Campylobacter-derived ligands induce cytokine and chemokine expression in chicken macrophages and cecal tonsil mononuclear cells.

Campylobacter jejuni colonizes the chicken gut at a high density without causing disease. However, consumption of poultry products contaminated with this bacterium causes gastroenteritis in humans. Therefore, it is critically important to reduce the Campylobacter burden in poultry products to prevent transmission to humans. Evidence indicates that enhancing intestinal mucosal immune responses is of paramount importance for preventing or reducing Campylobacter colonization in chickens. In view of this, the present study was undertaken to evaluate host responses to different C. jejuni-derived ligands, including lipooligosaccharide (LOS), outer membrane proteins (OMPs), and genomic DNA, with the ultimate goal of identifying a ligand with potent immunostimulatory capacity to serve as a mucosal vaccine adjuvant against enteric infections in chickens. The results revealed that C. jejuni pathogen-associated molecular patterns (PAMPs) varied in their ability to induce the expression of cytokines and chemokines in chicken macrophages and cecal tonsil mononuclear cells and nitric oxide production in macrophages. In addition, C. jejuni OMPs demonstrated superior activity over LOS and DNA ligands in eliciting cytokine expression associated with T helper (Th)1 and Th2 responses (interferon [IFN]-γ and interleukin [IL]-13, respectively), in addition to expression of pro-inflammatory cytokines (IL-1ß), chemokine (CXCLi2), and regulatory cytokines (IL-10 and TGFß1/4) in cecal tonsil cells. Importantly, in addition to their ability to induce innate responses, OMPs could also function as antigens to elicit C. jejuni-specific antibody responses and thereby confer dual protection against C. jejuni infection. Further studies are required to assess the protective efficacy of C. jejuni OMPs against C. jejuni infection in chickens.

Mechanism-based candidate inhibitors of uridine diphosphate galactopyranose mutase (UGM).

Uridine diphosphate-galactopyranose mutase (UGM), an enzyme found in many eukaryotic and prokaryotic human pathogens, catalyzes the interconversion of UDP-galactopyranose (UDP-Galp) and UDP-galactofuranose (UDP-Galf), the latter being used as the biosynthetic precursor of the galactofuranose polymer portion of the mycobacterium cell wall. We report here the synthesis of a sulfonium and selenonium ion with an appended polyhydroxylated side chain. These compounds were designed as transition state mimics of the UGM-catalyzed reaction, where the head groups carrying a permanent positive charge were designed to mimic both the shape and positive charge of the proposed galactopyranosyl cation-like transition state. An HPLC-based UGM inhibition assay indicated that the compounds inhibited about 25% of UGM activity at 500 µM concentration.

Synthesis and biological evaluation of nonionic substrate mimics of UDP-Galp as candidate inhibitors of UDP galactopyranose mutase (UGM).

The synthesis of 1-[5-O-(α-D-galactopyranosyl)-D-glucityl]pyrimidine-2,4(3H)-dione and 1-[(5-O-(ß-D-galactopyranosyl)-D-glucityl]pyrimidine-2,4(3H)-dione as non-ionic substrate mimics of UDP-Galp are described. UDP-Galp is a precursor of Galf, which is a primary component of the cell-wall glycans of several microorganisms. The interconversion of UDP-Galp and UDP-Galf is catalyzed by UDP galactopyranose mutase (UGM); its inhibition comprises a mode of compromising the microorganisms. The nonionic polyhydroxylated chain was intended to mimic the ionic pyrophosphate group and the ribose moiety in UDP-Galp and increase the bioavailabilities of the candidate inhibitors. Inhibition assays with UGM of Mycobacterium tuberculosis showed only weak inhibition of the enzyme by these compounds.

Synthesis and immunological activity of an oligosaccharide-conjugate as a vaccine candidate against Group A Streptococcus.

The synthesis and immunogenicity of a tetanus toxoid (TT)-conjugate of the hexasaccharide portion of the cell-wall polysaccharide (CWPS) of the Group A Streptococcus (GAS) is described. The synthesis relies on the reaction of an allyl glycoside of the hexasaccharide with cysteamine, followed by the reaction of the resultant amine with diethyl squarate to give the monoethyl squarate adduct. Subsequent reaction with the lysine ε-amino groups on TT gives the glycoconjugate containing 30 hexasaccharide haptens per TT molecule. The immunogenicity in mice is similar to that obtained with a native CWPS-TT conjugate, validating the glycoconjugate as a vaccine candidate against GAS infections.

Synthesis and immunological characterization of modified hyaluronic acid hexasaccharide conjugates.

The synthesis of a tetanus toxoid (TT)-conjugate of a hyaluronic acid (HA) hexasaccharide is described. The compound was intended for use in monitoring HA levels as a disease marker and as a potential vaccine against Group A Streptococcus (GAS) infections. We also report the synthesis of a chemically modified HA-hexasaccharide-TT conjugate in which the N-acetyl moiety of the N-acetyl-D-glucosamine residue is replaced with an N-propionyl unit in order to enhance immunogenicity. The oligosaccharides are synthesized in a convergent manner. The TT-conjugate syntheses rely on the reaction of the amines on the 6-aminohexyl aglycon of the hexasaccharides with diethyl squarate to give the monoethyl squarate adducts. Subsequent reactions with lysine ε-amino groups on TT then give the glycoconjugates containing an average of 8 hexasaccharide haptens per TT molecule. Immunological studies in mice show very similar antibody responses with both conjugates, suggesting that the N-acetyl groups of the glucosaminyl residues of the HA-hexasaccharide are not a critical part of the epitope recognized by the anti-HA polyclonal immune response. Furthermore, it would appear that the N-acyl moieties are not in close contact with the amino acid residues of the antibody combining sites.

Teorias/metodologias: trajetos de investigação com coletivos juvenis em São Paulo/Brasil / Theories/methodologies: research paths with young groups in São Paulo/Brazil / Teorías/metodologías: trayectos de investigación con colectivos juveniles en São Paulo/Brasil

Este artigo vincula-se a uma investigação mais ampla, cujo objetivo foi analisar novas práticas políticas juvenis e considerar as ações culturais protagonizadas por jovens, como lócus privilegiado de ações políticas. Destaca-se aqui a reflexão sobre as relações entre matrizes epistemológicas, teórico-conceituais e metodológicas para a construção e aplicação de um protocolo de investigação, centrado na etnografia de campo. Como sujeitos investigados, encontram-se coletivos juvenis que atuam nas periferias da cidade de São Paulo, regiões norte, sul, leste e oeste; são protagonistas de ações mediadas pela cultura, política, cotidianeidade e relações com as políticas públicas. A etnografia privilegiou a coleta de dados, com foco na observação etnográfica, entrevistas em profundidade, produção de acervos imagéticos e retorno de resultados aos jovens investigados.

Este artículo hace parte de una investigación que analiza prácticas políticas juveniles considera las acciones culturales como locus privilegiado de acciones políticas. Se destaca, aquí, la reflexión sobre las relaciones entre matrices epistemológicas, teóricas/conceptuales y metodológicas en la construcción de protocolos de investigación centrados en la etnografía. Como sujetos investigados, los colectivos juveniles de las periferias de la ciudad de San Pablo, regiones norte, sur, este y oeste, protagonistas de acciones mediadas por la cultura, la política, la cotidianeidad y las relaciones con las políticas públicas. La etnografía privilegia la recolección de datos, centrada en la observación, las encuestas, la producción de imágenes y el regreso de los resultados a los jóvenes investigados.

This article intend to do an investigation aimed to examine new practices and policies for collective young people, considering the cultural actions performed by young people as the privileged locus of political action. The highlight is based on the reflection on the relations between epistemological, theoretical, conceptual and methodological conceptions in constructing and implementing a research focus on an ethnographic field. As research subjects, are young groups that act in the suburb of different areas from Sao Paulo - north, south, east and west; these groups are the protagonists of actions mediated by culture, politics, everyday life related with public policy. The ethnography is focused in the data collection based on ethnographic observation, depth interviews, producing imagery collections and a feedback to the young investigated.

Antimycobacterial activity of UDP-galactopyranose mutase inhibitors.

The galactofuran region of the mycobacterial cell wall consists of alternating 5- and 6-linked beta-d-galactofuranose (beta-D-Galf) residues, essential for viability. UDP-galactofuranose (UDP-Galf), the donor for Galf, is synthesised from UDP-galactopyranose (UDP-Galp) by the enzyme UDP-galactopyranose mutase (UGM), which is not found in humans, rendering it a therapeutic target. The in vitro properties, i.e. enzymatic activity, antimycobacterial activity, cellular toxicity, activity in mycobacterial-infected macrophages and activity against non-replicating persistent mycobacteria, of (4-chlorophenyl)-[1-(4-chlorophenyl)-3-hydroxy-5-methyl-1H-pyrazol-4-yl]-methanone and 3-(4-iodophenyl)-2-[4-(3,4-dichlorophenyl)-thiazol-2-ylamino]-propionic acid were studied. The former compound, a pyrazole, was an inhibitor of UGM from Mycobacterium tuberculosis and Klebsiella pneumoniae and was effective against Mycobacterium smegmatis, Mycobacterium bovis BCG and M. tuberculosis but ineffective against other bacterial strains tested. This compound showed potency against mycobacteria in infected macrophages but exhibited moderate cellular toxicity and was ineffective against non-replicating persistent mycobacteria. This is the first report of a compound both with UGM inhibitory properties and broad antimycobacterial activities. The latter compound, an aminothiazole, was active against UGM from K. pneumoniae and M. tuberculosis but was ineffective against M. bovis BCG or M. tuberculosis as well as demonstrating higher cellular toxicity. These data validate the choice of UGM as a target for active antimycobacterial therapy and confirm the pyrazole compound as a viable lead candidate.

Design, synthesis, and immunochemical characterization of a chimeric glycopeptide corresponding to the Shigella flexneri Y O-polysaccharide and its peptide mimic MDWNMHAA.

Two glycopeptide chimeras corresponding to the Shigella flexneri Y O-polysaccharide and its peptide mimic were designed in an attempt to improve the binding affinity by increasing the entropy of binding relative to the original octapeptide mimic of the O-polysaccharide. The design was based on the X-ray crystal structures of a monoclonal antibody SYA/J6 in complex with its cognate ligands, a pentasaccharide corresponding to the S. flexneri Y O-polysaccharide and the octapeptide mimic, MDWNMHAA. Both chimeric molecules consist of a rhamnose trisaccharide linked through an alpha- or beta-thioglycosidic linkage to a MDW moiety in which the W unit has been modified. We predicted that omission of the NMHAA moiety would obviate the bound water molecules that provided complementarity with the antibody-combining site, and the conformational restriction resulting from imposition of an alpha-turn at the C-terminus of the peptide. The glycopeptides were then docked into the active site of SYA/J6 using the program autodock 3.0, and the structures were optimized. The best models obtained in each case showed that the chimeric molecules, with either an alpha- or beta-thioglycosidic linkage, might be reasonable surrogate ligands for the antibody. We report here the synthesis of the alpha-glycopeptide employing solution and solid-phase strategies. Immunochemical characterization indicated that the alpha-glycopeptide unfortunately did not inhibit binding of SYA/J6 to the S. flexneri Y lipopolysaccharide.

Immunological evidence for functional rather than structural mimicry by a Shigella flexneri Y polysaccharide-mimetic peptide.

An approach to vaccine design is the use of molecules that mimic the immunogenic element of interest. In this context, the interaction of MDWNMHAA, a peptide mimic of the Shigella flexneri Y O polysaccharide (PS), with an anti-carbohydrate monoclonal antibody, as studied previously by X-ray crystallography, suggested the presence of functional rather than structural mimicry and a bound peptide conformation that was not represented significantly in the free-ligand ensemble. The antibody response elicited by an MDWNMHAA-carrier protein (tetanus toxoid [TT]) conjugate has now been investigated in BALB/c mice. The mice were immunized following a homologous prime/boost strategy using MDWNMHAA-TT as the immunogen. The mice showed anti-peptide antibody (immunoglobulin G [IgG]) titers that increased after being boosted. High anti-lipopolysaccharide (LPS) (IgG) titers were observed after the last boost. A faster immune response, with cross-reactive titers, was observed with a peptide conjugate with 30% more copies of the peptide. The binding of anti-peptide polyclonal antibodies to LPS could be inhibited by LPS, PS, MDWNMHAA, and MDWNMHAA-bovine serum albumin, as assessed by inhibition enzyme-linked immunosorbent assay. Conversely, mice immunized with PS-TT showed IgG anti-peptide titers. These data demonstrate the cross-reactivity of the antibody response and support the hypothesis that functional, as opposed to structural, mimicry of the S. flexneri Y O PS by MDWNMHAA or the underrepresentation of the bound ligand conformation in the free-ligand ensemble does not compromise immunological cross-reactivity. Prime/boost strategies were performed with a heterologous boost of PS-TT or MDWNMHAA-TT. They led to high anti-LPS titers after only three injections, suggesting alternatives to improve the immunogenicity of the carbohydrate-mimetic peptide and confirming the antigenic mimicry.

Bioengineering of surface GD3 ganglioside for immunotargeting human melanoma cells.

N-Propionyl, N-butyryl (N-Bu), and N-benzoyl mannosamine, as precursors of sialic acid biosynthesis, were incubated with human melanoma SK-MEL-28 cells and resulted in the replacement of N-acetyl groups on the cell surface sialic acid residues, including those associated with GD3. Meanwhile, vaccines containing GD3 and modified GD3 tetrasaccharide-keyhole limpet hemocyanin conjugates were synthesized, and BALB/c mice were immunized with them together with monophosphoryl lipid A adjuvant. The GD3Bu-keyhole limpet hemocyanin conjugate raised the highest IgG titers without any cross-reactivity to unmodified GD3. Expression of GD3Bu epitopes on the surface of SK-MEL-28 cells was confirmed in vitro and in vivo by the binding of a polyclonal antiserum and monoclonal antibody (mAb) 2A, both of which specifically recognize GD3Bu, and by mass spectroscopic analysis of glycolipids extracted from cells. Following expression of GD3Bu on the surface of SK-MEL-28 cells, the cells could be lysed by mAb 2A and GD3Bu antiserum in the presence of complement. Although less effective in the control of existing large size tumors ( approximately 10 mm inner diameter) on BALB/c nu/nu mice, mAb 2A in combination with ManNBu effectively protected mice from SK-MEL-28 tumor grafting. This approach may provide a method to augment the immunogenicity of sialylated human antigens and to avoid generating an autoimmune response to them at same time.

Frequencies of lipopolysaccharide-defined epitopes in Haemophilus influenzae type b and non-typable isolates determined with monoclonal antibodies.

OBJECTIVE: To investigate the frequency of expression and stability of saccharide epitopes in 178 Haemophilus influenzae (39 type b and 138 non-typable) isolates from blood, cerebrospinal fluid, nasopharynx, pharynx, middle ear, conjunctiva, and pleural and bronchial fluid from symptomatic and asymptomatic children using five murine monoclonal antibodies (MAbs, MAHI 3, 4, 6, 8, 10) specific for the oligosaccharide moiety of the lipopolysaccharide (LPS) of H. influenzae, which recognize defined saccharide structures. METHODS: A whole bacteria enzyme immunoassay (EIA) and colony dot immunoblotting were used to determine the frequency of expression and stability of saccharide epitopes in the 178 H. influenzae isolates. RESULTS: Six main groups of strains were differentiated based on the EIA binding pattern with the MAbs: group A, reactive with all five MAbs (MAHI 3, 4, 6, 8 and 10); group B, reactive with four MAbs (MAHI 3, 6, 8 and 10); group C, reactive with three MAbs (MAHI 3, 6 and 8); group D, reactive with three MAbs (MAHI 3, 6 and 10); group E, reactive with two MAbs (MAHI 3 and 10); group F, reactive with MAb MAHI 3. Group B was the most common classification overall. None of the strains remained non-reactive. The frequencies of the binding patterns among the isolates obtained from different sources appeared to be statistically similar in most of the cases. Indications of phase variation of the LPS epitopes were observed with all the MAbs for strains obtained from all clinical sources as evaluated by colony dot immunoblotting. One of the epitopes displayed 22% phase variation, while four other epitopes were variably expressed, with about 50% on-off expression. CONCLUSIONS: This set of MAbs showed 100% reactivity among the isolates, in both EIA and colony dot immunoblotting, and allowed us to differentiate strains based on the LPS phenotype by whole bacteria EIA. Phase variation was indicated among all the isolates, independent of the source of isolation, and for all five MAbs. The LPS of isolates from different clinical sources often expressed some of the epitopes recognized by the MAbs, and most of the LPS phenotypes appeared at similar frequencies among isolates.