Effects of 5-hydroxytryptophan and m-hydroxybenzylhydrazine associated to Lactobacillus spp. on the humoral response of broilers challenged with Salmonella Enteritidis.

This study investigates the effects of different doses of serotonin, its precursor 5-hydroxytry-ptophan (5HTP), and m-hydroxybenzylhydrazine inhibitor (NSD1015), administered via intraperitoneal for 5 consecutive days, on behavior and average body weight of broilers. We also measured the humoral immune response and quantification of Salmonella Enteritidis in broilers chickens that received the drugs evaluated and a Lactobacillus pool. The study was divided into 3 experiments: Experiment 1--administration of pharmaceuticals with choice of dosage; Experiment 2--administration of pharmaceuticals and a Lactobacillus pool in birds that were not challenged with S. Enteritidis, and Experiment 3--administration of pharmaceuticals and a Lactobacillus pool in birds challenged with S. Enteritidis. The ELISA was used to scan dosages of intestinal IgA and serum IgY. We used colony-forming units to quantify S. Enteritidis. The concentrations of IgA and IgY did not show significant differences (P>0.05) in Experiment 2. In Experiment 3, NSD1015 associated with Lactobacillus determined higher IgA concentrations, promoting greater stimulus to the immune system than 5HTP. Regarding quantification of S. Enteritidis in the cecal content of birds, 5HTP associated to Lactobacillus determined the smallest number of bacteria, showing possible interaction of 5-hydroxytryptophan and Lactobacillus spp. with the immune system of broiler chickens.

Immune response of broiler chickens immunized orally with the recombinant proteins flagellin and the subunit B of cholera toxin associated with Lactobacillus spp.

This study investigated the immune response of broiler chickens with oral treatment of a Lactobacillus spp. pool (PL) associated with microencapsulated recombinant proteins flagellin (FliC) and the subunit B of cholera toxin (CTB). Immune responses were evaluated by measuring IgA from intestinal fluid, serum IgY, and immunostaining of CD8(+) T lymphocytes present in the cecum. The evaluations were performed on d 0, 7, 14, 21, and 28 posttreatment. A significant increase (P < 0.05) was observed in IgA levels in all immunized groups, especially 3 wk after immunization. Treatments 2 (recombinant CTB) and 3 (recombinant FliC+CTB) showed the highest concentrations. Similarly, serum concentrations IgY (µg/mL) increased along the experiment, and the means for treatments 2 and 3 showed significant differences (P < 0.05) compared with controls, reaching concentrations of 533 and 540 µg/mL, respectively. The number of CD8(+) T lymphocytes in all treatments greatly differed (P < 0.05) compared with the negative control at 21 d posttreatment. However, only treatment 2 (recombinant CTB), 4 (PL), and 5 (recombinant FliC+ recombinant CTB + PL) remained significantly (P < 0.05) different from the control at 28 d posttreatment. Thus, it is concluded that the microencapsulated recombinant proteins administered orally to broiler chickens are capable of stimulating humoral and cellular immune response, and the combinations of these antigens with Lactobacillus spp. can influence the population of CD8(+) T cells residing in the cecum.

Soropositividade para Mycoplasma hyopneumoniae em suínos abatidos em frigoríficos da região central do estado de São Paulo / Seropositivity for Mycoplasma hyopneumoniae in pigs at a slaughterhouse in the Central region of São Paulo

Mycoplasma hyopneumoniae is the etiologic agent of enzootic pneumonia in pigs and causes large economic losses in the swine industry. There is little data on the positivity of this disease in Brazil. The objective of this study was to evaluate the seropositivity for this agent in 200 serum samples collected from pigs in a slaughterhouse located in the central region of São Paulo. A high percentage (52%) of positivity was found indicating the presence of the agent and the need to implement control measures.

Multiple resistance to anthelmintics by Haemonchus contortus and Trichostrongylus colubriformis in sheep in Brazil.

The objective of this study was to determine the level of resistance of Haemonchus contortus and Trichostrongylus colubriformis in sheep to levamisole, albendazole, ivermectin, moxidectin, closantel and trichlorfon. The parasites were isolated from sheep naturally infected by gastrointestinal nematodes and were then kept in monospecifically-infected lambs for production of infective larvae (L3) of both species. Forty-two lambs, at three months of age, were simultaneously artificially infected with 4000 L3 of H. contortus and 4000 L3 of T. colubriformis. The animals were allocated into seven groups with six animals each that received one of the following treatments: Group 1--control, no treatment; Group 2--moxidectin (0.2mg/kg body weight (BW)); Group 3--closantel (10mg/kg BW); Group 4--trichlorfon (100mg/kg BW); Group 5--levamisole phosphate (4.7 mg/kg BW); Group 6--albendazole (5.0mg/kg BW); and Group 7--ivermectin (0.2mg/kg BW). Nematode fecal egg counts (FEC) were carried out on the day of treatment and again at 3, 7, 10 and 14 days post-treatment. On the same occasions, composite fecal cultures were prepared for each group for production of L3, which were identified into genus. The animals were sacrificed for worm counts at 14 days after treatment. The efficacy of each treatment was calculated from the arithmetic mean of the FEC or worm burden of the treated group, compared with the values of the control group. Only trichlorfon and moxidectin treatments resulted in a significant reduction of H. contortus recorded at necropsy (73% and 45% respectively). Moxidectin reduced T. colubriformis worm burdens by 82% and albendazole by 19%. All other anthelmintics resulted in no significant reduction in the numbers of worms found at necropsy. In conclusion, the isolates of H. contortus and T. colubriformis showed multiple resistance to all groups of anthelmintics tested. This is the first report, based on the controlled efficacy test, to show resistance of T. colubriformis to macrocyclic lactones in Brazil.

Novel factor V C2-domain mutation (R2074H) in two families with factor V deficiency and bleeding.

The molecular basis of Factor V deficiency has been defined in few patients only. We report a homozygous nucleotide change (G6395A) in two Tunisian probands with Factor V deficiency and bleeding episodes. This substitution results in the replacement of an arginine (R) by a histidine (H) in amino acid position 2074, located in the Factor V C2-domain. Mutations in this protein domain have not previously been described. Several lines of evidence support that this sequence variant is indeed disease causing: 1) Crystal structures of Factor V and molecular C2-domain modeling studies of H2074 suggest that the conserved R2074 is required for correct folding; 2) Structure-function studies of selective Factor V mutants (R2074A) demonstrate the importance of R2074 for structural stability of the Factor V C2-domain and for cofactor activity (1); 3) In Factor VIII, point mutations in codon 2209, which corresponds to position 2074 in Factor V, cause hemophilia A.

Promiscuous antigen presentation by the nonclassical MHC Ib Qa-2 is enabled by a shallow, hydrophobic groove and self-stabilized peptide conformation.

BACKGROUND: Qa-2 is a nonclassical MHC Ib antigen, which has been implicated in both innate and adaptive immune responses, as well as embryonic development. Qa-2 has an unusual peptide binding specificity in that it requires two dominant C-terminal anchor residues and is capable of associating with a substantially more diverse array of peptide sequences than other nonclassical MHC. RESULTS: We have determined the crystal structure, to 2.3 A, of the Q9 gene of murine Qa-2 complexed with a self-peptide derived from the L19 ribosomal protein, which is abundant in the pool of peptides eluted from the Q9 groove. The 9 amino acid peptide is bound high in a shallow, hydrophobic binding groove of Q9, which is missing a C pocket. The peptide makes few specific contacts and exhibits extremely poor shape complementarity to the MHC groove, which facilitates the presentation of a degenerate array of sequences. The L19 peptide is in a centrally bulged conformation that is stabilized by intramolecular interactions from the invariant P7 histidine anchor residue and by a hydrophobic core of preferred secondary anchor residues that have minimal interaction with the MHC. CONCLUSIONS: Unexpectedly, the preferred secondary peptide residues that exhibit tenuous contact with Q9 contribute significantly to the overall stability of the peptide-MHC complex. The structure of this complex implies a "conformational" selection by Q9 for peptide residues that optimally stabilize the large bulge rather than making intimate contact with the MHC pockets.

Allosteric activation of a spring-loaded natriuretic peptide receptor dimer by hormone.

Natriuretic peptides (NPs) are vasoactive cyclic-peptide hormones important in blood pressure regulation through interaction with natriuretic cell-surface receptors. We report the hormone-binding thermodynamics and crystal structures at 2.9 and 2.0 angstroms, respectively, of the extracellular domain of the unliganded human NP receptor (NPR-C) and its complex with CNP, a 22-amino acid NP. A single CNP molecule is bound in the interface of an NPR-C dimer, resulting in asymmetric interactions between the hormone and the symmetrically related receptors. Hormone binding induces a 20 angstrom closure between the membrane-proximal domains of the dimer. In each monomer, the opening of an interdomain cleft, which is tethered together by a linker peptide acting as a molecular spring, is likely a conserved allosteric trigger for intracellular signaling by the natriuretic receptor family.

Immunomodulation of experimental autoimmune encephalomyelitis with ordered peptides based on MHC-TCR binding motifs.

T cell-mediated destruction of the myelin sheath causes inflammatory damage of the CNS in multiple sclerosis (MS). The major T and B cell responses in MS patients who are HLA-DR2 (about two-thirds of MS patients) react to a region between residues 84 and 103 of myelin basic protein (1 ). The crystal structure of HLA-DR2 complexed with myelin basic protein(84-102) confirmed that Lys(91) is the major TCR contact site, whereas Phe(90) is a major anchor to MHC and binds the hydrophobic P4 pocket (2 ). We have tested peptides containing repetitive 4-aa sequences designed to bind critical MHC pockets and to interfere with T cell activation. One such sequence, EYYKEYYKEYYK, ameliorates experimental autoimmune encephalomyelitis in Lewis rats, an animal model of MS.

Norepinephrine prevents the adverse effects of lidocaine upon the heart. An experimental study in isolated guinea-pig hearts.

The adverse effects of local anesthetics associated with catecholamines upon the cardiovascular system, such as arterial pressure alterations and cardiac arrhythmias, have been highly discussed in pharmacological literature. When present in the blood circulation, the anesthetic salt promotes depressing effects at the cardiovascular system level, whilst the catecholaminic vasoconstrictors promote opposite effects upon the cardiovascular system. This paper aimed to verify which component is responsible for the initial effect upon the isolated heart of the guinea-pig by comparing the same anesthetic with and without vasoconstrictor. Guinea-pig hearts perfused using the Langendorff method received separate injections of lidocaine hydrochloride, without vasoconstrictor (LC), and with the vasoconstrictor norepinephrine at 1 : 50,000 (LCN) in doses of 3, 4 and 5 mg. LC was seen to significantly reduce the contraction force (CF) and the cardiac rate (CR) at all injected doses and at all times registered. LCN, though, significantly reduced CF in all doses only at 15 s after administration. At 30 and 60 s, LCN elevated CF to values close to those of the control. LCN in its 3 mg dose significantly increased CR at 30 and 60 s; with the 4 mg dose, significant alterations did not occur in CR and with the 5 mg dose CR was reduced significantly at 15 and 30 s, returning to values close to those of the control at 60 s. The electrocardiographic traces with LCN were similar to those with LC with a reduction occurring in the automatism of the sinus node, prolongation of the PR interval and widening of QRS, with all doses utilized at 15 s. Soon after the administration of LC and LCN ventricular asystole occurred, having a larger duration with LC. This duration was also increased when higher doses of anesthetics were used. The authors concluded that the initial cardio-depression effect of local anesthetics, with the vasoconstrictor norepinephrine, in the isolated guinea-pig heart is due to the anesthetic salt and that the vasoconstrictor exerts a protecting action against this effect.

In vitro reconstitution of recognition and activation complexes between interleukin-6 and gp130.

Gp130 is a shared signal-transducing receptor for a family of four-helix cytokines, of which interleukin-6 is a prototypic member. IL-6-type cytokines activate gp130 to elicit downstream intracellular JAK/STAT signaling cascades through formation of hetero-oligomeric receptor complexes. Interleukin-6 must first complex with its specific alpha-receptor (Ralpha) in order to bind and activate gp130. We have dissected the extracellular activation pathway of human gp130 by human IL-6 through reconstitution of soluble complexes representing intermediate and final states in the hierarchical assembly of the IL-6/IL-6Ralpha/gp130 signaling complex. To isolate these hetero-complexes, we have applied a protein engineering strategy of covalently linking IL-6 to its Ralpha, which results in a "hyperactive" single-chain complex (hyper-IL-6) which we express in both Escherichia coli and insect cells. We have determined that IL-6/IL-Ralpha and the cytokine-binding homology region (CHR) of gp130 (D2D3) form a stable trimolecular "recognition" complex (trimer) consisting of 1IL-6,1 IL-6Ralpha, and 1 gp130-CHR. Addition of the N-terminal (D1) Ig-like domain (IGD) of gp130 to the CHR results in a transition to a hexameric "activation" complex containing 2 IL-6, 2IL-6Ralpha, and 2 gp130. These results clearly demonstrate that the recognition and activation complexes are disparate hetero-oligomeric molecular species linked by the recruitment of the gp130 IGD by the unique site III epitope present on all gp130-class cytokines. The results of these studies are relevant to other members of the IL-6 family of gp130-cytokines and address a longstanding question concerning the respective roles of the gp130 CHR and IGD in assembly of the active signaling oligomer.

Kinetics and thermodynamics of T cell receptor- autoantigen interactions in murine experimental autoimmune encephalomyelitis.

In the current study, cellular and molecular approaches have been used to analyze the biophysical nature of T cell receptor (TCR)-peptide MHC (pMHC) interactions for two autoreactive TCRs. These two TCRs recognize the N-terminal epitope of myelin basic protein (MBP1-11) bound to the MHC class II protein, I-A(u), and are associated with murine experimental autoimmune encephalomyelitis. Mice transgenic for the TCRs have been generated and characterized in other laboratories. These analyses indicate that the mice either develop encephalomyelitis spontaneously (172.10 TCR) or only if immunized with autoantigen in adjuvant (1934.4 TCR). Here, we show that the 172.10 TCR binds MBP1-11:I-A(u) with a 4-5-fold higher affinity than the 1934.4 TCR. Consistent with the higher affinity, 172.10 T hybridoma cells are significantly more responsive to autoantigen than 1934.4 cells. The interaction of the 172.10 TCR with cognate ligand is more entropically unfavorable than that of the 1934.4 TCR, indicating that the 172.10 TCR undergoes greater conformational rearrangements upon ligand binding. The studies therefore suggest a correlation between the strength and plasticity of a TCR-pMHC interaction and the frequency of spontaneous disease in the corresponding TCR transgenic mice. The comparative analysis of these two TCRs has implications for understanding autoreactive T cell recognition and activation.

Structure of an extracellular gp130 cytokine receptor signaling complex.

The activation of gp130, a shared signal-transducing receptor for a family of cytokines, is initiated by recognition of ligand followed by oligomerization into a higher order signaling complex. Kaposi's sarcoma-associated herpesvirus encodes a functional homolog of human interleukin-6 (IL-6) that activates human gp130. In the 2.4 angstrom crystal structure of the extracellular signaling assembly between viral IL-6 and human gp130, two complexes are cross-linked into a tetramer through direct interactions between the immunoglobulin domain of gp130 and site III of viral IL-6, which is necessary for receptor activation. Unlike human IL-6 (which uses many hydrophilic residues), the viral cytokine largely uses hydrophobic amino acids to contact gp130, which enhances the complementarity of the viral IL-6-gp130 binding interfaces. The cross-reactivity of gp130 is apparently due to a chemical plasticity evident in the amphipathic gp130 cytokine-binding sites.

The interaction of neuropilin-1 with vascular endothelial growth factor and its receptor flt-1.

Neuropilin-1 (NP-1) was first identified as a semaphorin receptor involved in neuron guidance. Subsequent studies demonstrated that NP-1 also binds an isoform of vascular endothelial growth factor (VEGF) as well as several VEGF homologs, suggesting that NP-1 may also function in angiogenesis. Here we report in vitro binding experiments that shed light on the interaction between VEGF165 and NP-1, as well as a previously unknown interaction between NP-1 and one of the VEGF receptor tyrosine kinases, VEGFR1 or Flt-1. BIAcore analysis demonstrated that, with the extracellular domain (ECD) of NP-1 immobilized at low density, VEGF165 bound with low affinity (K(d) = 2 microm) and fast kinetics. The interaction was dependent on the heparin-binding domain of VEGF165 and increased the affinity of VEGF165 for its signaling receptor VEGFR2 or kinase insert domain-containing receptor. The affinity of VEGF165 for the NP-1 ECD was greatly enhanced either by increasing the density of immobilized NP-1 (K(d) = 113 nm) or by the addition of heparin (K(d) = 25 nm). We attribute these affinity enhancements to avidity effects mediated by the bivalent VEGF165 homodimer or multivalent heparin. We also show that the NP-1 ECD binds with high affinity (K(d) = 1.8 nm) to domains 3 and 4 of Flt-1 and that this interaction inhibits the binding of NP-1 to VEGF165. Based on these results, we propose that NP-1 acts as a coreceptor for various ligands and that these functions are dependent on the density of NP-1 on the cell membrane. Furthermore, Flt-1 may function as a negative regulator of angiogenesis by competing for NP-1.

A structural framework for deciphering the link between I-Ag7 and autoimmune diabetes.

Susceptibility to murine and human insulin-dependent diabetes mellitus correlates strongly with major histocompatibility complex (MHC) class II I-A or HLA-DQ alleles that lack an aspartic acid at position beta57. I-Ag7 lacks this aspartate and is the only class II allele expressed by the nonobese diabetic mouse. The crystal structure of I-Ag7 was determined at 2.6 angstrom resolution as a complex with a high-affinity peptide from the autoantigen glutamic acid decarboxylase (GAD) 65. I-Ag7 has a substantially wider peptide-binding groove around beta57, which accounts for distinct peptide preferences compared with other MHC class II alleles. Loss of Asp(beta57) leads to an oxyanion hole in I-Ag7 that can be filled by peptide carboxyl residues or, perhaps, through interaction with the T cell receptor.

A functional hot spot for antigen recognition in a superagonist TCR/MHC complex.

A longstanding question in T cell receptor signaling is how structurally similar ligands, with similar affinities, can have substantially different biological activity. The crystal structure of the 2C TCR complex of H-2Kb with superagonist peptide SIYR at 2.8 A elucidates a structural basis for TCR discrimination of altered peptide ligands. The difference in antigen potency is modulated by two cavities in the TCR combining site, formed mainly by CDRs 3alpha, 3beta, and 1beta, that complement centrally located peptide residues. This "functional hot spot" allows the TCR to finely discriminate amongst energetically similar interactions within different ligands for those in which the peptide appropriately stabilizes the TCR/pMHC complex and provides a new structural perspective for understanding differential signaling resulting from T cell cross-reactivity.

Structural basis of T cell recognition.

Exciting breakthroughs in the last two years have begun to elucidate the structural basis of cellular immune recognition. Crystal structures have been determined for full-length and truncated forms of alpha beta T cell receptor (TCR) heterodimers, both alone and in complex with their peptide-MHC (pMHC) ligands or with anti-TCR antibodies. In addition, a truncated CD8 coreceptor has been visualized with a pMHC. Aided in large part by the substantial body of knowledge accumulated over the last 25 years on antibody structure, a number of general conclusions about TCR structure and its recognition of antigen can already be derived from the relatively few TCR structures that have been determined. Small, but important, variations between TCR and antibody structures bear on their functional differences as well as on their specific antigen recognition requirements. As observed in antibodies, canonical CDR loop structures are already emerging for some of the TCR CDR loops. Highly similar docking orientations of the TCR V alpha domains in the TCR/pMHC complex appear to play a primary role in dictating orientation, but the V beta positions diverge widely. Similar TCR contact positions, but whose exact amino acid content can vary, coupled with relatively poor interface shape complementarity, may explain the flexibility and short half-lives of many TCR interactions with pMHC. Here we summarize the current state of this field, and suggest that the knowledge gap between the three-dimensional structure and the signaling function of the TCR can be bridged through a synthesis of molecular biological and biophysical techniques.

Molecular interactions between extracellular components of the T-cell receptor signaling complex.

The structural and biochemical basis of antigen recognition by the T-cell receptor (TCR)-CD3 signaling complex has been illuminated greatly over the past few years. Structural biology has contributed enormously to this understanding through the determination of crystal structures of many of the individual components of this complex, and some of the complexes. A number of general principles can be derived for the structure of the alpha beta TCR and its interaction with peptide-major histocompatibility complex (pMHC) in class I systems, as well as interaction of the CD8 co-receptor with MHC. Large buried surface areas within the protein-protein interfaces, and varying degrees of shape complementarity appear critical for modulating the stability of the multicomponent, low-affinity macromolecular complexes consisting of TCR, pMHC, CD8 or CD4, and CD3 gamma, delta, epsilon and zeta. Significant structural alterations in TCR and pMHC, upon complex formation, hint at an as yet unclear role for conformational change in both recognition and activation. Subtle chemical alterations in key peptide residues which contact the TCR can have dramatic agonist or antagonist effects on receptor activation, which correlate only loosely with the TCR/pMHC complex affinity, implying an ability of the signaling complex to "sense" fine differences in the interface. The stoichiometry of an activated TCR signaling complex is still an unresolved issue, as is the structure and disposition of the CD3 components. However, functional experiments are bridging this gap and providing us with preliminary working models of the multimeric assemblies.

Emerging principles for T cell receptor recognition of antigen in cellular immunity.

The structural basis of antigen recognition in cellular immunity has been elucidated through the determination of crystal structures of major histocompatibility complex (MHC) molecules bound to antigenic peptides, T cell receptors (TCR), CD8 and CD4 co-receptors and, most recently, TCRs in complex with peptide-MHC (pMHC). The mechanisms that generate the diversity of the immune response to invading microorganisms were first realized at a genetic level and are necessary in order to cope with the enormous number of potential antigens. This diversity is manifested in the protein products of the genes which code for the components of the TCR signalling complex. The structure of the TCR reveals both striking similarities with and fundamental differences from its functional counterpart, the antibody, in the humoral immune system. The conserved manner in which the TCR recognizes and interacts with its peptide-MHC ligand allows the TCR great latitude in its potential to form productive interactions with antigen-presenting cells that bear numerous ligands to which the TCR has not been previously exposed. This phenomenon of cross-, or alloreactivity arises from a combination of conserved structural features across all MHC molecules, both self and foreign, and some degree of molecular mimicry. Non-classical MHC ligands presenting either modified or specialized peptides, lipids, carbohydrates, or no ligand at all, are now thought to play increasingly important roles in cellular immunity. We review some of the recent structural results and our current state of knowledge about TCR structure, and how this relates to its function.

Probing the activation requirements for naive CD8+ T cells with Drosophila cell transfectants as antigen presenting cells.

Activation of T cells involves multiple receptor-ligand interactions between T cells and antigen presenting cells (APC). At least two signals are required for T-cell activation: Signal 1 results from recognition of MHC/peptide complexes on the APC by cell surface T-cell receptors (TCR), whereas Signal 2 is induced by the interactions of co-stimulatory molecules on APC with their complementary receptors on T cells. This review focuses on our attempts to understand these various signals in a model system involving the 2C TCR. The structural basis of Signal 1 was investigated by determining the crystal structure of 2C TCR alone and in complex with MHC/peptide. Analysis of these structures has provided some basic rules for how TCR and MHC/peptide interact; however, the critical question of how this interaction transduces Signal 1 to T cells remains unclear. The effects of Signal 1 and Signal 2 on T-cell activation were examined with naive T cells from the 2C TCR transgenic mice, defined peptides as antigen and transfected Drosophila cells as APC. The results suggest that, except under extreme conditions, Signal 1 alone is unable to activate naive CD8 T cells despite the induction of marked TCR downregulation. Either B7 or intercellular adhesion molecule (ICAM)-1 can provide the second signal for CD8 T-cell activation. However, especially at low MHC/peptide densities, optimal activation and differentiation of CD8 T cells required interaction with both B7 and ICAM-1 on the same APC. Thus, the data suggest that at least two qualitatively different co-stimulation signals are required for full activation of CD8 T cells under physiological conditions.