RESUMO
Some insoluble aluminum salts are commonly used in injectable vaccines as adjuvants to accelerate, prolong, or enhance the antigen-specific immune responses. Data from previous studies testing the nasal mucosal vaccine adjuvant activity of aluminum salts are conflicting. The present study is designed to further assess the feasibility of using aluminum salts in injectable vaccines as nasal mucosal vaccine adjuvants. Using Alhydrogel®, the international scientific standard of aluminum (oxy)hydroxide gels, and ovalbumin or 3 × M2e-HA2, a synthetic influenza virus fusion protein, as antigens, we showed in a mouse model that when dosed intranasally Alhydrogel® enables antigens adsorbed on it to induce stronger antigen-specific immune responses in both serum samples (e.g., specific IgG) and nasal and lung mucosal secretions (i.e., specific IgA) in all immunized mice, as compared with nasal immunization with the antigens alone. Rerouting insoluble aluminum salts in injectable vaccines may represent a viable approach for (nasal) mucosal vaccine adjuvant discovery.
Assuntos
Hidróxido de Alumínio/imunologia , Formação de Anticorpos/imunologia , Antígenos Virais/imunologia , Imunidade nas Mucosas , Proteínas Virais de Fusão/imunologia , Adjuvantes Imunológicos/administração & dosagem , Adjuvantes Imunológicos/química , Administração Intranasal , Adsorção , Hidróxido de Alumínio/administração & dosagem , Hidróxido de Alumínio/química , Animais , Antígenos Virais/química , Antígenos Virais/genética , Feminino , Imunoglobulina A/análise , Imunoglobulina A/biossíntese , Imunoglobulina A/imunologia , Imunoglobulina G/biossíntese , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Mucosa Nasal/imunologia , Vacinação , Proteínas Virais de Fusão/administração & dosagemRESUMO
Insoluble aluminum salts such as aluminum oxyhydroxide have been used for decades as adjuvants in human vaccines, and many vaccines contain aluminum salts as adjuvants. Aluminum salt-adjuvanted vaccines must be managed in cold-chain (2-8° C) during transport and storage, as vaccine antigens in general are too fragile to be stable in ambient temperatures, and unintentional slowing freezing causes irreversible aggregation and permanent damage to the vaccines. Previously, we reported that thin-film freeze-drying can be used to convert vaccines adjuvanted with an aluminum salt from liquid suspension into dry powder without causing particle aggregation or decreasing in immunogenicity following reconstitution. In the present study, using ovalbumin (OVA)-adsorbed Alhydrogel® (i.e. aluminum oxyhydroxide, 2% w/v) as a model vaccine, we showed that the immunogenicity of thin-film freeze-dried OVA-adsorbed Alhydrogel® vaccine powder was not significantly changed after it was exposed for an extended period of time in temperatures as high as 40° C or subjected to repeated slow freezing-and-thawing. It is expected that immunization programs can potentially benefit by integrating thin-film freeze-drying into vaccine preparations.
Assuntos
Adjuvantes Imunológicos/efeitos da radiação , Hidróxido de Alumínio/efeitos da radiação , Liofilização , Temperatura , Potência de Vacina , Vacinas/imunologia , Vacinas/efeitos da radiação , Adjuvantes Imunológicos/administração & dosagem , Hidróxido de Alumínio/administração & dosagem , Animais , Feminino , Camundongos Endogâmicos BALB C , Ovalbumina/administração & dosagem , Ovalbumina/imunologia , Ovalbumina/efeitos da radiação , Vacinas/administração & dosagemRESUMO
There has been growing interest in utilizing small interfering RNA (siRNA) specific to pro-inflammatory cytokines, such as tumor necrosis factor-α ( TNF-α), in chronic inflammation therapy. However, delivery systems that can increase the distribution of the siRNA in chronic inflammation sites after intravenous administration are needed. Herein we report that innovative functionalization of the surface of siRNA-incorporated poly (lactic-co-glycolic) acid (PLGA) nanoparticles significantly increases the delivery of the siRNA in the chronic inflammation sites in a mouse model. The TNF-α siRNA incorporated PLGA nanoparticles were prepared by the standard double emulsion method, but using stearoyl-hydrazone-polyethylene glycol 2000, a unique acid-sensitive surface active agent, as the emulsifying agent, which renders (i) the nanoparticles PEGylated and (ii) the PEGylation sheddable in low pH environment such as that in chronic inflammation sites. In a mouse model of lipopolysaccharide-induced chronic inflammation, the acid-sensitive sheddable PEGylated PLGA nanoparticles showed significantly higher accumulation or distribution in chronic inflammation sites than PLGA nanoparticles prepared with an acid-insensitive emulsifying agent (i.e., stearoyl-amide-polyethylene glycol 2000) and significantly increased the distribution of the TNF-α siRNA incorporated into the nanoparticles in inflamed mouse foot.
RESUMO
Aluminum salts such as aluminum oxyhydroxide and aluminum hydroxyphosphate are commonly used human vaccine adjuvants. In an effort to improve the adjuvant activity of aluminum salts, we previously showed that the adjuvant activity of aluminum oxyhydroxide nanoparticles is significantly more potent than that of aluminum oxyhydroxide microparticles. The present study was designed to (i) understand the mechanism underlying the potent adjuvant activity of aluminum oxyhydroxide nanoparticles, relative to microparticles, and (ii) to test whether aluminum hydroxyphosphate nanoparticles have a more potent adjuvant activity than aluminum hydroxyphosphate microparticles as well. In human THP-1 myeloid cells, wild-type and NLRP3-deficient, both aluminum oxyhydroxide nanoparticles and microparticles stimulate the secretion of proinflammatory cytokine IL-1ß by activating NLRP3 inflammasome, although aluminum oxyhydroxide nanoparticles are more potent than microparticles, likely related to the higher uptake of the nanoparticles by the THP-1 cells than the microparticles. Aluminum hydroxyphosphate nanoparticles also have a more potent adjuvant activity than microparticles in helping a model antigen lysozyme to stimulate specific antibody response, again likely related to their stronger ability to activate the NLRP3 inflammasome.
Assuntos
Adjuvantes Imunológicos/farmacologia , Compostos de Alumínio/farmacologia , Células Mieloides/imunologia , Nanopartículas/química , Animais , Formação de Anticorpos , Linhagem Celular , Feminino , Humanos , Inflamassomos/imunologia , Interleucina-1beta/imunologia , Camundongos Endogâmicos BALB C , Proteína 3 que Contém Domínio de Pirina da Família NLR/imunologiaRESUMO
Botulinum neurotoxin (BoNT) is a lethal neurotoxin, for which there is currently not an approved vaccine. Recent efforts in developing vaccine candidates against botulism have been directed at the heavy chain fragment of BoNT, because antibodies against this region have been shown to prevent BoNT from binding to its receptor and thus to nerve cell surface, offering protection against BoNT intoxication. In the present study, it was shown that immunization with plasmid DNA that encodes the 50 KDa C-terminal fragment of the heavy chain of BoNT serotype C (i.e., BoNT/C-Hc50) and is carried by cationic poly (lactic-co-glycolic) acid (PLGA) nanoparticles induces stronger BoNT/C-specific antibody responses, as compared to immunization with the plasmid alone. Importantly, the antibodies have BoNT/C-neutralizing activity, protecting the immunized mice from a lethal dose of BoNT/C challenge. A plasmid DNA vaccine encoding the Hc50 fragments of BoNT serotypes that cause human botulism may represent a viable vaccine candidate for protecting against botulinum neurotoxin intoxication.
Assuntos
Anticorpos Antibacterianos/biossíntese , Anticorpos Neutralizantes/biossíntese , Toxinas Botulínicas/imunologia , Ácido Láctico , Nanopartículas , Ácido Poliglicólico , Vacinas de DNA/imunologia , Animais , Anticorpos Antibacterianos/sangue , Anticorpos Antibacterianos/imunologia , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/imunologia , Vacinas Bacterianas/imunologia , Toxinas Botulínicas/genética , Botulismo/prevenção & controle , Humanos , Imunização , Imunoglobulina G/sangue , Injeções Intramusculares , Camundongos , Plasmídeos , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Sorogrupo , Vacinas de DNA/administração & dosagem , Vacinas de DNA/genéticaRESUMO
Hepatitis C virus (HCV) is a positive-strand RNA virus within the Flaviviridae family. The viral "spike" of HCV is formed by two envelope glycoproteins, E1 and E2, which together mediate viral entry by engaging host receptors and undergoing conformational changes to facilitate membrane fusion. While E2 can be readily produced in the absence of E1, E1 cannot be expressed without E2 and few reagents, including monoclonal antibodies (mAbs), are available for study of this essential HCV glycoprotein. A human mAb to E1, IGH526, was previously reported to cross-neutralize different HCV isolates, and therefore, we sought to further characterize the IGH526 neutralizing epitope to obtain information for vaccine design. We found that mAb IGH526 bound to a discontinuous epitope, but with a major component corresponding to E1 residues 314-324. The crystal structure of IGH526 Fab with this E1 glycopeptide at 1.75Å resolution revealed that the antibody binds to one face of an α-helical peptide. Single mutations on the helix substantially lowered IGH526 binding but did not affect neutralization, indicating either that multiple mutations are required or that additional regions are recognized by the antibody in the context of the membrane-associated envelope oligomer. Molecular dynamics simulations indicate that the free peptide is flexible in solution, suggesting that it requires stabilization for use as a candidate vaccine immunogen.
Assuntos
Epitopos/ultraestrutura , Anticorpos Anti-Hepatite C/imunologia , Fragmentos Fab das Imunoglobulinas/imunologia , Proteínas do Envelope Viral/imunologia , Proteínas do Envelope Viral/ultraestrutura , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Sítios de Ligação de Anticorpos , Linhagem Celular , Cristalografia por Raios X , Mapeamento de Epitopos , Epitopos/imunologia , Células HEK293 , Hepacivirus/imunologia , Humanos , Simulação de Dinâmica MolecularRESUMO
Many currently licensed and commercially available human vaccines contain aluminum salts as vaccine adjuvants. A major limitation with these vaccines is that they must not be exposed to freezing temperatures during transport or storage such that the liquid vaccine freezes, because freezing causes irreversible coagulation that damages the vaccines (e.g., loss of efficacy). Therefore, vaccines that contain aluminum salts as adjuvants are formulated as liquid suspensions and are required to be kept in cold chain (2-8°C) during transport and storage. Formulating vaccines adjuvanted with aluminum salts into dry powder that can be readily reconstituted before injection may address this limitation. Spray freeze-drying of vaccines with low concentrations of aluminum salts and high concentrations of trehalose alone, or a mixture of sugars and amino acids, as excipients can convert vaccines containing aluminum salts into dry powder, but fails to preserve the particle size and/or immunogenicity of the vaccines. In the present study, using ovalbumin as a model antigen adsorbed onto aluminum hydroxide or aluminum phosphate, a commercially available tetanus toxoid vaccine adjuvanted with potassium alum, a human hepatitis B vaccine adjuvanted with aluminum hydroxide, and a human papillomavirus vaccine adjuvanted with aluminum hydroxyphosphate sulfate, it was shown that vaccines containing a relatively high concentration of aluminum salts (i.e., up to ~1%, w/v, of aluminum hydroxide) can be converted into a dry powder by thin-film freezing followed by removal of the frozen solvent by lyophilization while using low levels of trehalose (i.e., as low as 2% w/v) as an excipient. Importantly, the thin-film freeze-drying process did not cause particle aggregation, nor decreased the immunogenicity of the vaccines. Moreover, repeated freezing-and-thawing of the dry vaccine powder did not cause aggregation. Thin-film freeze-drying is a viable platform technology to produce dry powders of vaccines that contain aluminum salts.
Assuntos
Adjuvantes Farmacêuticos/química , Compostos de Alúmen/química , Vacinas contra Hepatite B , Tecnologia Farmacêutica/métodos , Toxoide Tetânico , Hidróxido de Alumínio/química , Animais , Varredura Diferencial de Calorimetria , Composição de Medicamentos , Estabilidade de Medicamentos , Feminino , Liofilização , Vacinas contra Hepatite B/química , Vacinas contra Hepatite B/imunologia , Imunoglobulina G/sangue , Camundongos Endogâmicos BALB C , Microscopia Eletrônica de Varredura , Ovalbumina/imunologia , Tamanho da Partícula , Fosfatos/química , Pós , Toxoide Tetânico/química , Toxoide Tetânico/imunologiaRESUMO
Dioxides of methylthiourea (methylaminoiminomethanesulfinic acid, MAIMSA) and dimethylthiourea (dimethylaminoiminomethanesulfinic acid, DMAIMSA) were synthesized and, together with thiourea dioxide (aminoiminomethanesulfinic acid, AIMSA), were studied with respect to their decompositions and hydrolyses in basic aqueous media. All three were stable in acidic media and existed as zwitterions with the positive charge spread out on the 4-electron 3-center N-C-N skeleton and the negative charge delocalized over the two oxygen atoms. All three are characterized by long and weak C-S bonds that are easily cleaved in polar solvents through a nucleophilic attack on the positively disposed carbon center, followed by cleavage of the C-S bond. The sulfur moiety leaving groups are highly unstable, reducing, and rapidly oxidized to S(IV) as hydrogen sulfite in the presence of oxidant. In aerobic conditions, molecular oxygen is a sufficient and efficient oxidant that can oxidize, at diffusion-controlled limits, the highly reducing sulfur species in one-electron steps, thus opening up a cascade of possibly genotoxic reactive oxygen species, commencing with the superoxide anion radical. Radical formation in these decompositions was confirmed by electron paramagnetic resonance techniques. In strongly basic media, decomposition of the dioxides to yield sulfoxylate (SO2(2-), HSO2(-)) is irreversible and, in anaerobic environments, will disproportionate to yield more stable sulfur species from HS(-) to SO4(2-). Decomposition products were dependent on concentrations of molecular oxygen. Solutions open to the atmosphere, with availability to excess oxygen, gave the urea analogue of the thiourea and sulfate, while in limited oxygen conditions hydrogen sulfite and other mixed oxidation states sulfur oxoanions are obtained. DMAIMSA has the longest C-S bond at 0.188 nm and was the most reactive. MAIMSA, with the shortest at 0.186 nm, was the least reactive. Electrospray ionization-mass spectrometry data managed to detect all of the formerly postulated intermediates.
Assuntos
Tioureia/análogos & derivados , Concentração de Íons de Hidrogênio , Hidrólise , Cinética , Oxigênio/química , Tioureia/químicaRESUMO
UNLABELLED: The hepatitis C virus (HCV) envelope glycoprotein E1E2 complex is a candidate vaccine antigen. Previous immunization studies of E1E2 have yielded various results on its ability to induce virus-neutralizing antibodies in animal models and humans. The murine model has become a vital tool for HCV research owing to the development of humanized mice susceptible to HCV infection. In this study, we investigated the antibody responses of mice immunized with E1E2 and a novel soluble form of E1E2 (sE1E2) by a DNA prime and protein boost strategy. The results showed that sE1E2 elicited higher antibody titers and a greater breadth of reactivity than the wild-type cell-associated E1E2. However, immune sera elicited by either immunogen were only weakly neutralizing. In order to understand the contrasting results of binding and serum neutralizing activities, epitopes targeted by the polyclonal antibody responses were mapped and monoclonal antibodies (MAbs) were generated. The results showed that the majority of serum antibodies were directed to the E1 region 211 to 250 and the E2 regions 421 to 469, 512 to 539, 568 to 609, and 638 to 651, instead of the well-known immunodominant E2 hypervariable region 1 (HVR1). Unexpectedly, in MAb analysis, â¼ 12% of MAbs isolated were specific to the conserved E2 antigenic site 412 to 423, and 85% of them cross-neutralized multiple HCV isolates. The epitopes recognized by these MAbs are similar but distinct from the previously reported HCV1 and AP33 broadly neutralizing epitopes. In conclusion, E1E2 can prime B cells specific to conserved neutralizing epitopes, but the levels of serum neutralizing antibodies elicited are insufficient for effective virus neutralization. The sE1E2 constructs described in this study can be a useful template for rational antigen engineering. IMPORTANCE: Hepatitis C virus infects 2 to 3% of the world's population and is a leading cause of liver failures and the need for liver transplantation. The virus envelope glycoprotein complex E1E2 produced by detergent extraction of cells overexpressing the protein was evaluated in a phase I clinical trial but failed to induce neutralizing antibodies in most subjects. In this study, we designed a novel form of E1E2 which is secreted from cells and is soluble and compared it to wild-type E1E2 by DNA immunization of mice. The results showed that this new E1E2 is more immunogenic than wild-type E1E2. Detailed mapping of the antibody responses revealed that antibodies to the conserved E2 antigenic site 412 to 423 were elicited but the serum concentrations were too low to neutralize the virus effectively. This soluble E1E2 provides a new reagent for studying HCV and for rational vaccine design.
Assuntos
Hepacivirus/imunologia , Anticorpos Anti-Hepatite C/imunologia , Hepatite C/imunologia , Proteínas do Envelope Viral/imunologia , Motivos de Aminoácidos , Animais , Mapeamento de Epitopos , Feminino , Hepacivirus/química , Hepacivirus/genética , Hepatite C/virologia , Humanos , Imunização , Camundongos , Camundongos Endogâmicos BALB C , Testes de Neutralização , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genéticaRESUMO
Accurate and in-depth mapping of antibody responses is of great value in vaccine and antibody research. Using hepatitis C virus (HCV) as a model, we developed an affordable and high-throughput microarray-based assay for mapping antibody specificities to continuous antibody epitopes of HCV at high resolution. Important parameters in the chemistry for conjugating peptides/antigens to the array surface, the array layout, fluorophore choice and the methods for data analysis were investigated. Microscopic glass slide pre-coated with N-Hydroxysuccinimide (NHS)-ester (Slide H) was the preferred surface for conjugation of aminooxy-tagged peptides. This combination provides a simple chemical means to orient the peptides to the conjugation surface via an orthogonal covalent linkage at the N- or C-terminus of each peptide. The addition of polyvinyl alcohol to printing buffer gave uniform spot morphology and improved sensitivity and specificity of binding signals. Libraries of overlapping peptides covering the HCV E1 and E2 glycoprotein polypeptides (15-mer, 10 amino acids overlap) of 6 major HCV genotypes and the entire polypeptide sequence of the prototypic strain H77 were synthesized and printed in quadruplets in the assays. The utility of the peptide arrays was confirmed using HCV monoclonal antibodies (mAbs) specific to known continuous epitopes and immune sera of rabbits immunized with HCV antigens. The methods developed here can be easily adapted to studying antibody responses to antigens relevant in vaccine and autoimmune research.
Assuntos
Antígenos Virais/imunologia , Epitopos , Hepacivirus/imunologia , Anticorpos Anti-Hepatite C/sangue , Análise Serial de Proteínas , Proteínas do Envelope Viral/imunologia , Animais , Especificidade de Anticorpos , Biomarcadores/sangue , Soluções Tampão , Ésteres/química , Ensaios de Triagem em Larga Escala , Biblioteca de Peptídeos , Álcool de Polivinil/química , Coelhos , Reprodutibilidade dos Testes , Succinimidas/químicaRESUMO
Diisocyanates (dNCOs) are highly reactive low molecular weight chemicals used in the manufacture of polyurethane products and are the most commonly reported cause of occupational asthma. Mechanistic disease studies and development of biomonitoring and research tools, such as monoclonal antibodies (mAbs) have been hampered by dNCOs' ability to self-polymerize and to cross-link biomolecules. Toluene diisocyanate (TDI)-specific monoclonal antibodies (mAbs), with potential use in immunoassays for exposure and biomarker assessments, were produced and reactivities characterized against mono- and diisocyanate and dithioisocyanate protein conjugates. In general, TDI reactive mAbs displayed stronger recognition of isocyanate haptenated proteins when the NCO was in the ortho position relative to the tolyl group, and were capable of discriminating between isocyanate and isothiocyanate conjugates and between aromatic and aliphatic dNCOs. Preliminary studies using TDI vapor exposed cells suggest potential utility of these mAbs for both research and biomonitoring.
Assuntos
Anticorpos Monoclonais/imunologia , Haptenos/imunologia , Proteínas/imunologia , Tolueno 2,4-Di-Isocianato/imunologia , Animais , Anticorpos Monoclonais/metabolismo , Afinidade de Anticorpos/imunologia , Especificidade de Anticorpos/imunologia , Western Blotting , Linhagem Celular , Linhagem Celular Tumoral , Ensaio de Imunoadsorção Enzimática , Feminino , Haptenos/química , Humanos , Hibridomas/imunologia , Hibridomas/metabolismo , Cinética , Camundongos , Camundongos Endogâmicos BALB C , Ligação Proteica , Proteínas/química , Tolueno 2,4-Di-Isocianato/químicaRESUMO
Consumer and medical products can contain leachable chemical allergens which can cause skin sensitization. Recent efforts have been directed at the development of non-animal based tests such as in vitro cell activation assays for the identification of skin sensitizers. Prohapten identification by in vitro assays is still problematic due to the lack of prohapten bioactivation. The present study evaluated the effect of hapten and prohapten exposure on cell surface markers expression (CD86, CD54 and CD40) in the human monocytic leukemia, THP-1, cell line. Upregulation of activation and costimulatory markers are key events in the allergic sensitization process and have been reported to serve as indicators of skin sensitization. Cells were exposed to the prohaptens benzo(a)pyrene (BaP), 7,12-dimethylbenz(a)anthracene (DMBA), carvone oxime (COx), cinnamic alcohol (CA) and isoeugenol (IEG) at concentrations ranging from 1 to 10 µM for 24 and 48 h. The direct-binding haptens dinitrochlorobenzene (DNCB), benzoquinone (BQ), hydroxylethyl acrylate (HEA) and benzylbromide (BB) were used as positive controls. Cells were also exposed to the irritants sodium dodecyl sulfate (SDS) and sulfanilamide (SFA). Bioactivation of prohaptens was achieved by adding aroclor-induced rat liver microsomes (S9) to the cell cultures. Consistent upregulation of surface expressions of CD86, CD54 (ICAM-1) and CD40 was observed in THP-1 cells treated with direct-acting haptens (±S9) or prohapten (+S9). Upregulation of these markers was not observed after exposure to skin irritants or prohaptens in the absence of exogenously added S9. In conclusion, modification of in vitro cell culture assays to include co-incubation with microsomes enhances identification of prohaptens and allows them to be clearly distinguished from direct-binding haptens.
Assuntos
9,10-Dimetil-1,2-benzantraceno/toxicidade , Benzo(a)pireno/toxicidade , Eugenol/análogos & derivados , Haptenos/toxicidade , Microssomos Hepáticos/efeitos dos fármacos , Propanóis/toxicidade , 9,10-Dimetil-1,2-benzantraceno/classificação , 9,10-Dimetil-1,2-benzantraceno/metabolismo , Animais , Benzo(a)pireno/classificação , Benzo(a)pireno/metabolismo , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Eugenol/classificação , Eugenol/metabolismo , Eugenol/toxicidade , Haptenos/classificação , Haptenos/metabolismo , Humanos , Microssomos Hepáticos/metabolismo , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Propanóis/classificação , Propanóis/metabolismo , RatosRESUMO
Toluene diisocyanate (TDI) is an industrially important polymer cross-linker used in the production of polyurethane. Workplace exposure to TDI and other diisocyanates is reported to be a leading cause of low molecular weight-induced occupational asthma (OA). Currently we have a limited understanding of the pathogenesis of OA. Monoclonal antibodies (MAbs) that recognize TDI bound proteins would be valuable tools/reagents, both in exposure monitoring and in TDI-induced asthma research. We sought to develop toluene diisocyanate (TDI)-specific MAbs for potential use in the development of standardized immunoassays for exposure and biomarker assessments. Mice were exposed 4 h/day for 12 consecutive weekdays to 50 ppb, 2,4;2,6 TDI vapor (80/20 mixture). Splenocytes were isolated 24 h after the last exposure for hybridoma production. Hybridomas were screened in a solid-phase indirect enzyme-linked immunosorbent assay (ELISA) against a 2,4 TDI-human serum albumin (2,4 TDI-HSA) protein conjugate. Three hybridomas producing 2,4 TDI-HSA reactive IgM MAbs were obtained. The properties of these MAbs (isotype and reactivity to various protein-isocyanate conjugate epitopes) were characterized using ELISA, dot blot, and Western blot analyses. Western blot analyses demonstrated that some TDI conjugates form inter- and intra-molecular links, resulting in multimers and a change in the electrophoretic mobility of the conjugate. These antibodies may be useful tools for the isolation of endogenous diisocyanate-modified proteins after natural or experimental exposures and for characterization of the toxicity of specific dNCOs.
Assuntos
Anticorpos Monoclonais/imunologia , Haptenos/imunologia , Isocianatos/imunologia , Tolueno 2,4-Di-Isocianato/imunologia , Administração por Inalação , Animais , Anticorpos Monoclonais/classificação , Especificidade de Anticorpos , Asma/induzido quimicamente , Asma/diagnóstico , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Hibridomas , Immunoblotting , Imunoglobulina M , Camundongos , Camundongos Endogâmicos C57BL , Albumina Sérica , Tolueno 2,4-Di-Isocianato/administração & dosagem , Tolueno 2,4-Di-Isocianato/toxicidadeRESUMO
The need for alternatives to animal-based skin sensitization testing has spurred research on the use of in vitro, in silico, and in chemico methods. Glutathione and other select peptides have been used to determine the reactivity of electrophilic allergens to nucleophiles, but these methods are inadequate to accurately measure rapid kinetics observed with many chemical sensitizers. A kinetic spectrophotometric assay involving the reactivity of electrophilic sensitizers to nitrobenzenethiol was evaluated. Stopped-flow techniques and conventional UV spectrophotometric measurements enabled the determination of reaction rates with half-lives ranging from 0.4 ms (benzoquinone) to 46.2 s (ethyl acrylate). Rate constants were measured for seven extreme, five strong, seven moderate, and four weak/nonsensitizers. Seventeen out of the 23 tested chemicals were pseudo-first order, and three were second order. In three out of the 23 chemicals, deviations from first and second order were apparent where the chemicals exhibited complex kinetics whose rates are mixed order. The reaction rates of the electrophiles correlated positively with their EC3 values within the same mechanistic domain. Nonsensitizers such as benzaldehyde, sodium lauryl sulfate, and benzocaine did not react with nitrobenzenethiol. Cyclic anhydrides, select diones, and aromatic aldehydes proved to be false negatives in this assay. The findings from this simple and rapid absorbance model show that for the same mechanistic domain, skin sensitization is driven mainly by electrophilic reactivity. This simple, rapid, and inexpensive absorbance-based method has great potential for use as a preliminary screening tool for skin allergens.
Assuntos
Alérgenos/química , Alternativas aos Testes com Animais , Testes Cutâneos , Compostos de Sulfidrila/química , Alérgenos/toxicidade , Animais , Dermatite Alérgica de Contato/patologia , Meia-Vida , Cinética , Espectrofotometria Ultravioleta , Compostos de Sulfidrila/toxicidadeRESUMO
Diisocyanates are highly reactive chemical compounds widely used in the manufacture of polyurethanes. Although diisocyanates have been identified as causative agents of allergic respiratory diseases, the specific mechanism by which these diseases occur is largely unknown. To better understand the chemical species produced when isocyanates are reacted with model peptides, tandem mass spectrometry was employed to unambiguously identify the binding site of four commercially-relevant isocyanates on model peptides. In each case, the isocyanates react preferentially with the N-terminus of the peptide. No evidence of side-chain/isocyanate adduct formation exclusive of the N-terminus was observed. However, significant intra-molecular diisocyanate crosslinking was observed between the N-terminal amine and a side-chain amine of arginine, when Arg was located within two residues of the N-terminus. Addition of multiple isocyanates to the peptide occurs via polymerization of the isocyanate at the N-terminus, rather than via addition of multiple isocyanate molecules to varied residues within the peptide. The direct observation of isocyanate binding to the N-terminus of peptides under these experimental conditions is in good agreement with previous studies on the relative reaction rate of isocyanate with amino acid functional groups.
Assuntos
Isocianatos/análise , Isocianatos/química , Peptídeos/análise , Peptídeos/química , Mapeamento de Interação de Proteínas/métodos , Espectrometria de Massas por Ionização por Electrospray/métodos , Sítios de Ligação , Ligação ProteicaRESUMO
The effect of treatment with either oxamniquine or praziquantel on S.mansoni specific IFN-gamma, IL-4, IL-5 and IL-10 was compared on PBMC which were collected pretreatment, 6 and 18 weeks post treatment. Using sandwich ELISA on the supernatants harvested from the PBMC stimulation by crude S. mansoni SEA and SWAP antigens after 5 days the levels of PBMC proliferation and cytokine production were similar according to treatment with either praziquantel or oxamniquine. Before treatment, infected groups showed low ratios, of IL-4:IFN-gamma, IL-5:IFNgamma and IL-10:IFN-gamma, indicating that IFN-gamma was high in the infected individuals. The general increase in immuno-modulation was observed post-treatment with elevated immune reactivity and cytokine production in both treatment groups. Treatment induced significant increases in levels of IL-4 (p < 0.05), IL-5 (p < 0.0001) and IL-10 (p < 0.05) cytokines 6 and 18 weeks after treatment. There were no significant differences in the increase in IL-4, IL-5 and IL-10 between children treated with praziquantel or oxamniquine. Pre-treatment IFN-gamma and IL-5 levels were positively correlated with infection (p < 0.001), while post treatment IL-4 cytokine levels were negatively correlated with baseline infection status (p < 0.001). The results suggest that treatment-induced immune responses are similar for both common anti-schistosome drugs praziquantel or oxamniquine having similar and immunizing effect.
Assuntos
Citocinas/efeitos dos fármacos , Oxamniquine/farmacologia , Praziquantel/farmacologia , Schistosoma mansoni/isolamento & purificação , Esquistossomose mansoni/diagnóstico , Esquistossomose mansoni/tratamento farmacológico , Adolescente , Animais , Antígenos de Helmintos/imunologia , Criança , Estudos de Coortes , Citocinas/imunologia , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Leucócitos Mononucleares/imunologia , Masculino , Oxamniquine/uso terapêutico , Praziquantel/uso terapêutico , Schistosoma mansoni/imunologia , Resultado do Tratamento , Zimbábue/epidemiologiaRESUMO
The oxidation of trimethylthiourea (TMTU) by acidic bromate has been studied. The reaction mimics the dynamics observed in the oxidation of unsubstituted thiourea by bromate with an induction period before formation of bromine. The stoichiometry of the reaction was determined to be 4:3, thus 4BrO(3)- + 3R(1)R(2)C=S+ 3H(2)O --> 4Br- + 3R(1)R(2)C=O + 3SO(4)(2-) + 6H+. This substituted thiourea is oxidized at a much faster rate than the unsubstituted thiourea. The oxidation mechanism of TMTU involves initial oxidations through sulfenic and sulfinic acids. At the sulfinic acid stage, the major oxidation pathway is through the cleavage of the C-S bond to form a reducing sulfur leaving group, which is easily oxidized to sulfate. The minor pathway through the sulfonic acid produces a very stable intermediate that is oxidized only very slowly to urea and sulfate. The direct reaction of aqueous bromine with TMTU was faster than reactions that form bromine, with a bimolecular rate constant of (1.50 +/- 0.04) x 10(2) M(-1) s(-1). This rapid reaction ensured that no oligooscillatory bromine formation was observed. The oxidation of TMTU was modeled by a simple reaction scheme containing 20 reactions.