Immunological responses and cytokine gene expression analysis to Cooperia punctata infections in resistant and susceptible Nelore cattle

Cellular and humoral immune response, as well as cytokine gene expression, was assessed in Nelore cattle with different degrees of resistance to Cooperia punctata natural infection. One hundred cattle (male, weaned, 11-12 months old), kept together on pasture, were evaluated. Faecal and blood samples were collected for parasitological and immunological assays. Based on nematode faecal egg counts (FEC) and worm burden, the seven most resistant and the eight most susceptible animals were selected. Tissue samples of the small intestine were collected for histological quantification of inflammatory cells and analysis of cytokine gene expression (IL-2, IL-4, IL-8, IL-1 2p35, IL-13, TNF-alpha, IFN-gamma, MCP-1, MCP-2, and MUC- 1) using real-time RT-PCR. Mucus samples were also collected for IgA levels determination. Serum IgG1 mean levels against C. punctata antigens were higher in the resistant group, but significant differences between groups were only observed 14 days after the beginning of the experiment against infective larvae (1-3) and 14 and 84 days against adult antigens. The resistant group also presented higher IgA levels against C. punctata (L3 and adult) antigens with significant difference 14 days after the beginning of the trial (P < 0.05). In the small-intestine mucosa, levels of IgA anti-L3 and anti-adult C. punctata were higher in the resistant group, compared with the susceptible group (P < 0.05). Gene expression of both T(H)2 cytokines (IL-4 and IL-13) in the resistant group and T(H)1 cytokines (IL-2, IL-1 2p35, IFN-gamma and MCP-1) in the susceptible group was up-regulated. Such results suggested that immune response to C. punctata was probably mediated by TH2 cytokines in the resistant group and by T(H)1 cytokines in the susceptible group. (C) 2008 Elsevier B.V. All rights reserved.

Sex determination of murine and bovine embryos using cytotoxicity and immunofluorescence assays

Murine and bovine embryos were sexed by using polyclonal and monoclonal anti-H-Y antibodies in cytotoxic and immunofluorescence assays. The specificity of 3 different sources of antibodies are compared. Sexing was accomplished by complement-mediated cytotoxicity or by H-Y antigen detection using a anti-mouse Ig fluorescein conjugate. Cytogenetic ascertainment for correct sex identification yielded a score of about 85% in both species, while the immunofluorescence assay yielded a score of 83%. © 1993.

Molecular profiling of sepsis in mice using Fourier Transform Infrared Microspectroscopy

Sepsis is a life threatening condition resulting from a high burden of infection. It is a major health care problem and associated with inflammation, organ dysfunction and significant mortality. However, proper understanding and delineating the changes that occur during this complex condition remains a challenge. A comparative study involving intra-peritoneal injection of BALB/c mice with Salmonella Typhimurium (infection), lipopolysaccharide (endotoxic shock) or thioglycollate (sterile peritonitis) was performed. The changes in organs and sera were profiled using immunological assays and Fourier Transform Infrared (FTIR) micro-spectroscopy. There is a rapid rise in inflammatory cytokines accompanied with lowering of temperature, respiratory rate and glucose amounts in mice injected with S. Typhimurium or lipopolysaccharide. FTIR identifies distinct changes in liver and sera: decrease in glycogen and protein/lipid ratio and increase in DNA and cholesteryl esters. These changes were distinct from the pattern observed in mice treated with thioglycollate and the differences in the data obtained between the three models are discussed. The combination of FTIR spectroscopy and other biomarkers will be valuable in monitoring molecular changes during sepsis. GRAPHICS] Intra-peritoneal infection with high dose of Salmonella Typhimurium leads to rapid increase in inflammatory cytokines, e.g. Tnf alpha (A). FTIR analysis of liver (B) and sera (C) identifies several metabolic changes: glycogen, protein/lipid, cholesteryl esters and DNA.

Paralytic shellfish poisoning (PSP) toxin binders for optical biosensor technology: problems and possibilities for the future: a review

This review examines the developments in optical biosensor technology, which uses the phenomenon of surface plasmon resonance, for the detection of paralytic shellfish poisoning (PSP) toxins. Optical biosensor technology measures the competitive biomolecular interaction of a specific biological recognition element or binder with a target toxin immobilised onto a sensor chip surface against toxin in a sample. Different binders such as receptors and antibodies previously employed in functional and immunological assays have been assessed. Highlighted are the difficulties in detecting this range of low molecular weight toxins, with analogues differing at four chemical substitution sites, using a single binder. The complications that arise with the toxicity factors of each toxin relative to the parent compound, saxitoxin, for the measurement of total toxicity relative to the mouse bioassay are also considered. For antibodies, the cross-reactivity profile does not always correlate to toxic potency, but rather to the toxin structure to which it was produced. Restrictions and availability of the toxins makes alternative chemical strategies for the synthesis of protein conjugate derivatives for antibody production a difficult task. However, when two antibodies with different cross-reactivity profiles are employed, with a toxin chip surface generic to both antibodies, it was demonstrated that the cross-reactivity profile of each could be combined into a single-assay format. Difficulties with receptors for optical biosensor analysis of low molecular weight compounds are discussed, as are the potential of alternative non-antibody-based binders for future assay development in this area.

Correlates of protective immunity against hepatitis C virus

Le virus de l’hépatite C (VHC) infecte ~185 millions d’individus dans le monde. Malgré le développement des nouvelles thérapies dirigées contre le VHC, on compte deux millions de nouvelles infections chaque année, en particulier dans les pays sous-développés et les populations marginalisées. Comme pour la plupart des virus à infection chronique, le développement d’un vaccin prophylactique efficace est limité par le manque de caractérisation des déterminants de la mémoire immunitaire protectrice lors des épisodes de réinfection naturelle chez les êtres humains. Le VHC représente un modèle unique au sein des virus à infection chronique pour étudier l’immunité protectrice. En effet ~30% des patients infectés par le VHC peuvent être guéris suite à un premier épisode d’infection spontanément. Dans cette thèse, nous avons étudié l’immunité protectrice contre le VHC dans une cohorte d’utilisateurs de drogues par injection qui sont à risque d’être infectés ou réinfectés. Notre hypothèse est que la majorité des patients qui ont résolu une première infection par le VHC sont protégés contre le développement d’une infection chronique s’ils sont réexposés. Cette immunité protectrice est associée à la présence des cellules T CD4 et CD8 polyfonctionnelles qui possèdent des fréquences, magnitudes et avidités élevées. La capacité protectrice des cellules T mémoire contre les séquences variables du VHC est dépendante de la diversité et flexibilité du répertoire de leurs récepteurs de cellules T (TCR), qui reconnaissent les séquences variables des épitopes ciblés. Notre premier objectif était de définir et détailler les déterminants de l’immunité protectrice conférée par les cellules T spécifiques du VHC. Nos résultats ont montré que la protection pendant l’épisode de réinfection était associée à une augmentation de la magnitude et du spectre des réponses spécifiques par les cellules T CD4 et CD8 polyfonctionnelles, ainsi que par l’apparition d’une population de cellules T tétramère+ CD8+ effectrices qui expriment faiblement le marqueur CD127 (CD127lo) lors du pic de la réponse. Chez les patients qui ont développé une infection chronique pendant l’épisode de réinfection, nous avons observé une expansion très limitée des cellules T CD4 et CD8. Le séquençage des épitopes ciblés par les cellules T CD8 chez ces patients qui sont non-protégés a montré que les séquences de ces épitopes sont différentes des séquences de référence qui étaient utilisées pour tous les essais immunologiques de cette étude. Le deuxième objectif était d’analyser la dynamique du répertoire des TCRs des cellules T CD8 spécifiques chez les patients protégés versus les patients non-protégés. Nos résultats ont montré que le répertoire des cellules T CD8 spécifiques est plus focalisé que chez les patients protégés. En plus, nous avons observé que les clonotypes qui forment le répertoire chez les patients protégés sont distincts de ceux chez les patients non-protégés. Ces clonotypes chez les patients protégés ont montré de plus grandes avidité et polyfonctionnalité que leurs homologues chez les patients non-protégés. En conclusion, nos résultats suggèrent que la protection contre le développement d’une infection chronique pendant l’épisode de réinfection par le VHC est associée à une augmentation de la magnitude, du spectre et de la fonctionnalité des réponses des cellules T spécifiques, ainsi qu’à un répertoire des TCRs plus focalisé composé des clonotypes distincts qui possèdent de plus grandes avidité et polyfonctionnalité que chez les patients non-protégés. L’homologie des séquences des souches virales entre les différents épisodes de l’infection est un déterminant majeur de l’établissement d’une protection efficace. Ces résultats ont donc des implications très importantes pour le développement d’un vaccin prophylactique contre le VHC et d’autres virus à infection chronique.

SBTX, a new toxic protein distinct from soyatoxin and other toxic soybean [Glycine max] proteins, and its inhibitory effect on Cercospora sojina growth

SBTX, a novel toxin from soybean, was purified by ammonium sulfate fractionation followed by chromatographic steps DEAE-Cellulose, CM-Sepharose and Superdex 200 HR fast-protein liquid chromatography (FPLC). Lethality of SBTX to mice (LD50 5.6 mg/kg) was used as parameter in the purification steps. SBTX is a 44-kDa basic glycoprotein composed of two polypeptide chains (27 and 17 kDa) linked by a disulfide bond. The N-terminal sequences of the 44 and 27 kDa chains were identical (ADPTFGFTPLGLSEKANLQIMKAYD), differing from that of 17 kDa (PNPKVFFDMTIGGQSAGRIVMEEYA). SBTX contains high levels of Glx, Ala, Asx, Gly and Lys and showed maximum absorption at 280 nm, epsilon(1 cm) (1%) of 6.3, and fluorescence emission in the 290-450nm range upon excitation at 280nm. The secondary structure content was 35% alpha-helix, 13% beta-strand and beta-sheet, 27% beta-turn, 25% unordered, and 1% aromatic residues. Immunological assays showed that SBTX was related to other toxic proteins, such as soyatoxin and canatoxin, and cross-reacted weekly with soybean trypsin inhibitor and agglutinin, but it was devoid of protease-inhibitory and hemagglutinating activities. The inhibitory effect of SBTX on growth of Cercospora sojina, fungus causing frogeye leaf spot in soybeans, was observed at 50 mu g/ml, concentration 112 times lesser than that found to be lethal to mice. This effect on phytopathogenic fungus is a potential attribute for the development of transgenic plants with enhanced resistance to pathogens. (c) 2007 Elsevier Ltd. All rights reserved.

Haematologic and immunologic parameters of bullfrogs, Lithobates catesbeianus, fed probiotics

The effects of two probiotics (P(1) - Lactobacillus acidophilus, Bifidobacterium bifidum and Enterococcus faecium and P(2) - Bacillus subtilis) supplemented to commercial feed (40% crude protein) on the haematological and immunological parameters of the bullfrog Lithobates catesbeianus were studied. Two doses of each probiotic (5 and 10 g kg-1 of food) were added to the diets and fed to frogs, totalling five treatments over 112 days. Haematological analyses consisted of total and differential leucocyte counts, erythrocyte and thrombocyte counts, haematocrit, haemoglobin levels and RBC indices (mean corpuscular volume, mean corpuscular haemoglobin - and mean corpuscular haemoglobin concentration) and the immunological parameters included phagocytic capacity and phagocytic index of peritoneal phagocytes. The results showed that the probiotics did not significantly influence any of the haematological parameters measured. However, immunological assays showed that the probiotics had an immunostimulating effect. The greatest effects were seen with probiotic P(1) fed at a dose of 10 g kg-1 of diet and probiotic P(2) fed at 5 g kg-1 of diet.

Propolis and the immune system: a review

Propolis has been used empirically for centuries and it was always mentioned as an immunomodulatory agent. In recent years, in vitro and in vivo assays provided new information concerning its mechanisms of action, thus a review dealing with propolis and the immune system became imperative. This review compiles data from our laboratory as well as from other researchers, focusing on its chemical composition and botanical sources, the seasonal effect on its composition and biological properties, its immunomodulatory and antitumor properties, considering its effects on antibody production and on different cells of the immune system, involving the innate and adaptive immune response. In vitro and in vivo assays demonstrated the modulatory action of propolis on murine peritoneal macrophages, increasing their microbicidal activity. Its stimulant action on the lytic activity of natural killer cells against tumor cells, and on antibody production was demonstrated. Propolis inhibitory effects on lymphoproliferation may be associated to its anti-inflammatory property. In immunological assays, the best results were observed when propolis was administered over a short-term to animals. Propolis antitumor property and its anticarcinogenic and antimutagenic potential are discussed. Since humans have used propolis for different purposes and propolis-containing products have been marketed, the knowledge of its properties with scientific basis is not only of academic interest but also of those who use propolis as well. This review opens a new perspective on the investigation of propolis biological properties, mainly with respect to the immune system. (c) 2007 Elsevier B.V.. All rights reserved.

Cytokine gene expression in response to Haemonchus placei infections in Nelore cattle

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

In situ expression of mononuclear cell markers and interleukin-2 receptor in renal allograft biopsies of acute rejection and borderline cases

The correct diagnosis of renal allograft rejection may be difficult using only clinical and/or histopathological criteria. Immunological assays should be considered in order to evaluate the phenotype of inflammatory infiltrate in renal allograft biopsies. Immunohistochemical studies were performed to detect mononuclear cells, CD4 and CD8 T lymphocytes, B lymphocytes, macrophages, null cells, and positive cells for interleukin-2 receptors. A total of 41 allograft biopsies classified into three groups were studied: acute cellular rejection (28 biopsies/22 patients), borderline (7 biopsies/5 patients) and control (6 biopsies/6 patients). In the rejection group (RG), increased cellularity was found mainly at the tubulo-interstitial level. Expression of CD8 positive cells was higher in RG when compared to borderline (BG) and control (CG) groups, respectively (0.9 vs. 0.0 vs. 0.35 cells/mm2; p < 0.001). Expression of macrophages was not statistically significant among the three groups (RG = 0.6 vs. BG = 0.2 vs. CG = 0.0 cells/mm2; p < 0.02). In the BG, CD4 + cells predominated (BG = 0.2 vs. RG = 0.05 vs. CG = 0.0 cells/mm2; p < 0.05). Clinically these patients were treated as cases of acute rejection. The numbers and different types of infiltrating cells did not correlate with patient's clinical outcome. Copyright © Informa Healthcare.

Inhibition of function in Xenopus oocytes of the inwardly rectifying G-protein-activated atrial K channel (GIRK1) by overexpression of a membrane-attached form of the C-terminal tail.

Coexpression in Xenopus oocytes of the inwardly rectifying guanine nucleotide binding (G)-protein-gated K channel GIRK1 with a myristoylated modification of the (putative) cytosolic C-terminal tail [GIRK1 aa 183-501 fused in-frame to aa 1-15 of p60src and denoted src+ (183-501)] leads to a high degree of inhibition of the inward G-protein-gated K+ current. The nonmyristoylated segment, src- (183-501), is not active. Although some interference with assembly is not precluded, the evidence indicates that the main mechanism of inhibition is interference with functional activation of the channel by G proteins. In part, the tail functions as a blocking particle similar to a "Shaker ball"; it may also function by competing for the available supply of free G beta gamma liberated by hormone activation of a seven-helix receptor. The non-G-protein-gated weak inward rectifier ROMK1 is less effectively inhibited, and a Shaker K channel was not inhibited. Immunological assays show the presence of a high concentration of src+ (183-501) in the plasma membrane and the absence of any membrane forms for the nonmyristoylated segment.

Antibody detection of Burkholderia pseudommallei and Burkholderia mallei

Monoclonal and polyclonaI antibodies have been produced for use in immunological assays for the detection of Burkholderia pseudomallei and Burkholderia mallei. Monoclonal antibodies recognising a high molecular weight polysaccharide material found in some strains of both species have been shown to be effective in recognising B. pseudomallei and B. mallei and distinguishing them from other organisms. The high molecular weight polysaccharide material is thought to be the capsule of B. pseudomallei and B. mallei and may have important links with virulence. B. pseudomallei and B. mallei are known to be closely related, sharing many epitopes, but antigenic variation has been demonstrated within both the species. The lipopolysaccharide from strains of B. pseudomal/ei and B. mallei has been isolated and the silver stain profiles found to be visually very similar. A monoclonal antibody raised to B. mallei LPS has been found to recognise both B. mallei and B. pseudomallei strains. However, in a small number of B. pseudomallei strains a visually atypical LPS profile has been demonstrated. A monoclonal ant ibody rai sed against this atypical LPS showed no recognition of the typical LPS profile of either B. mallei or B. pseudomallei. This atypical LPS structure has not been reported and may be immunologically distinct from the typical LPS. Molecular biology and antibody engineering techniques have been used in an attempt to produce single-chain antibody fragments reactive to B. pseudomallei. Sequencing of one of the single-chain antibody fragments produced showed high homology with murine immunoglobulin genes, but none of the single-chain antibody fragments were found to be specific to B. pselldomallei.

An evaluation of antigen capture assays for detecting active filarial antigens

Lymphatic filariasis is a parasitic disease of tropical countries. This is a disfiguring and painful disease contracted in childhood, but the symptoms become apparent only in later years. Diagnosis of filarial infection is very crucial for the management of the disease. The main objective of this study was to develop a filarial antigen-based immunological assay for the diagnosis and surveillance of the disease. Monoclonal and polyclonal antibodies were raised to the recombinant protein Brugia malayi vespid allergen homologue (VAH). Capture enzyme-linked immunosorbent assay (ELISA) was standardized utilizing various combinations of antibodies and evaluated with serum samples of endemic normal (EN, n = 110), microfilaraemic (MF, n = 65), chronic pathology (CP, n = 45) and non-endemic normal (NEN, n = 10) individuals. Of the 230 samples tested, VAHcapture assay detected circulating antigen in 97.91% of bancroftian and 100% of brugian microfilaraemic individuals, and 5% of endemic normal individuals, comparable to the earlier reported SXP-1 antigen detection assay. However, the combination of VAH and SXP-1 (VS) capture ELISA was found to be more robust, detecting 100% of microfilaraemic individuals and with higher binding values. Thus an antigen capture immunoassay has been developed, which can differentiate active infection from chronic infection by detecting circulating filarial antigens in clinical groups of endemic areas.

Development and standardization of solid phase assays for the detection of anti-neutrophil cytoplasmic antibodies (ANCA). A report on the second phase of an international cooperative study on the standardization of ANCA assays

Anti-neutrophil cytoplasmic antibodies (ANCA) are diagnostic markers for systemic vasculitis. They are classically I detected by an indirect immunofluorescence test using normal donor neutrophils as substrate. This assay lacks antigenic specificity and is not quantitative. The 'EC/BCR Project for ANCA Assay Standardization' is an international collaboration study with the aim to develop and standardize solid phase assays for ANCA detection. In this part of the study the isolation and characterization of proteinase-3 and myeloperoxidase, the two main target molecules for ANCA, and the development and standardization of ELISAs with these antigens are described. Six laboratories successfully isolated purified proteinase-3 preparations that could be used. Three of these preparations, together with one myeloperoxidase preparation, were subsequently used for ANCA testing by ELISA. The ELISA technique was standardized in two rounds of testing in the 14 participating laboratories. The coefficient of variation of these new assays decreased from values of approx. 50% in the first round to approx. 20% in the second round. We conclude that purified proteinase-3 and myeloperoxidase can be used in standardized ELISAs for ANCA detection. Whether such procedures offer advantages over the IIF test will be determined in a prospective clinical study.

Inflammatory and immunological biomarkers are not related to survival in adults with Cystic Fibrosis

Background
Chronic Pseudomonas aeruginosa pulmonary infection is associated with a decline in lung function and reduced survival in people with Cystic Fibrosis (CF). Damaging inflammatory and immunological mediators released in the lungs can be used as markers of chronic infection, inflammation and lung tissue damage.

Methods
Clinical samples were collected from CF patients and healthy controls. Serum IgG and IgA anti-Pseudomonas antibodies, sputum IL-8 and TNFα, plasma IL-6 and urine TNFr1 were measured by ELISA. Sputum neutrophil elastase (NE), cathepsin S and cathepsin B were measured by spectrophotometric and fluorogenic assays. The relationship between IgG and IgA, inflammatory mediators and long-term survival was determined.

Results
IgG and IL-6 positively correlated with mortality. However, multivariate analysis demonstrated that after adjusting for FEV1, IgG was not independently related to mortality. A relationship was observed between IgG and IL-6, TNFα, TNFr1 and between IgA and IL8, cathepsin S and cathepsin B.

Conclusions
These data indicate that biomarkers of inflammation are not independent predictors of survival in people with CF.