-soluble egg antigen (SEA) antibodies were analyzed by ELISA. In mice orally tolerant to ovalbumin the concomitant ip injection of DNP-Ova resulted in significantly lower anti-SEA antibodies (ELISA*: 1395 ± 352 in non-tolerant and 462 ± 146 in tolerant mice) and affected granuloma formation around eggs, significantly decreasing granuloma size (area: 22,260 ± 2478 to 12,993 ± 3242 µm²). Active mechanisms triggered by injection of tolerated antigen (ovalbumin) reduce granuloma formation.

Dietary cellulose has no effect on the regeneration of hemoglobin in growing rats with iron deficiency anemia

The objective of the present study was to determine the effect of cellulose on intestinal iron absorption in rats during recovery from iron deficiency anemia. Twenty-one-day-old male Wistar-EPM rats were fed an iron-free ration for two weeks to induce anemia. At 5 weeks of age, the rats were divided into two groups (both groups receiving 35 mg of elemental iron per kg diet): cellulose group (N = 12), receiving a diet containing 100 g of cellulose/kg and control (N = 12), receiving a diet containing no cellulose. The fresh weight of the feces collected over a 3-day period between the 15th and 18th day of dietary treatment was 10.7 ± 3.5 g in the group receiving cellulose and 1.9 ± 1.2 g in the control group (P<0.001). Total food intake was higher in the cellulose group (343.4 ± 22.0 g) than in the control (322.1 ± 13.1 g, P = 0.009) during the 3 weeks of dietary treatment. No significant difference was observed in weight gain (cellulose group = 132.8 ± 19.2, control = 128.0 ± 16.3 g), hemoglobin increment (cellulose group = 8.0 ± 0.8, control = 8.0 ± 1.0 g/dl), hemoglobin level (cellulose group = 12.3 ± 1.2, control = 12.1 ± 1.3 g/dl) or in hepatic iron levels (cellulose group = 333.6 ± 112.4, control = 398.4 ± 168.0 µg/g dry tissue). We conclude that cellulose does not adversely affect the regeneration of hemoglobin, hepatic iron level or the growth of rats during recovery from iron deficiency anemia.

Interleukin-5 mediates peritoneal eosinophilia induced by the F1 cell wall fraction of Histoplasma capsulatum

An alkali-insoluble fraction 1 (F1), which contains mainly ß-glucan isolated from the cell wall of Histoplasma capsulatum, induces eosinophil recruitment into the peritoneal cavity of mice. The present study was carried out to determine the participation of interleukin-5 (IL-5) in this process. Inbred C57BL/6 male mice weighing 15-20 g were treated ip with 100 µg of anti-IL-5 monoclonal antibody (TRFK-5, N = 7) or an isotype-matched antibody (N = 7), followed by 300 µg F1 in 1 ml PBS ip 24 h later. Controls (N = 5) received only 1 ml PBS. Two days later, cells from the peritoneal cavity were harvested by injection of 3 ml PBS and total cell counts were determined using diluting fluid in a Neubauer chamber. Differential counts were performed using Rosenfeld-stained cytospin preparations. The F1 injection induced significant (P < 0.01) leukocyte recruitment into the peritoneal cavity (8.4 x 10(6) cells/ml) when compared with PBS alone (5.5 x 10(6) cells/ml). Moreover, F1 selectively (P < 0.01) induced eosinophil recruitment (1 x 10(6) cells/ml) when compared to the control group (0.07 x 10(6) cells/ml). Treatment with TRFK-5 significantly (P < 0.01) inhibited eosinophil recruitment (0.18 x 10(6) cells/ml) by F1 without affecting recruitment of mononuclear cells or neutrophils. We conclude that the F1 fraction of the cell wall of H. capsulatum induces peritoneal eosinophilia by an IL-5-dependent mechanism. Depletion of this cytokine does not have effect on the recruitment of other cell types induced by F1.

Comparison of the homeostasis model assessment and quantitative insulin sensitivity check index with data from forearm metabolic studies for the in vivo assessment of insulin sensitivity

The present study was designed to compare the homeostasis model assessment (HOMA) and quantitative insulin sensitivity check index (QUICKI) with data from forearm metabolic studies of healthy individuals and of subjects in various pathological states. Fifty-five healthy individuals and 112 patients in various pathological states, including type 2 diabetes mellitus, essential hypertension and others, were studied after an overnight fast and for 3 h after ingestion of 75 g of glucose, by HOMA, QUICKI and the forearm technique to estimate muscle uptake of glucose combined with indirect calorimetry (oxidative and non-oxidative glucose metabolism). The patients showed increased HOMA (1.88 ± 0.14 vs 1.13 ± 0.10 pmol/l x mmol/l) and insulin/glucose (I/G) index (1.058.9 ± 340.9 vs 518.6 ± 70.7 pmol/l x (mg/100 ml forearm)-1), and decreased QUICKI (0.36 ± 0.004 vs 0.39 ± 0.006 (µU/ml + mg/dl)-1) compared with the healthy individuals. Analysis of the data for the group as a whole (patients and healthy individuals) showed that the estimate of insulin resistance by HOMA was correlated with data obtained in the forearm metabolic studies (glucose uptake: r = -0.16, P = 0.04; non-oxidative glucose metabolism: r = -0.20. P = 0.01, and I/G index: r = 0.17, P = 0.03). The comparison of QUICKI with data of the forearm metabolic studies showed significant correlation between QUICKI and non-oxidative glucose metabolism (r = 0.17, P = 0.03) or I/G index (r = -0.37, P < 0.0001). The HOMA and QUICKI are good estimates of insulin sensitivity as data derived from forearm metabolic studies involving direct measurements of insulin action on muscle glucose metabolism.

Effect of the Escherichia coli EMO strain on experimental infection by Salmonella enterica serovar Typhimurium in gnotobiotic mice

An experimental infection with Salmonella enterica subsp. enterica serovar Typhimurium was evaluated in gnotobiotic mice previously exposed to a plasmid-free non-pathogenic Escherichia coli (EMO strain). Mice were exposed to EMO (experimental) or not (control) 10 days before challenge with Salmonella Typhimurium (10² colony forming units (CFU)/mouse). Survival after challenge was higher (P < 0.05) in the experimental group (16%) than in the control animals (0%). Histopathological examination of the colon and ileum mucosa of the experimental group showed less extensive lesions such as edema, cell inflammatory infiltration and hyperemia. The epithelial cells of the mucosal surface and the production of the mucous layer were also better preserved in the experimental group. The population levels of Salmonella Typhimurium in the feces were initially 10-fold lower (P < 0.05) in the experimental groups. However, 3 days after challenge both experimental and control groups showed similar population levels ranging from 10(8) to()10(9) CFU/g of feces. The intestinal contents of total and anti-Salmonella Typhimurium sIgA were higher in the experimental groups 10 days after inoculation of E. coli EMO strain. Translocation of Salmonella Typhimurium to the spleen was 10-fold lower (P < 0.05) in the experimental group only on day 3 after infection. This was not related to an increase in the bacterial blood clearance of the animals, as shown by experimental venous challenge with E. coli B41. In conclusion, treatment of mice with E. coli EMO strain promoted a relative protection against experimental infection with Salmonella Typhimurium. This protection was not due to the reduction of the population of pathogens in the intestine but was probably related to stimulation of the immune response.

Effects of autacoid inhibitors and of an antagonist on malaria infection in mice

The effects of p-chlorophenylalanine, an inhibitor of serotonin synthesis, indomethacin, an inhibitor of prostaglandin synthesis, cyproheptadine, a serotonin, bradykinin and histamine antagonist, were assessed separately and in combination with chloroquine (CQ) in Vom strains of Swiss albino mice (18-22 g) of either sex infected intraperitoneally with 1 x 10(7) Plasmodium yoelii nigeriensis-induced malaria. As prophylactic, these agents reduced from 31.9 ± 4.5 to 16.1 ± 8.1% the level of parasitemia relative to control but had no appreciable activity as curative agents when administered subcutaneously once daily for 4 days after 72 h of parasites innoculum in vivo. However, CQ alone and the combination of these agents with CQ in curative and prophylactic treatments significantly reduced (from 50.3 ± 5.8 to 4.9 ± 0.75%) the level of parasitemia (P < 0.05), which was taken only once 72 h after the parasites innoculum. The prophylactic result was shown to produce better results than the curative treatment. The data indicate that inhibitors and an antagonist can reduce the parasitemia load (the extent of damage and the severity of infection) as well as enhance the effects of CQ when combined with it for malaria therapy. The study reveals that the production of autacoids in established infection renders autacoid inhibitors and an antagonist ineffective for radical cure in malarial mice; however, selective inhibition of local hormones implicated in the pathological manifestations of malaria infection by autacoid inhibitors and an antagonist may be a possible pathway to reduce the severity of infection and the associated tissue damage and to enhance the efficacy of available anti-malarials.

Preclinical evaluation of the antidiabetic effect of Eugenia jambolana seed powder in streptozotocin-diabetic rats

The world is facing an explosive increase in the incidence of diabetes mellitus and cost-effective complementary therapies are needed. The effects of Eugenia jambolana, a household remedy for diabetes, were studied. Streptozotocin diabetic female albino Wistar rats weighing 150-200 g (N = 6) were fed E. jambolana seed powder (250, 500 or 1000 mg/kg) for 15 days. Diabetic rats fed 500 and 1000 mg/kg seed powder showed an increase in body weight on day 20 in relation to day 5 (6 ± 4.7, 9 ± 7.8 vs diabetic control -16 ± 7.1 g, P < 0.001), a decrease in fasting blood glucose (75 ± 11.9, 123 ± 14.4 vs diabetic control -34 ± 12.1 mg/dl, P < 0.001), a difference in post-treatment fasting and peak blood glucose (38 ± 11.9, 36 ± 14.2 vs diabetic control 78 ± 11.9 mg/dl, P < 0.001), and a difference in liver glycogen (50 ± 6.8, 52 ± 7.5 vs normal control 90 ± 6.6 µg/g of liver tissue, P < 0.001). Tri-terpenoids, tannins, gallic acid, and oxalic acid were the chemical constituents detected in E. jambolana seed. The best results were obtained with an oral dose of 500 mg/kg. Subacute toxicity studies with a single administration of 2.5 and 5.0 g/kg seed powder showed no mortality or abnormality. These data on the antidiabetic effect of E. jambolana seed are adequate for approval of phase 2 clinical trials to evaluate this seed powder as complementary therapy in type 2 and type 1 diabetes.

Effect of the consumption of tomato paste on plasma prostate-specific antigen levels in patients with benign prostate hyperplasia

The consumption of tomatoes and tomato products has been associated with a reduced risk of prostate cancer. We observed a decrease of 10.77% in prostate-specific antigen (PSA) levels in patients with benign prostate hyperplasia who were submitted to daily ingestion of tomato paste. This was an experimental rather than a controlled study with a sample of 43 men ranging in age from 45 to 75 years, all with histological diagnoses of benign prostate hyperplasia and plasma PSA levels of 4-10 ng/mL. All patients received 50 g of tomato paste once a day for 10 consecutive weeks and PSA levels were analyzed before, during and after the consumption of tomato paste. ANOVA for repeated measures was used to compare PSA levels before, during and after the consumption of tomato paste. The mean ± SD PSA level was 6.51 ± 1.48 ng/mL at baseline and 5.81 ± 1.58 ng/mL (P = 0.005) after 10 weeks. Acceptance was good in 88.3, regular in 9.3, and poor in 2.3% of the patients. Dietary ingestion of 50 g of tomato paste per day for 10 weeks significantly reduced mean plasma PSA levels in patients with benign prostate hyperplasia, probably as a result of the high amount of lycopene in tomato paste. This was not a prostate cancer prevention study, but showed some action of tomato paste in prostate biology. The development of prostate cancer is typically accompanied by an increase in plasma PSA levels, thus any intervention that affects plasma PSA levels can suggest an impact in the progression of disease.

Partial purification of tightly bound mitochondrial hexokinase from maize (Zea mays L.) root membranes

In mammals, hexokinase (HK) is strategically located at the outer membrane of mitochondria bound to the porin protein. The mitochondrial HK is a crucial modulator of apoptosis and reactive oxygen species generation. In plants, these properties related to HK are unknown. In order to better understand the physiological role of non-cytosolic hexokinase (NC-HK) in plants, we developed a purification strategy here described. Crude extract of 400 g of maize roots (230 mg protein) contained a specific activity of 0.042 µmol G6P min-1 mg PTN-1. After solubilization with detergent two fractions were obtained by DEAE column chromatography, NC-HK 1 (specific activity = 3.6 µmol G6P min-1 mg PTN-1 and protein recovered = 0.7 mg) and NC-HK 2. A major purification (yield = 500-fold) was obtained after passage of NC-HK 1 through the hydrophobic phenyl-Sepharose column. The total amount of protein and activity recovered were 0.04 and 18%, respectively. The NC-HK 1 binds to the hydrophobic phenyl-Sepharose matrix, as observed for rat brain HK. Mild chymotrypsin digestion did not affect adsorption of NC-HK 1 to the hydrophobic column as it does for rat HK I. In contrast to mammal mitochondrial HK, glucose-6-phosphate, clotrimazole or thiopental did not dissociate NC-HK from maize (Zea mays) or rice (Oryza sativa) mitochondrial membranes. These data show that the interaction between maize or rice NC-HK to mitochondria differs from that reported in mammals, where the mitochondrial enzyme can be displaced by modulators or pharmacological agents known to interfere with the enzyme binding properties with the mitochondrial porin protein.

Low expression of antigen-presenting and costimulatory molecules by lung cells from tuberculosis patients

Costimulatory and antigen-presenting molecules are essential to the initiation of T cell immunity to mycobacteria. The present study analyzed by immunocytochemistry, using monoclonal antibodies and alkaline phosphatase-anti-alkaline phosphatase method, the frequency of costimulatory (CD86, CD40, CD40L, CD28, and CD152) and antigen-presenting (MHC class II and CD1) molecules expression on human lung cells recovered by sputum induction from tuberculosis (TB) patients (N = 22) and non-TB controls (N = 17). TB cases showed a statistically significant lower percentage of HLA-DR+ cells than control subjects (21.9 ± 4.2 vs 50.0 ± 7.2%, P < 0.001), even though similar proportions of TB cases (18/22) and control subjects (16/17, P = 0.36) had HLA-DR-positive-stained cells. In addition, fewer TB cases (10/22) compared to control subjects (16/17) possessed CD86-expressing cells (P = 0.04; OR: 0.05; 95%CI = 0.00-0.51), and TB cases expressed a lower percentage of CD86+ cells (P = 0.04). Moreover, TB patients with clinically limited disease (£1 lobe) on chest X-ray exhibited a lower percentage of CD86-bearing cells compared to patients with more extensive lung disease (>1 lobe) (P = 0.02). The lower expression by lung cells from TB patients of HLA-DR and CD86, molecules involved in antigen presentation and activation of T cells, may minimize T cell recognition of Mycobacterium tuberculosis, fostering an immune dysfunctional state and active TB.

Aflatoxin M1 in samples of "minas" cheese commercialized in the city of Belo Horizonte - Minas Gerais/Brazil

Milk products such as cheeses may be contaminated by aflatoxin M1 when dairy cattle have consumed feeds contaminated with aflatoxin B1. Samples of "Minas" cheeses (fresh, canastra and standard) were collected by the Inspection Service in the Mercado Central in Belo Horizonte city, Minas Gerais - Brazil. A purified extract was obtained by extraction with dichloromethane followed by a washing with n-hexane and immunoaffinity column purification. The quantification of aflatoxin M1 was done by high performance liquid chromatography (HPLC) using a fluorescence detector. Recoveries were about 75%. In 56 of the 75 samples (74.7%), the presence of aflatoxin M1 was detected in concentrations ranging between 0.02 and 6.92ng/g of cheese. In the positive cases ( > or = 0.02ng/g) the mean contamination level of aflatoxin M1 was 0.08ng/g in fresh cheese, 0.36ng/g in canastra cheese and 0.62ng/g in standard cheese. No aflatoxin M1 maximum tolerance level in cheese has been established in Brazil.

Comparison of total phenolic content and antiradical capacity of powders and "chocolates" from cocoa and cupuassu

Polyphenolic compounds seem to be related to the health benefits produced by the cocoa due to their antioxidant properties. Cupuassu powder, prepared from Theobroma grandiflorum seeds, is a very promising cocoa powder substitute. In order to assess the potential health benefits of the cupuassu powder, a comparison was performed between cocoa, chocolate, and cupuassu powders in relation to the content of total phenolic compounds, flavonoids and DPPH scavenging capacity of methanolic extracts. Cupuassu "chocolates" (milk, dark, and white) were also analyzed. Mineral, lipid, protein, and moisture determinations were made in cocoa and cupuassu powders and in cupuassu "chocolates". Results showed that the phenolic contents of cocoa and chocolate powders are more than three times higher than those of cupuassu powder; however, flavonoid contents were significantly lower. The DPPH scavenging capacity varied hugely among the different samples, from 0.5 (white cupuassu "chocolate") to 120 (cocoa powder) μg of Trolox equivalent per 100 g (FW), and presented a significant correlation (r = 0.977) with the total phenolic contents but not with the flavonoid contents (r = -0.035).