Toxocariasis/cysticercosis seroprevalence in a long-term rural settlement, Sao Paulo, Brazil

Seroprevalence of Toxocara and Taenia solium and risk factors for infection with these parasites were explored in a long-term rural settlement in Sao Paula state, Brazil. An ELISA for the detection of anti-Toxocara IgG and IgE and anti-T. solium cysticerci was standardized using Toxocara excretory-secretory antigens (TES) obtained from the cultured second-stage larvae of T. canis and by vesicular fluid antigen from Taenia crassiceps cysticerci (VC). For cysticercosis, the reactive ELISA samples were assayed by Western blot using 18 kDa and 14 kDa proteins purified from VF. Out of 182 subjects, 25 (13.7%,) presented anti-Toxocora IgG and it positive correlation between total IgE and the reactive index of specific anti-TES IgE (P=0.0265) was found amongst the subjects found seropositive for anti-Toxocara IgG. In these individuals 38.0%. showed ocular manifestations. The frequency of anti-T. solium cysticerci confirmed by Western blot wits 0.6%, Seropositivity for Toxocara was correlated with low educational levels and the owning of dogs. Embryonated eggs of Toxocara spp. were found in 43.3% of the analysed areas.

Sero-epidemiology of toxocariasis in a rural settlement in Sao Paulo state, Brazil

The seroprevalence of Toxocara canis and risk factors for infection with this parasite were explored in a rural settlement in Sao Paulo state, Brazil. Total IgA and IgE levels in 79 subjects were determined by turbidimetry and chemiluminescence, respectively. Total counts of leucocytes and erythrocytes and differential counts of leucocytes were made by flow cytometry. ELISA for the detection of anti-Toxocara IgG, IgA and IgE were standardized using Toxocara excretory-secretory antigens (TES) obtained from the cultured second-stage larvae of T. canis. Seventeen (21.5%) of the subjects were found positive for anti-Toxocara IgG, with no significant differences in such seropositivity with age or gender. Thirty (38%) of the subjects showed eosinophilia and 70 (89%) had elevated levels of total IgE. Among the 17 subjects found seropositive for anti-Toxocara IgG, the percentage of leucocytes represented by eosinophils (P=0.0069) and total levels of IgE (P=0.0452) were positively correlated with the levels of anti-TES IgE. Although anti-TES IgA was detected in 10 (59%) of the subjects, there was no significant correlation between the levels of total IgA and those of Toxocara-specific IgA. Only one of the 17 subjects found positive for anti-Toxocara IgG had attended a secondary school and all but two belonged to households with monthly incomes of < U.S.$100. In the study community at least, seropositivity may be related to poor living standards and lack of basic sanitary conditions. The presence of anti-Toxocara IgE and IgA may facilitate the diagnosis of toxocariasis and may well be useful for monitoring the success of treatment.

Structural characterization of exopolysaccharides from biofilm of a cariogenic streptococci

Soluble (EPS-SOL), as well as insoluble extracellular polysaccharide (EPS-INSOL), extracted from biofilm of Streptococcus mutans, were analyzed by nuclear magnetic resonance spectroscopy, methylation analysis, and a controlled Smith degradation. EPS-SOL was a branched alpha-glucan containing a (1 -> 6)-and (1 -> 3)-linkages. EPS-INSOL was a branched alpha-glucan with similar linkages, but with a (1 -> 3)-linked main-chain partially substituted at O-6 with Glcp-(1 -> 6)-Glcp-side chains. Biofilm EPS had a distinct chemical structure compared with those synthesized by plankton cells or by purified enzymes from S. mutans, which could indicate different mechanisms for its degradation. (C) 2011 Published by Elsevier Ltd.

Trypanocidal structure-activity relationship for cis- and trans-methylpluviatolide

The trypanocidal activity of racemic mixtures of cis- and trans-methylpluviatolides was evaluated in vitro against trypomastigote forms of two strains of Trypanosoma cruzi, and in the enzymatic assay of T. cruzi gGAPDH. The cytotoxicity of the compounds was assessed by the MTT method using LLC-MK2 cells. The effect of the compounds on peroxide and NO production were also investigated. The mixture of the trans stereoisomers displayed trypanocidal activity (IC(50) similar to 89.3 mu M). Therefore, it was separated by chiral HPLC, furnishing the and (+) (-)-enantiomers. Only the (-)-enantiomer was active against the parasite (IC(50) similar to 18.7 mu M). Despite being inactive, the (+)-enantiomer acted as an antagonistic competitor. Trans-methylpluviatolide displayed low toxicity for LLC-MK(2) cells, with an IC(50) of 6.53 mM. Furthermore, methylpluviatolide neither inhibited gGAPDH activity nor hindered peroxide and NO production at the evaluated concentrations. (C) 2008 Elsevier Ltd. All rights reserved.

Leukotrienes Are Potent Adjuvant during Fungal Infection: Effects on Memory T Cells

Leukotrienes (LTs) are potent lipid mediators involved in the control of host defense. LTB(4) induces leukocyte accumulation, enhances phagocytosis and bacterial clearance, and increases NO synthesis. LTB(4) is also important in early effector T cell recruitment that is mediated by LTB(4) receptor 1, the high-affinity receptor for LTB(4). The aims of this study were to evaluate whether LTs are involved in the secondary immune response to vaccination in a murine model of Histoplasma capsulatum infection. Our results demonstrate that protection of wild-type mice immunized with cell-free Ags from H. capsulatum against histoplasmosis was associated with increased LTB(4) and IFN-gamma production as well as recruitment of memory T cells into the lungs. In contrast, cell-free Ag-immunized mice lacking 5-lipoxygenase(-/-), a critical enzyme involved in LT synthesis, displayed a marked decrease on recruitment of memory T cells to the lungs associated with increased synthesis of TGF-beta as well as IL-10. Strikingly, these effects were associated with increased mortality to 5-lipoxygenase(-/-)-infected mice. These data establish an important immunomodulatory role of LTs, in both the primary and secondary immune responses to histoplasmosis. The Journal of Immunology, 2008,181: 8544-8551.

Strongyloides venezuelensis: The antigenic identity of eight strains for the immunodiagnosis of human strongyloidiasis

The antigens of eight strains of Strongyloides venezuelensis were identified by means of the indirect immunofluorescence antibody (IFAT), enzyme-linked immunosorbent assay (ELISA) and immunoblot (IB) tests. Infective larvae (L3) from these strains were obtained from Rattus norvegicus feces cultures. For IFAT, sections of U were used while the ELISA and IB, tests were conducted with alkaline extract. Ninety serum samples were tested: 30 from patients with S. stercoralis, 30 from patients with other parasitic diseases, and 30 from healthy subjects (free of parasites). Average sensitivity and specificity among all eight strains, both for IFAT and ELISA, were, respectively, 93% and 100%. In the IB, anti-S. stercoralis IgG recognized a single antigenic fraction with 45 kDa. Serum samples from patients with S. stercoralis revealed antigens from different strains of S. venezuelensis, indicating antigenic identity for possible use in the synthesis of recombinant antigen that could be useful in immunodiagnosis and vaccine against this parasite. (C) 2008 Elsevier Inc. All rights reserved.

Evaluation of Environmental Mycobacteria Contamination in a Specific Pathogen Free Animal Facility from a Tropical Country

P>With the evidence showing the protection variability of bacille Calmette-Guerin, new potential vaccines for tuberculosis have been tested around the world. One of the general concerns in tuberculosis vaccine development is the possibility of priming the host immune system with prior exposure to environmental mycobacteria antigens, which can change the efficacy of subsequent vaccination. As there is a great homology between the species from Mycobacterium genera, the previous contact of experimental animals with environmental mycobacteria could sensitize the mice and, in this way, could influence subsequent vaccine research. The aim of our study was to investigate critical points in an animal facility to search for environmental mycobacteria that eventually could be in direct or indirect contact with the experimental animals. Samples were collected from surfaces of walls, floor, animal cages and shelves and analysed using the Ogawa-Kudoh decontamination method. Samples of drinking water, food and sawdust were collected for analysis by the NALC/NaOH decontamination method. Also, the samples were cultivated directly in broth medium, without any method for decontamination. After decontamination methods, we observed bacterial colony growth in 4.31% of the total of samples analysed. These samples were stained with Ziehl-Neelsen and we did not detect any acid-fast bacilli, suggesting that the animal facility analysed is free from contamination by environmental mycobacteria and is not a source of mycobacterial antigens. Furthermore, our study showed a new paradigm in tuberculosis vaccine development: concern about the animal facility environment in terms of immune system priming of experimental animals by nascent bacterial contaminants.

Innate immune lectins kill bacteria expressing blood group antigen

The expression of ABO(H) blood group antigens causes deletion of cells that generate self-specific antibodies to these antigens but this deletion limits adaptive immunity toward pathogens bearing cognate blood group antigens. To explore potential defense mechanisms against such pathogens, given these limitations in adaptive immunity, we screened for innate proteins that could recognize human blood group antigens. Here we report that two innate immune lectins, galectin-4 (Gal-4) and Gal-8, which are expressed in the intestinal tract, recognize and kill human blood group antigen-expressing Escherichia coli while failing to alter the viability of other E. coli strains or other Gram-negative or Gram-positive organisms both in vitro and in vivo. The killing activity of both Gal-4 and Gal-8 is mediated by their C-terminal domains, occurs rapidly and independently of complement and is accompanied by disruption of membrane integrity. These results demonstrate that innate defense lectins can provide immunity against pathogens that express blood group-like antigens on their surface.

Expression of Human Recombinant Antibody Fragments Capable of Partially Inhibiting the Phospholypase Activity of Crotalus durissus terrificus Venom

Crotoxin is the main toxic component of the South American rattlesnake Crotalus durissus terrificus venom. It is composed of two different subunits: CA, crotapotin, and CB (basic subunit of cortoxin isolated from C. d. terrificus), a weakly toxic phospholipase A(2) with high enzymatic activity. The phospholipases A(2) are abundant in snake venoms and are responsible for disruption of cell membrane integrity via hydrolysis of its phospholipids. However, in addition to their normal digestive action, a wide range of pharmacological activities, such as neurotoxic, myotoxic, oedema-inducing, hypotensive, platelet-aggregating, cardiotoxic, and anticoagulant effects have been attributed to venom phospholipases A(2). In this study, we used a non-immune human single-chain fragment variable library, Griffin.1 (Medical Research Council, Cambridge, UK) for selection of recombinant antibodies against antigens present in C. d. terrificus venom and identification of specific antibodies able to inhibit the phospholipase activity. Two clones were identified as capable of inhibiting partially this activity in vitro. These clones were able to reduce in vivo the myotoxic and oedema-inducing activity of CB and the lethality of C. d. terrificus venom and crotoxin, but had no effect on the in vitro anticoagulant activity of CB. These results demonstrate the potential of using recombinant single-chain fragment variable libraries in the production of antivenoms.

An alpha-type phospholipase A(2) inhibitor from Bothrops jararacussu snake plasma: Structural and functional characterization

An inhibitory protein that neutralizes the enzymatic, toxic and pharmacological activities of several phospholipases A(2) from Bothrops venoms was isolated from B. jararacussu snake plasma by affinity chromatography using the immobilized myotoxin BthTX-I on Sepharose gel. Biochemical characterization of this inhibitory protein, denominated alpha BjussuMIP, showed it to be an oligomeric glycoprotein with M-r of 24,000 for the monomeric subunit. Secondary structural analysis by circular dichroism revealed 44% alpha-helix, 18% beta-sheet, 10% beta-turn and 28% random coil structures. Circular dichroism spectroscopy indicated that no significant alterations in the secondary structure of either alpha BjussuMIP or the target protein occur following their interaction. The product from the reaction with reverse transcriptase produced a cDNA fragment of 432 bp that codifies for a mature protein of 144 amino acid residues. The first 21 amino acid residues from the N-terminal and five tryptic peptides were characterized by mass spectrometry of the mature protein and confirmed by the nucleotide sequence. Alignment of alpha BjussuMIP with other snake inhibitors showed a sequence similarity of 73-92% with these alpha PLIs. alpha BjussuMIP was relatively stable within the pH range of 6-12 and temperatures from 0 degrees C to 80 degrees C, even after deglycosylation. The results showed effects against Bothrops phospholipase A(2) activities (enzymatic, edema inducing, myotoxic, cytotoxic and bactericidal), suggesting that alpha BjussuMIP may prove useful in the treatment of snakebite envenomations. (C) 2008 Elsevier Masson SAS. All rights reserved.

Immunopathology and Pathophysiology of Experimental Autoimmune Encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory demyelinating disease of the central nervous system (CNS), and is widely studied as an animal model of the human CNS demyelinating diseases, including multiple sclerosis (Raine, 1984). EAE can be induced by inoculation with whole CNS tissue, purified myelin basic protein (MBP) or myelin proteolipid protein (PLP), together with adjuvants. It may also be induced by the passive transfer of T cells specifically reactive to these myelin antigens. EAE may have either an acute or a chronic relapsing course. Acute EAE closely resembles the human disease acute disseminated encephalomyelitis, while chronic relapsing EAE resembles multiple sclerosis. EAE is also the prototype for T-cell-mediated autoimmune disease in general. This chapter will focus on the immunopathology and pathophysiology of EAE, which are the subjects of investigation in my laboratory.

Characterization of serum antibodies to Porphyromonas gingivalis in individuals with and without periodontitis

Although Porphyromonas gingivalis is a defined pathogen in periodontal disease, many subjects control the infection without experiencing loss of attachment. Differences in host susceptibility to the disease may be reflected in the pattern of humoral antibodies against specific P. gingivalis antigens. The aim of this study was to determine the presence of antibodies against immunodominant P. gingivalis antigens as well as the isotype and subclass of anti-P. gingivalis antibodies against outer membrane antigens in four groups of patients: P. gingivalis-positive, 1) with and 2) without periodontitis, and P. gingivalis-negative, 3) with and 4) without periodontitis. Antigens of molecular weight 92, 63, and 32 kDa and lipopolysaccharide were found to be immunodominant. Group 1 subjects showed a significantly higher response to the 92 and 63 kDa antigens compared with other groups. The response to lipopolysaccharide was significantly higher in group 1, and lower in group 4 than in groups 2, 3. Immunoglobulin G(1) (IgG(1)), IgG(2) and IgM antibodies against P. gingivalis outer membrane were present in all subjects, while only some subjects were seropositive for IgG(3), IgG(4) and IgA. There were no differences in concentrations for IgG(1), IgG(3) and IgM. The IgG(2) concentration in group 4 was significantly higher than in groups 1 and 2, while the IgG(4) concentration in group 4 was significantly lower than in other groups. The frequency of seropositivity for IgG(4) and IgA was lowest in group 4, while IgG; seropositivity was almost exclusively seen in healthy patients iii groups 2, 4. These findings suggest that the presence of IgG(3) may reflect non-susceptibility to the disease, while lack of IgG(4) may be indicative of periodontal health and lack of infection.

Vascular leakage stimulates phenotype alteration in ocular cells, contributing to the pathology of proliferative vitreoretinopathy

Plasma leaking from damaged retinal blood vessels can have a significant impact on the pathologies of the posterior segment of the eye. Inflammation in the eye and metabolic change resulting from diabetes mellitus causes vascular leakage with alteration of the phenotype of retinal pigment epithelial (RPE) cells and fibrocytes, resulting in changes in cell function. Phenotypically altered cells then significantly contribute to the pathogenesis of retinopathies by being incorporated into tractional membranes in the vitreous, where they secrete matrix molecules, such as fibronectin, and express altered cell surface antigens. We hypothesize that there is a direct relationship between the leaking of plasma and the proliferation and phenotypic change of RPE cells and fibroblasts, thus exacerbating the pathology of retinal disease. If the hypothesis is correct, control of vascular leakage becomes an important target of therapy in proliferative vitreoretinopathy.

Structure and distribution of N-glycans on the S-7-allele stylar self-incompatibility ribonuclease of Nicotiana alata

S-RNases are the stylar products of the self-incompatibility (S)-locus in solanaceous plants (including Nicotiana alata), and as such, are involved in the prevention of self-pollination. All cDNA sequences of S-RNase products of functional S-alleles contain potential N-glycosylation sites, with one site being conserved in all cases, suggesting that N-glycosylation is important in self-incompatibility. In this study, we report on the structure and localization of the N-glycans on the S-7-allele RNase of N, alata, A total of nine N-glycans, belonging to the high-mannose- and xylosylated hybrid-classes, were identified and characterized by a combination of electrospray-ionization mass-spectrometry (ESI-MS), H-1-NMR spectroscopy, and methylation analyses. The glycosylation pattern of individual glycosylation sites was determined by ESI-MS of the glycans released from isolated chymotryptic glycopeptides, All three N-glycosylation sites showed microheterogeneity and each had a unique complement of N-glycans, The N-glycosylation pattern of the S-7-RNase is significantly different to those of the S-1- and S-2-RNases.

Anthocyanin synthesis, growth and nutrient uptake in suspension cultures of strawberry cells

Strawberry (Fragaria ananassa cv. Shikinari) cell suspension cultures carried out in shake flasks for 18 d were closely examined for cell growth, anthocyanin synthesis and the development of pigmented cells in relation to the uptake of carbohydrate, extracellular PO4, NO3, NH4, and calcium. Cell viability, extracellular anthocyanin content, pH and electrical conductivity of the broth were also monitored. The specific growth rate of strawberry cells at exponential phase was 0.27 and 0.28 d(-1) based on fresh and dry weight, respectively. Anthocyanin synthesis was observed to increase continuously to a maximum value of 0.86 mg/g fresh cell weight (FCW) at day 6, and was partially growth-associated. Anthocyanin synthesis was linearly related to the increase in pigmented cell ratio, which increased with time and reached a maximum value of ca. 70% at day 6 due to reduction in cell viability and depletion of substrate. Total carbohydrate uptake was closely associated with increase in cell growth, and glucose was utilized in preference to fructose. Nitrate and ammonia were consumed until 9 d of culture, but phosphate was completely absorbed within 4 d. Calcium was assimilated throughout the growth cycle. After 9 d, cell lysis was observed which resulted in the leakage of intracellular substances and a concomitant pH rise. Anthocyanin was never detected in the broth although the broth became darkly pigmented during the lysis period. This suggests that anthocyanin was synthesized only by viable pigmented cells, and degraded rapidly upon cell death and lysis. Based on the results of kinetic analysis, a model was developed by incorporating governing equations for the ratio of pigmented cells into a Bailey and Nicholson's model. This was verified by comparison with the experimental data. The results suggest Bat the model satisfactorily describes the strawberry cell culture process, and may thus be used for process optimization.