BnP1, a novel P-I metalloproteinase from Bothrops neuwiedi venom: Biological effects benchmarking relatively to jararhagin, a P-IIISVMP

Snake venom metalloproteinases (SVMPs) have been extensively studied and their effects associated with the local bleeding observed in human accidents by viper snakes. Representatives of P-I and P-III classes of SVMPs similarly hydrolyze extracellular matrix proteins or coagulation factors while only P-III SVMPs induce significant hemorrhage in experimental models. In this work, the effects of P-I and P-III SVMPs on plasma proteins and cultures of muscle and endothelial cells were compared in order to enlighten the mechanisms involved in venom-induced hemorrhage. To reach this comparison, BnP1 was isolated from B. neuwiedi venom and used as a weakly hemorrhagic P-I SVMPs and jararhagin was used as a model of potently hemorrhagic P-III SVMP. BnP1 was isolated by size exclusion and anion-exchange chromatographies, showing apparent molecular mass of approximately 24kDa and sequence similarity with other members of SVMPs, which allowed its classification as a group P-I SVMP. The comparison of local effects induced by SVMPs showed that BnP1 was devoid of significant myotoxic and hemorrhagic activities and jararhagin presented only hemorrhagic activity. BnP1 and jararhagin were able to hydrolyze fibrinogen and fibrin, although the latter displayed higher activity in both systems. Using HUVEC primary cultures, we observed that BnP1 induced cell detachment and a decrease in the number of viable endothelial cells in levels comparable to those observed by treatment with jararhagin. Moreover, both BnP1 and jararhagin induced apoptosis in HUVECs while only a small increase in LDH supernatant levels was observed after treatment with jararhagin, suggesting that the major mechanism involved in endothelial cell death is apoptosis. Jararhagin and BnP1 induced little effects on C2C12 muscle cell cultures, characterized by a partial detachment 24h after treatment and a mild necrotic effect as evidenced by a small increase in the supernatants LDH levels. Taken together, our data show that P-I and P-III SVMPs presented comparable effects except for the hemorrhagic activity, suggesting that hydrolysis of coagulation factors or damage to endothelial cells are not sufficient for induction of local bleeding. (C) 2007 Elsevier Ltd. All rights reserved.

Functional Characterization of LcpA, a Surface-Exposed Protein of Leptospira spp. That Binds the Human Complement Regulator C4BP

We have previously shown that pathogenic leptospiral strains are able to bind C4b binding protein (C4BP). Surface-bound C4BP retains its cofactor activity, indicating that acquisition of this complement regulator may contribute to leptospiral serum resistance. In the present study, the abilities of seven recombinant putative leptospiral outer membrane proteins to interact with C4BP were evaluated. The protein encoded by LIC11947 interacted with this human complement regulator in a dose-dependent manner. The cofactor activity of C4BP bound to immobilized recombinant LIC11947 (rLIC11947) was confirmed by detecting factor I-mediated cleavage of C4b. rLIC11947 was therefore named LcpA (for leptospiral complement regulator-acquiring protein A). LcpA was shown to be an outer membrane protein by using immunoelectron microscopy, cell surface proteolysis, and Triton X-114 fractionation. The gene coding for LcpA is conserved among pathogenic leptospiral strains. This is the first characterization of a Leptospira surface protein that binds to the human complement regulator C4BP in a manner that allows this important regulator to control complement system activation mediated either by the classical pathway or by the lectin pathway. This newly identified protein may play a role in immune evasion by Leptospira spp. and may therefore represent a target for the development of a human vaccine against leptospirosis.

PAS-1, a protein from Ascaris suum, modulates allergic inflammation via IL-10 and IFN-gamma, but not IL-12

Helminths and their products have a profound immunomodulatory effect upon the inductive and effector phases of inflammatory responses, including allergy. We have demonstrated that PAS-1, a protein isolated from Ascaris strum worms, has an inhibitory effect on lung allergic inflammation due to its ability to down-regulate eosinophilic inflammation, Th2 cytokine release and IgE antibody production. Here, we investigated the role of IL-12, IFN-gamma and IL-10 in the PAS-1-induced inhibitory mechanism using a murine model of asthma. Wild type C57BL/6, IL-12(-/-), IFN-gamma(-/-) and IL-10(-/-) mice were immunized with PAS-1 and/or OVA and challenged with the same antigens intranasally. The suppressive effect of PAS-I was demonstrated on the cellular influx into airways, with reduction of eosinophil number and eosinophil peroxidase activity in OVA + PAS-1-immunized wild type mice. This effect well correlated with a significant reduction in the levels of IL-4, IL-5, IL-13 and eotaxin in BAL fluid. Levels of IgE and IgG1 antibodies were also impaired in serum from these mice. The inhibitory activity of PAS-I was also observed in IL-12(-/-) mice, but not in IFN-gamma(-/-) and IL-10(-/-) animals. These data show that IFN-gamma and IL-10, but not IL-12, play an important role in the PAS-1 modulatory effect. (C) 2008 Elsevier Ltd. All rights reserved.

New malaria vaccine candidates based on the Plasmodium vivax Merozoite Surface Protein-1 and the TLR-5 agonist Salmonella Typhimurium FliC flagellin

The present study evaluated the immunogenicity of new malaria vaccine formulations based on the 19 kDa C-terminal fragment of Plasmodium vivax Merozoite Surface Protein-1 (MSP1(19)) and the Salmonella enterica serovar Typhimurium flagellin (FIiC), a Toll-like receptor 5 (TLR5) agonist. FHC was used as an adjuvant either admixed or genetically linked to the P. vivax MSP1(19) and administered to C57BL/6 mice via parenteral (s.c.) or mucosal (i.n.) routes. The recombinant fusion protein preserved MSP1(19) epitopes recognized by Sera collected from P. vivax infected humans and TLR5 agonist activity. Mice parenterally immunized with recombinant P vivax MSPI 19 in the presence of FliC, either admixed or genetically linked, elicited strong and long-lasting MSP1 (19)-specific systemic antibody responses with a prevailing IgG1 subclass response. Incorporation of another TLR agonist, CpG ODN 1826, resulted in a more balanced response, as evaluated by the IgG1/IgG2c ratio, and higher cell-mediated immune response measured by interferon-gamma secretion. Finally, we show that MSPI 19-specific antibodies recognized the native protein expressed on the surface of P. vivax parasites harvested from infected humans. The present report proposes a new class of malaria vaccine formulation based on the use of malaria antigens and the innate immunity agonist FliC. it contains intrinsic adjuvant properties and enhanced ability to induce specific humoral and cellular immune responses when administered alone or in combination with other adjuvants. (C) 2008 Elsevier Ltd. All rights reserved.

Induction of neutralizing antibodies in mice immunized with an amino-terminal polypeptide of Streptococcus mutans P1 protein produced by a recombinant Bacillus subtilis strain

The oral pathogen Streptococcus mutans expresses a surface protein, P1, which interacts with the salivary pellicle on the tooth surface or with fluid-phase saliva, resulting in bacterial adhesion or aggregation, respectively. P1 is a target of protective immunity. Its N-terminal region has been associated with adhesion and aggregation functions and contains epitopes recognized by efficacious antibodies. In this study, we used Bacillus subtilis, a gram-positive expression host, to produce a recombinant N-terminal polypeptide of P1 (P1(39-512)) derived from the S. mutans strain UA159. Purified P1(39-512) reacted with an anti-full-length P1 antiserum as well as one raised against intact S. mutans cells, indicating preserved antigenicity. Immunization of mice with soluble and heat-denatured P1(39-512) induced antibodies that reacted specifically with native P1 on the surface of S. mutans cells. The anti-P1(39-512) antiserum was as effective at blocking saliva-mediated aggregation of S. mutans cells and better at blocking bacterial adhesion to saliva-coated plastic surfaces compared with the anti-full-length P1 antiserum. In addition, adsorption of the anti-P1 antiserum with P1(39-512) eliminated its ability to block the adhesion of S. mutans cells to abiotic surfaces. The present results indicate that P1(39-512), expressed and purified from a recombinant B. subtilis strain, maintains important immunological features of the native protein and represents an additional tool for the development of anticaries vaccines.

Functional and immunological characterization of a natural polymorphic variant of a heat-labile toxin (LT-I) produced by enterotoxigenic Escherichia coli (ETEC)

Heat-labile toxins (LT) encompass at least 16 natural polymorphic toxin variants expressed by wild-type enterotoxigenic Escherichia coli (ETEC) strains isolated from human beings, but only one specific form, produced by the reference ETEC H10407 strain (LT1), has been intensively studied either as a virulence-associated factor or as a mucosal/transcutaneous adjuvant. In the present study, we carried out a biological/immunological characterization of a natural LT variant (LT2) with four polymorphic sites at the A subunit (S190L, G196D, K213E, and S224T) and one at the B subunit (T75A). The results indicated that purified LT2, in comparison with LT1, displayed similar in vitro toxic activities (adenosine 3`,5`-cyclic monophosphate accumulation) on mammalian cells and in vivo immunogenicity following delivery via the oral route. Nonetheless, the LT2 variant showed increased adjuvant action to ovalbumin when delivered to mice via the transcutaneous route while antibodies raised in mice immunized with LT2 displayed enhanced affinity and neutralization activity to LT1 and LT2. Taken together, the results indicate that the two most frequent LT polymorphic forms expressed by wild ETEC strains share similar biological features, but differ with regard to their immunological properties.

Neonatal mitochondrial encephaloneuromyopathy due to a defect of mitochondrial protein synthesis

Mitochondrial diseases are clinically and genetically heterogeneous disorders due to primary mutations in mitochondrial DNA (mtDNA) or nuclear DNA (nDNA). We studied a male infant with severe congenital encephalopathy, peripheral neuropathy, and myopathy. The patient`s lactic acidosis and biochemical defects of respiratory chain complexes I, III, and IV in muscle indicated that he had a mitochondrial disorder while parental consanguinity suggested autosomal recessive inheritance. Cultured fibroblasts from the patient showed a generalized defect of mitochondrial protein synthesis. Fusion of cells from the patient with 143B206 rho(0) cells devoid of mtDNA restored cytochrome c oxidase activity confirming the nDNA origin of the disease. Our studies indicate that the patient has a novel autosomal recessive defect of mitochondrial protein synthesis. (C) 2008 Elsevier B.V. All rights reserved.

CD8(+) T cell adjuvant effects of Salmonella FliCd flagellin in live vaccine vectors or as purified protein

Salmonella flagellin, the flagellum structural subunit, has received particular interest as a vaccine adjuvant conferring enhanced immunogenity to soluble proteins or peptides, both for activation of antibody and cellular immune responses. In the present study, we evaluated the Salmonella enterica FliCd flagellin as a T cell vaccine adjuvant using as model the 9-mer (SYVPSAEQI) synthetic H2(d)-restricted CD8(+) T cell-specific epitope (CS(280-288)) derived from the Plasmodium yoelii circumsporozoite (G) protein. The FliCd adjuvant effects were determined under two different conditions: (i) as recombinant flagella, expressed by orally delivered live S. Dublin vaccine strains expressing the target CS(280-288) peptide fused at the central hypervariable domain, and (ii) as purified protein in acellular vaccines in which flagellin was administered to mice either as a recombinant protein fused or admixed with the target CS(280-288) peptide. The results showed that CS(280-288)-specific cytotoxic CD8(+) T cells were primed when BALB/c mice were orally inoculated with the expressing the CS280-288 epitope S. Dublin vaccine strain. In contrast, mice immunized with purified FliCd admixed with the CS280-288 peptide and, to a lesser extent, fused with the target peptide developed specific cytotoxic CD8(+) T cell responses without the need of a heterologous booster immunization. The CD8(+) T cell adjuvant effects of flagellin, either fused or not with the target peptide, correlated with the in vivo activation of CD11c(+) dendritic cells. Taken together, the present results demonstrate that Salmonella flagellins are flexible adjuvant and induce adaptative immune responses when administered by different routes or vaccine formulations. (C) 2009 Elsevier Ltd. All rights reserved.

Islet neogenesis-associated protein signaling in neonatal pancreatic rat islets: involvement of the cholinergic pathway

Islet neogenesis associated protein (INGAP) increases islet mass and insulin secretion in neonatal and adult rat islets. lit the Present Study, we measured the short- and long-term effects of INGAP-PP (a pentadecapeptide having the 104-118 amino acid sequence of INGAP) upon islet protein expression and phosphorylation of components of the PI3K, MAPK and cholinergic pathways, and on insulin secretion. Short-term exposure of neonatal islets to INGAP-PP (90 s, 5, 15, and 30 min) significantly increased Akt1(-Ser473) and MAPK3/1(-Thr202/Tyr204) phosphorylation and INGAP-PP also acutely increased insulin secretion from islets perifused with 2 and 20 mM glucose. Islets cultured for 4 days in the presence of INGAP-PP showed an increased expression of Akt1, Frap1, and Mapk1 mRNAs as well as of the muscarinic M3 receptor subtype, and phospholipase C (PLC)-beta 2 proteins. These islets also showed increased Akt1 and MAPK3/1 protein phosphorylation. Brief exposure of INGAP-P-treated islets to carbachol (Cch) significantly increased P70S6K(-Thr389) and MAPK3/1 phosphorylation and these islets released more insulin when challenged with Cch that was prevented by the M3 receptor antagonist 4-DAMP in a concentration-dependent manner. In conclusion, these data indicate that short- and long-term exposure to INGAP-PP significantly affects the expression and the phosphorylation of proteins involved in islet PI3K and MAPK signaling pathways. The observations of INGAPP-PP-stimulated up-regulation of cholinergic M3 receptors and PLC-beta 2 proteins, enhanced P70S6K and MAIIK3/1 phosphorylation and Cch-induced insulin secretion suggest a participation of the cholinergic pathway in INGAP-PP-mediated effects.

Deficiency of the human complement regulatory protein factor H associated with low levels of component C9

We identified a 4-year-old Brazilian boy from a family of Japanese descent and history of consanguinity, who suffered from severe recurrent pneumonia. He carries factor H (FH) deficiency associated with reduced levels of component C9 and low serum levels of C3 and factor B. His mother also presented low levels of these proteins and factor I, while his father and sister had only lower levels of FH. Western blot assays confirmed the complete absence of FH and FHL-1 polypeptides in this patient. Sequencing of the proband`s FH cDNA revealed a homozygous G453A substitution, encoding an Arg(127)His change. His mother, father and sister are heterozygous for this substitution. Despite the absence of FH in the plasma, this protein was detected in the patient`s fibroblasts, suggesting that Arg(127) may be important for FH secretion. Low concentrations of C9 were detected in the proband serum but no mutations in the patient`s C9 gene or promoter have been identified, suggesting that this is a consequence of uncontrolled complement activation and high C9 consumption.

The crystal structure of the leptospiral hypothetical protein LIC12922 reveals homology with the periplasmic chaperone SurA

Leptospirosis is a world spread zoonosis caused by members of the genus Leptospira. Although leptospires were identified as the causal agent of leptospirosis almost 100 years ago, little is known about their biology, which hinders the development of new treatment and prevention strategies. One of the several aspects of the leptospiral biology not yet elucidated is the process by which outer membrane proteins (OMPs) traverse the periplasm and are inserted into the outer membrane. The crystal structure determination of the conserved hypothetical protein LIC12922 from Leptospira interrogans revealed a two domain protein homologous to the Escherichia coli periplasmic chaperone SurA. The LIC12922 NC-domain is structurally related to the chaperone modules of E. coli SurA and trigger factor, whereas the parvulin domain is devoid of peptidyl prolyl cis-trans isomerase activity. Phylogenetic analyses suggest a relationship between LIC12922 and the chaperones PrsA, PpiD and SurA. Based on our structural and evolutionary analyses, we postulate that LIC12922 is a periplasmic chaperone involved in OMPs biogenesis in Leptospira spp. Since LIC12922 homologs were identified in all spirochetal genomes sequenced to date, this assumption may have implications for the OMPs biogenesis studies not only in leptospires but in the entire Phylum Spirochaetes. (C) 2010 Elsevier Inc. All rights reserved.

Time-dependent increases in ouabain-sensitive Na(+), K(+)-ATPase activity in aortas from diabetic rats: The role of prostanoids and protein kinase C

Aims: Na(+), K(+)-ATPase activity contributes to the regulation of vascular contractility and it has been suggested that vascular Na(+), K(+)-ATPase activity may be altered during the progression of diabetes; however the mechanisms involved in the altered Na(+), K(+)-ATPase activity changes remain unclear. Thus, the aim of the present study was to evaluate ouabain-sensitive Na(+), K(+)-ATPase activity and the mechanism(s) responsible for any alterations on this activity in aortas from 1- and 4-week streptozotocin-pretreated (50 mg kg(-1), i.v.) rats. Main methods: Aortic rings were used to evaluate the relaxation induced by KCl (1-10 mM) in the presence and absence of ouabain (0.1 mmol/L) as an index of ouabain-sensitive Na(+), K(+)-ATPase activity. Protein expression of COX-2 and p-PKC-beta II in aortas were also investigated. Key findings: Ouabain-sensitive Na(+), K(+)-ATPase activity was unaltered following 1-week of streptozotocin administration, but was increased in the 4-week diabetic aorta (27%). Endothelium removal or nitric oxide synthase inhibition with L-NAME decreased ouabain-sensitive Na(+), K(+)-ATPase activity only in control aortas. In denuded aortic rings, indomethacin. NS-398, ridogrel or Go-6976 normalized ouabain-sensitive Na(+), K(+)-ATPase activity in 4-week diabetic rats. In addition, COX-2 (51%) and p-PKC-beta II (59%) protein expression were increased in 4-week diabetic aortas compared to controls. Significance: In conclusion, diabetes led to a time-dependent increase in ouabain-sensitive Na(+), K(+)-ATPase activity. The main mechanism involved in this activation is the release of TxA(2)/PGH(2) by COX-2 in smooth muscle cells, linked to activation of the PKC pathway. (C) 2010 Elsevier Inc. All rights reserved.

S100 beta Protein Expression: Gender- and Age-Related Daily Changes

S100 beta is a soluble protein released by glial cells mainly under the activation of the 5-HT1A receptor. It has been reported as a neuro-trophic and -tropic factor that promotes neurite maturation and outgrowth during development. This protein also plays a role in axonal stability and the plasticity underlying long-term potentiation in adult brains. The ability of S100 beta to rapidly regulate neuronal morphology raises the interesting point of whether there are daily rhythm or gender differences in S100 beta level in the brain. To answer this question, the S100 beta expression in adult female and male rats, as well as in adult female CD-21 and S100 beta -/- female mice, were investigated. Scintillation counting and morphometric analysis of the immunoreactivity of S100 beta, showed rhythmic daily expression. The female and male rats showed opposite cycles. Females presented the highest value at the beginning of the rest phase (5:00 h), while in males the maximum value appeared in the beginning of the motor activity period (21:00 h). These results confirm previous S100 beta evaluations in human serum and cerebrospinal fluid reporting the protein`s function as a biomarker for brain damage (Gazzolo et al. in Clin Chem 49:967-970, 2003; Clin Chim Acta 330:131-133, 2003; Pediatr Res 58:1170-1174, 2005), similar behavior was also observed for GFAP in relation to Alzheimer Disease (Fukuyama et al. in Eur Neurol 46:35-38, 2001). The data should be taken into account when considering S100 beta as a biomarker of health condition. In addition, the results raise questions on which structure or condition imposes these rhythms as well as on the physiological meaning of the observed gender differences.

Naturally acquired inhibitory antibodies to Plasmodium vivax Duffy binding protein are short-lived and allele-specific following a single malaria infection

The Duffy binding protein of Plasmodium vivax (DBP) is a critical adhesion ligand that participates in merozoite invasion of human Duffy-positive erythrocytes. A small outbreak of P. vivax malaria, in a village located in a non-malarious area of Brazil, offered us an opportunity to investigate the DBP immune responses among individuals who had their first and brief exposure to malaria. Thirty-three individuals participated in the five cross-sectional surveys, 15 with confirmed P. vivax infection while residing in the outbreak area (cases) and 18 who had not experienced malaria (non-cases). In the present study, we found that only 20% (three of 15) of the individuals who experienced their first P. vivax infection developed an antibody response to DBP; a secondary boosting can be achieved with a recurrent P. vivax infection. DNA sequences from primary/recurrent P. vivax samples identified a single dbp allele among the samples from the outbreak area. To investigate inhibitory antibodies to the ligand domain of the DBP (cysteine-rich region II, DBP(II)), we performed in vitro assays with mammalian cells expressing DBP(II) sequences which were homologous or not to those from the outbreak isolate. In non-immune individuals, the results of a 12-month follow-up period provided evidence that naturally acquired inhibitory antibodies to DBP(II) are short-lived and biased towards a specific allele.

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.