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Benzoquinone (BQ) is an extremely potent electrophilic contact allergen that haptenates endogenous proteins through Michael addition (MA). It is also hypothesized that BQ may haptenate proteins via free radical formation. The objective of this study was to assess the inductive effects (activating and deactivating) of substituents on BQ reactivity and the mechanistic pathway of covalent binding to nucleophilic thiols. The BQ binding by Cys34 on human serum albumin was studied, and for reactivity studies, nitrobenzenethiol (NBT) was used as a surrogate for protein binding of the BQ and benzoquinone derivatives (BQD). Stopped flow techniques were used to determine pseudo-first order rate constants (k) of methyl-, t-butyl-, and chlorine-substituted BQD reactions with NBT, whereas electron pair resonance (EPR) studies were performed to investigate the possible free radical mediated binding mechanism of BQD. Characterization of adducts was performed using mass spectrometry (MS) and nuclear magnetic resonance spectroscopy (NMR). The rate constant values demonstrated the chlorine substituted (activated) BQD to be more reactive toward NBT, than the methyl and t-butyl-substituted (deactivated) BQD, and this correlated with the respective EPR intensities. The EPR signal, however, was quenched in the presence of NBT suggesting MA as the dominant reaction pathway. MS and NMR results confirmed adduct formation to be a result of MA of NBT onto the BQ ring with vinylic substitution also occurring for chlorine-substituted derivatives. The binding positions on BQ and NBT/BQD stoichiometric ratios were affected by whether the inductive effects of the substituents on the ring were positive or negative. The reactivity of BQ and BQD is discussed in terms of the potential relationship to allergenic potency. Hammett and Taft (HT) constants were then used to estimate the influence of these substituents on chemical reactivity. HT values demonstrated chlorine substituted BQD to be more reactive than methyl substituted BQD. BQ and BQD dermal allergenicity, as evaluated in the murine local lymph node assay, (LLNA) was consistent with that predicted by reactivity and HT parameters. These results demonstrate the effect of substituents on BQ reactivity and dermal allergic sensitization, and suggest the potential utility of chemical reactivity data and HT values for electrophilic allergen identification and potency ranking.

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Thesis (MTech (Biomedical Technology))--Cape Technikon, 2000. / Professionals in a healthcare setting use latex gloves on a daily basis, primarily to prevent transmission of microbial and viral organisms to and from patients and specimens. Repeated exposure to latex proteins (through direct skin contact or mucous membrane absorption) leads to the formation of circulating latex-specific antibodies and increases the risk of sensitisation. Among all known risk groups, healthcare workers have the highest risk to develop latex hypersensitivity. Early detection of antibodies or predisposing factors (e.g. atopy or impaired skin barrier function), could assist in the identification and management of risk groups and limit possible sensitisation. An experimental group with high occupational latex exposure is compared to a control group with low or no occupational latex exposure at Tygerberg Hospital, Cape Town. A questionnaire was completed by all subjects to obtain a thorough history of past and present latex exposure and to identify other risk factors. A complete physical examination was done to evaluate clinical signs and symptoms of risk factors and latex hypersensitivity. Atopy was evaluated by means of the United Kingdom's Diagnostic Criteria for Atopy, personal and lor family history of atopy, haematogram and total serum IgE analyses. Latex-specific IgE antibodies were measured immunometrically. Skin prick tests were performed on subjects with negative in vitro results, but with predefined clinical symptoms suggestive of latex hypersensitivity. An

Wong Ho Wan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 140-153). / Abstracts in English and Chinese. / Thesis Committee --- p.I / Statement --- p.II / Acknowledgements --- p.III / Abstract --- p.IV / 摘要 --- p.VI / List of Tables --- p.VIII / List of Figures --- p.IX / List of Abbreviations --- p.XI / Table of Contents --- p.XIII / Chapter 1 --- General introduction --- p.1 / Chapter 2 --- Literature reviews --- p.4 / Chapter 2.1 --- LRP and winged bean --- p.4 / Chapter 2.1.1 --- Nutritional values of crop plants --- p.4 / Chapter 2.1.2 --- Lysine-rich protein (LRP) --- p.7 / Chapter 2.1.2.1 --- Identification of lysine-rich protein (LRP) --- p.7 / Chapter 2.1.2.2 --- Cloning cDNA for WBLRP --- p.7 / Chapter 2.1.2.3 --- Transgenic Expression of LRP in other plants --- p.8 / Chapter 2.1.3 --- Unknowns remained --- p.8 / Chapter 2.2 --- Food allergy and gastro-immunity --- p.10 / Chapter 2.2.1 --- What is allergy? 一 A brief introduction --- p.10 / Chapter 2.2.2 --- Food allergy and its symptoms --- p.12 / Chapter 2.2.3 --- Gastrointestinal immunity --- p.13 / Chapter 2.2.4 --- Possible mechanism of food allergy --- p.16 / Chapter 2.2.5 --- Available tests and limitations --- p.18 / Chapter 2.2.6 --- Radioallergosorbent test (RAST) --- p.19 / Chapter 2.2.7 --- Digestibility test --- p.20 / Chapter 2.2.8 --- Betv-1 Allergen Family --- p.21 / Proteins --- p.23 / Chapter 2.3 --- Pathogenesis-related proteins --- p.23 / Chapter 2.3.1 --- Defense-related proteins and pathogenesis-related proteins (PRs) --- p.23 / Chapter 2.3.2 --- Class 10 PR proteins (PR-10s) --- p.25 / Chapter 2.3.3 --- The expression patterns of PR-10s --- p.27 / Chapter 2.3.3.1 --- Pathogens-induced and signal-induced expression --- p.27 / Chapter 2.3.3.2 --- Spatially- and developmentally-regulated expression --- p.28 / Chapter 2.3.3.3 --- Other induction patterns --- p.29 / Chapter 2.3.4 --- Functions ofPR-10s --- p.30 / Chapter 2.4 --- Development of hypotheses and experiments --- p.32 / Chapter 3 --- Materials and methods --- p.36 / Chapter 3.1 --- Introduction --- p.36 / Chapter 3.2 --- Materials --- p.38 / Chapter 3.2.1 --- Chemicals --- p.38 / Chapter 3.2.2 --- Apparatus and commercial kits --- p.39 / Chapter 3.2.3 --- Vectors and bacterial strains --- p.39 / Chapter 3.2.4 --- Plant and animal materials --- p.40 / Chapter 3.2.5 --- Computer software --- p.40 / Chapter 3.3 --- Purification of LRP --- p.41 / Chapter 3.3.1 --- Purification of LRP from winged bean --- p.41 / Chapter 3.3.1.1 --- Extraction of total protein --- p.41 / Chapter 3.3.1.2 --- Differential pI precipitation --- p.41 / Chapter 3.3.1.3 --- Determination of the pI point of LRP --- p.42 / Chapter 3.3.1.4 --- Native tricine-PAGE and gel elution --- p.42 / Chapter 3.3.2 --- Purification from E. coli --- p.45 / Chapter 3.3.2.1 --- Construction of pET vector expressing recombinant LRP (rLRP) --- p.45 / Chapter 3.3.2.2 --- Expression of rLRP --- p.50 / Chapter 3.3.2.3 --- Purification by gel electrophoresis and gel band elution --- p.50 / Chapter 3.4 --- Anti-serum production --- p.52 / Chapter 3.5 --- Allergy tests --- p.53 / Chapter 3.5.1 --- Pepsin digestion --- p.53 / Chapter 3.5.1.1 --- Determination of optimal concentration of pepsin --- p.53 / Chapter 3.5.1.2 --- Pepsin digestion of allergenic and non-allergenic model proteins --- p.55 / Chapter 3.5.1.3 --- Pepsin digestion of LRP and immunodetection --- p.55 / Chapter 3.5.2 --- Trypsin digestion --- p.56 / Chapter 3.5.2.1 --- Determination of optimal trypsin concentration --- p.56 / Chapter 3.5.2.2 --- Trypsin digestion of allergenic and non-allergenic model proteins --- p.57 / Chapter 3.5.2.3 --- Trypsin digestion of LRP and immuno-detection --- p.57 / Chapter 3.5.3 --- Pepsin and trypsin digestion --- p.58 / Chapter 3.5.3.1 --- Digestions of allergenic model proteins --- p.58 / Chapter 3.5.3.2 --- Digestion of LRP --- p.58 / Chapter 3.5.4 --- IgE binding tests --- p.58 / Chapter 3.6 --- Physiology studies --- p.59 / Chapter 3.6.1 --- Preparation for the studies --- p.59 / Chapter 3.6.1.1 --- Growing winged bean in the field --- p.59 / Chapter 3.6.1.2 --- Growing winged bean in sterile conditions --- p.60 / Chapter 3.6.1.3 --- Production ofLRP-cDNA probe --- p.60 / Chapter 3.6.2 --- Detecting the expression of LRP in winged bean --- p.61 / Chapter 3.6.2.1 --- RNA extraction --- p.61 / Chapter 3.6.2.2 --- RT-PCR and DNA sequencing --- p.62 / Chapter 3.6.2.3 --- RNA electrophoresis and northern blot analysis --- p.63 / Chapter 3.6.2.4 --- Protein extraction --- p.63 / Chapter 3.6.2.5 --- Western blot and immuno-detection --- p.63 / Chapter 3.6.3 --- Expression of LRP in germinating winged bean seeds --- p.64 / Chapter 3.6.3.1 --- Seed germination --- p.64 / Chapter 3.6.3.2 --- Detection of LRP in germinating seeds --- p.64 / Chapter 3.6.4 --- RNase activity test --- p.65 / Chapter 4 --- Results --- p.67 / Chapter 4.1 --- Purification of LRP --- p.67 / Chapter 4.1.1 --- Purification from winged bean --- p.67 / Chapter 4.1.1.1 --- Identification of pI point of LRP --- p.67 / Chapter 4.1.1.2 --- Native tricine PAGE and gel elution --- p.70 / Chapter 4.1.2 --- Purification from E. coli --- p.71 / Chapter 4.1.2.1 --- Construction of pET-LRP vector --- p.71 / Chapter 4.1.2.2 --- Expression of rLRP and gel purification --- p.74 / Chapter 4.2 --- Antiserum production --- p.76 / Chapter 4.3 --- Allergy tests --- p.81 / Chapter 4.3.1 --- Pepsin digestion --- p.81 / Chapter 4.3.2 --- Trypsin digestion --- p.89 / Chapter 4.3.3 --- Pepsin and trypsin digestion --- p.96 / Chapter 4.3.4 --- Human serum IgE binding test --- p.104 / Chapter 4.4 --- Physiological studies --- p.105 / Chapter 4.4.1 --- Samples preparation --- p.105 / Chapter 4.4.2 --- RT-PCR and DNA sequencing --- p.105 / Chapter 4.4.3 --- Expression profile of WBLRP in winged bean somatic organs --- p.108 / Chapter 4.4.4 --- Expression profile ofWBLRP in winged bean flower --- p.111 / Chapter 4.4.5 --- Expression profile ofWBLRP in winged bean maturing seeds --- p.114 / Chapter 4.4.6 --- Expression profile of WBLRP gene in winged bean germinating seeds --- p.117 / Chapter 4.4.7 --- Functional assay of LRP --- p.121 / Chapter 5 --- Discussion --- p.124 / Chapter 5.1 --- LRP purification and antibody production --- p.124 / Chapter 5.2 --- Allergy tests --- p.125 / Chapter 5.3 --- Expression of LRP in WB --- p.131 / Chapter 5.4 --- Functional assay of LRP --- p.134 / Chapter 5.5 --- Hypothesis Testing --- p.135 / Chapter 5.6 --- Future prospective， --- p.136 / Chapter 6 --- Conclusion --- p.138 / Chapter 7 --- References --- p.140

Gluten-related disorders (GRDs) refer to a group of conditions that are caused by the ingestion of the gluten proteins present in wheat, barley, and rye. The global prevalence of GRDs is estimated to range from 0.6% to 10.6% of the general population, making it a significant global health issue. Treatment of GRDs requires dietary gluten avoidance. In China, there is believed to be a growing number of people with GRDs associated with changing eating patterns, increasing awareness, and better detection methods of these conditions in China. However, there is a lack of research about how to help this population maintain a restrictive diet and navigate food and social environment. The main purpose of this study was to explore the feasibility and acceptability of a culturally adapted low-gluten diet intervention among young adults in southeastern China. This study was a pre-post study design to investigate whether the intervention is effective in helping participants maintain a low-gluten diet for eight weeks. Participants were 62 young adults living on campus in southeastern China. Image-based food records and questionnaires were used to assess their dietary adherence, dietary quality, satisfaction, knowledge, self-efficacy, and other related determinants of following a low-gluten diet. Results suggested good feasibility of this dietary intervention. Only 1.9% of the total items consumed during the intervention was high-gluten or likely-high-gluten items, such as processed meat, mixed dishes, and fried food, and traditional noodles, suggesting an overall good compliance to the low-gluten diet. Specifically, over 95% of participants were found to be compliant with the diet based on all adherence measures. Females had better compliance than males (p=0.005 based on frequency, p=0.039 based on grams of gluten intake). Results also suggested good acceptability of this dietary intervention. All participants found the dietary intervention to be satisfactory with group communication and reminders rated to be the most helpful components. The perceived difficulty level of maintaining the low-gluten diet was 6.34 out of 10 (10 being the most difficult). The most rated barriers were fewer food choices and change of eating habits. Participants reported having more perceived barriers at the end of study compared to the beginning of the study, mean (SD) 17.19 (5.82) vs. 16.13 (4.19) out of 32, p = 0.285. Motivation scores were significantly lower at the end of study compared to the beginning of the study, mean (SD) 11.66 (2.21) vs. 13.40 (2.37) out of 16, p < 0.001. Increased perceived barriers and decreased motivation may suggest that they experienced more challenges in maintaining the low-gluten diet at the end of this two-month intervention. During the intervention, participants had significantly lower calories, carbohydrates, and vitamin B1 (thiamin) intake compared to baseline (p <0.05). Participants’ average dietary diversity score had no significant difference compared to baseline, 7.68 (1.10) vs. 7.69 (1.35), p=0.96. Participants had increased objective knowledge (p<0.0001), subjective knowledge (p< 0.0001), and behavioral capability (p<0.0001) compared to baseline. However, univariate and multivariate regression analyses did not find significant predicting effects of any determinants on dietary adherence. Our dietary assessment method, the image-based food records, was shown to be a reasonably valid and reliable tool to estimate the dietary intake among Chinese young adults based on comparison to weighted food records with the Bland–Altman plot and inter-rater reliability test (Cohen’s kappa=0.875). These findings suggested that a culturally adapted low-gluten dietary intervention was feasible and acceptable among Chinese young adults. Improvement on long-term dietary adherence and more research on determinants is needed. This study may inform health practitioners and policy makers to provide better culturally tailored support to patients who need to follow a low-gluten diet in China.

Orientador: Flavia Maria Netto / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-10T02:53:32Z (GMT). No. of bitstreams: 1 VillasBoas_MarianaBattaglin_M.pdf: 1570328 bytes, checksum: 8264ab45a2d69c56c77f60117495628a (MD5) Previous issue date: 2008 / Resumo: O leite bovino contém várias proteínas que são consideradas antigênicas e capazes de induzir resposta imune, dentre elas, a ß-lactoglubulina (ß-Lg) é uma das mais antigênicas. A enzima transglutaminase (TG) é a única enzima utilizada comercialmente que catalisa reação de ligação cruzada inter ou intramolecular em diversas proteínas, formando polímeros de alta massa molar. Até o momento, pouco se conhece sobre a modificação dos epítopos antigênicos na estrutura das proteínas do soro de leite modificada ou polimerizada pela enzima TG. O presente trabalho teve como objetivo estudar o efeito da reação de polimerização induzida pela TG na atividade antigênica da ß-Lg. A modificação da ß-Lg com a TG foi realizada em dois experimentos: (1) a ß-Lg foi tratada termicamente (80 °C/60 min) em diferentes concentrações (3, 5 e 7%) e polimerizada com TG (5, 10, 25 e 50 U/g de proteína) e (2) a ß-Lg (3, 5 e 7%) foi polimerizada com TG 25 U/g de proteína na presença de agente redutor Cys nas concentrações 0,05, 0,1, 0,25 e 0,4 mol/L. A caracterização da ß-Lg polimerizada foi realizada por eletroforese (SDS-PAGE) e cromatografia líquida de alta eficiência de exclusão molecular (CLAE-EM). A antigenicidade da proteína foi avaliada por métodos imunoquímicos, utilizando soro de camundongos BALB/c e técnicas de Immunoblotting e ELISA. Quando a ß-Lg 3% foi tratada termicamente e polimerizada com TG, 40% da ß-Lg permaneceu nas formas monomérica e dimérica. Já as amostras tratadas com maior concentração de proteína (5% e 7%) apresentaram de 70% a 87% de material com massa molar (MM) > 100 kDa, sendo que ß-Lg 7% polimerizada com 25U TG/g foi a que apresentou maior grau de polimerização. Na presença de Cys, observou-se maior percentual de produtos com MM > 100 kDa que o obtido com o tratamento térmico. Nas amostras ß-Lg 5% e ß-Lg 7%, a presença da Cys possivelmente aumentou a susceptibilidade da ß-Lg à polimerização. As amostras selecionadas para avaliação da antigenicidade foram ß-Lg nativa, ß-Lg 7% tratada termicamente e polimerizada com 25U TG/g de proteína (ß-Lg 7% TT 25TG) e ß-Lg 7% polimerizada com 25 U/g de proteína na presença de Cys 0,25 mol/L (ß-Lg 7% 0,25Cys TG). Os resultados mostraram que os animais sensibilizados com ß-Lg polimerizada na presença de agente redutor Cys apresentaram níveis séricos de IgE e IgG menores, comparados aos grupos imunizados com ß-Lg nativa e ß-Lg tratada termicamente e polimerizada com TG, sugerindo que a polimerização na presença de agente redutor Cys pode reduzir o potencial antigênico da proteína ao modificar .e/ou ocultar regiões de epítopos na proteína / Abstract: The bovine milk contains several proteins that are considered antigenic and capable of inducing immune responses, among them ß-lactoglubulin (ß-Lg) is one of the most antigenic protein. The enzyme transglutaminase (TG) is the only enzyme commercially used that catalyzes reaction of crosslinking inter or intramolecular in several proteins, forming polymer of high molecular mass. Up to now, little is known about modification of epitopes of whey proteins by the TG. The present study examines the effect of the polymerization reaction induced by TG on the antigenic activity of ß-Lg. The ß-Lg was modified by TG in two experiments: (1) ß-Lg was heat treated (80 °C/60 min) at different concentrations (3, 5 and 7%) and modified with TG (5, 10 , 25 and 50U / g protein), and (2) ß-Lg (3, 5 and 7%) was modified with TG 25U / g of protein in the presence of reducing agent Cys ( 0.05, 0.1, 0.25, 0.4 mol/L). The characterization of the modified ß-Lg was performed by electrophoresis (SDSPAGE) and high performance liquid chromatography molecular exclusion (HPLCMS). The antigenicity of the protein was measured by Immunoblotting and ELISA, using serum from BALB/c mices. When ß-Lg 3% was heat treated and modified by TG, 40% of the ß-Lg remained as monomeric and dimeric forms. However, the samples treated at higher protein concentration (5% and 7%) showed 70% to 87% of material with molecular weigh > 100 kDa. ß-Lg 7% modified with 25U TG / g presented the greatest degree of polymerization. In the presence of Cys, higher percentage of products with MW > 100 kDa was obtained than with the previous heat treatment. The presence of Cys increased the susceptibility of ß-Lg for polymerization, especially those treated at protein 5 and 7% concentrations. The samples selected for evaluation of the antigenicity were native ß-Lg, 7% ß-Lg treated with 25U TG / g of protein (ß-Lg 7% TT 25TG) and 7% ß-Lg treated with 25U TG / g protein in the presence of Cys 0.25 mol / L (ß-Lg 7% 0.25 Cys TG). The results showed that animals sensitized with ß-Lg in the presence of reducing the agent Cys had lower levels of IgG and IgE, compared to the groups immunized with native ß-Lg and ß-Lg treated with high temperature and modified with TG, suggesting that the polymerization in the presence of reducing agent Cys can modify or hide epitopes and reduce the potential antigenic of the protein / Mestrado / Nutrição Experimental e Aplicada à Tecnologia de Alimentos / Mestre em Alimentos e Nutrição

FundaÃÃo Cearense de Apoio ao Desenvolvimento Cientifico e TecnolÃgico / O lÃtex da planta lactÃfera Calotropis procera (Ait.) R. Br. à um composto biologicamente ativo importante que apresenta propriedades relevantes como as atividades antiinflamatÃria e antidiarrÃica, embora jà tenha sido previamente mostrado que esse lÃtex produz efeitos tÃxicos considerÃveis em animais. Um outro fato à que nÃo à sabido, ainda, se as proteÃnas do lÃtex de C. procera produzem efeitos alergÃnicos, assim como as proteÃnas do lÃtex de Hevea brasiliensis. O potencial do lÃtex como um fÃrmaco, por essa razÃo, depende da separaÃÃo das propriedades curativas das propriedades tÃxicas e alergÃnicas, mas nÃo existe investigaÃÃo cientÃfica nessa Ãrea. O presente estudo teve como objetivo investigar aspectos bioquÃmicos, toxicolÃgicos e alergÃnicos do lÃtex da planta C. procera, atravÃs do estudo da digestibilidade in vitro e in vivo, avaliaÃÃo da toxicidade subcrÃnica e aguda por via oral e anÃlise da induÃÃo de resposta imune pelas vias subcutÃnea e oral das suas fraÃÃes. O lÃtex foi fracionado em trÃs fraÃÃes distintas de acordo com a sua solubilidade em Ãgua e tamanho molecular. As fraÃÃes foram assim denominadas: proteÃnas do lÃtex (PL), correspondendo Ãs principais proteÃnas do lÃtex; proteÃnas da diÃlise (PD), representando as substÃncias de baixa massa molecular; e borracha do lÃtex (BL) que à altamente insolÃvel em Ãgua. A fraÃÃo PL foi investigada, com relaÃÃo a alguns aspectos bioquÃmicos, como seu perfil protÃico por PAGE-SDS e anÃlise da composiÃÃo de seus aminoÃcidos. As proteÃnas do lÃtex tambÃm foram submetidas à digestÃo in vitro com as enzimas proteolÃticas pepsina, tripsina, quimiotripsina e protease de Streptomyces griseus e a digestibilidade in vitro foi avaliada por cromatografia de filtraÃÃo em gel ou PAGE-SDS. A digestibilidade in vivo de PL foi analisada quando animais experimentais ingeriram essa fraÃÃo por 35 dias. O volume de PL consumido foi registrado diariamente e amostras das fezes dos animais coletadas, tratadas e submetidas à PAGE-SDS e ensaios de imunodifusÃo radial dupla de Ouchterlony, com anticorpos policlonais contra PL. Com relaÃÃo aos aspectos toxicolÃgicos, a fraÃÃo PL foi oralmente administrada a animais experimentais por 35 dias, para a avaliaÃÃo da toxicidade subcrÃnica. Aumentos na massa corpÃrea foram registrados e amostras sangÃÃneas foram analisadas semanalmente. ApÃs o perÃodo experimental, os animais foram sacrificados e um nÃmero de parÃmetros bioquÃmicos e fisiolÃgicos foram determinados. Esses incluÃram determinaÃÃes sÃricas de glicose sangÃÃnea, colesterol total, HDL-colesterol, triglicerÃdeos, testes da funÃÃo hepÃtica (proteÃnas totais, albumina, alanina aminotransferase â ALT e aspartato aminotransferase â AST), testes da funÃÃo renal (urÃia e creatinina), contagens total e diferencial de leucÃcitos sangÃÃneos e do fluido peritonial e anÃlise das proteÃnas sÃricas por PAGE-SDS. Os animais tiveram seus ÃrgÃos vitais (fÃgado, rins, baÃo, intestino delgado, intestino grosso, pÃncreas e estÃmago) dissecados e as massas frescas relativas foram determinadas. Na avaliaÃÃo da toxicidade aguda, a fraÃÃo PL foi administrada por via oral a animais experimentais e a monitoraÃÃo de alteraÃÃes comportamentais desses animais tambÃm foi realizada. Quanto aos aspectos alergÃnicos, respostas imunolÃgicas do lÃtex de C. procera foram investigadas pelas vias oral e subcutÃnea em camundongos. Anti-soros contra as fraÃÃes PL, PD e BL foram analisados quanto a IgG e IgA por ELISA, enquanto IgE e IgG1 foram monitoradas por anafilaxia cutÃnea passiva (PCA) em ratos e camundongos, respectivamente. Os perfis protÃicos das fraÃÃes PL e BL foram analisados por PAGE-SDS. Com relaÃÃo aos resultados dos aspectos bioquÃmicos, a fraÃÃo PL possui uma quantidade apreciÃvel de proteÃnas, baixo conteÃdo de aminoÃcidos sulfurados (metionina e cisteÃna) e um nÃmero considerÃvel de aminoÃcidos (arginina, lisina, fenilalanina e tirosina) que representam sÃtios de clivagem para as enzimas proteolÃticas animais usadas. A fraÃÃo PL foi digerida pela aÃÃo da pepsina, tripsina e quimiotripsina como revelado por anÃlises de filtraÃÃo em gel e PAGE-SDS. A digestÃo completa das proteÃnas do lÃtex foi facilmente obtida pelo tratamento com a protease de S. griseus. Anticorpos policlonais de coelho produzidos contra PL nÃo apresentaram reaÃÃo cruzada com as molÃculas presentes nas fezes dos ratos experimentais. PadrÃes semelhantes de eletroforese foram observados para as quantidades desprezÃveis de proteÃna observadas nos materiais fecais dos animais controle e experimentais. Quanto aos resultados da avaliaÃÃo da toxicidade subcrÃnica, nenhuma morte foi observada durante o experimento. Animais controle e experimentais apresentaram taxa de crescimento semelhante e os ÃrgÃos vitais exibiram massas frescas relativas similares. As funÃÃes hepÃtica e renal, parÃmetros glicÃmicos e lipidÃmicos sÃricos situaram-se em nÃveis normais. Os padrÃes protÃicos eletroforÃticos dos soros dos animais controle e experimental exibiram perfis muito similares. A fraÃÃo PL nÃo induziu inflamaÃÃo aguda na cavidade peritonial dos ratos, como determinado pelas contagens total e diferencial de leucÃcitos. O resultado mais relevante detectado foi um aparente efeito proliferativo das proteÃnas do lÃtex sobre os linfÃcitos sangÃÃneos que tenderam a aumentar, enquanto os neutrÃfilos permaneceram em nÃvel normal. Deve ser enfatizado que os animais experimentais exibiram comportamento, aspectos morfolÃgicos e bioquÃmicos normais, indicando ser improvÃvel que os mesmos estivessem sob qualquer condiÃÃo patolÃgica ou infecciosa. Portanto, o incremento da populaÃÃo de linfÃcitos no soro sangÃÃneo deve ser atribuÃdo a um efeito notÃvel das proteÃnas do lÃtex e nÃo a qualquer evento prejudicial. A fraÃÃo PL foi incapaz de induzir toxicidade aguda, pois nenhuma mudanÃa comportamental foi observada nos animais experimentais. Com relaÃÃo aos resultados dos aspectos alergÃnicos, nenhuma das fraÃÃes induziu aumentos nos nÃveis de anticorpos, quando os camundongos receberam as fraÃÃes do lÃtex por via oral e, portanto, nÃo desenvolveram alergia. Entretanto, anti-soros de camundongos sensibilizados com PL e BL por administraÃÃo subcutÃnea apresentaram resposta imunolÃgica considerÃvel, e PD nÃo induziu sÃntese de anticorpos. O nÃvel de IgG aumentou consistentemente contra PL e BL, enquanto a resposta de IgA foi detectada unicamente contra PL. PL e BL induziram reaÃÃes de PCA muito fortes, sugerindo que ambas as fraÃÃes contÃm substÃncias do lÃtex envolvidas na alergenicidade. AlÃm disso, a anÃlise protÃica de PL e BL sugere que BL ainda retÃm proteÃnas residuais, co-precipitadas com a borracha, abundantemente encontradas na fraÃÃo PL, que poderiam explicar as alergenicidades semelhantes. Nenhuma reaÃÃo de IgG1 foi detectada em quaisquer dos anti-soros testados. Conclui-se que as proteÃnas do lÃtex foram parcialmente susceptÃveis à proteÃlise em ensaios in vitro e, ou foram digeridas e absorvidas, ou foram absorvidas Ãntegras, quando ingeridas em ensaios in vivo. Eventos tÃxicos ou letalidade associados ao lÃtex, como descritos na literatura, nÃo estÃo relacionados com a fraÃÃo PL. A fraÃÃo PL produziu efeito proliferativo parcial sobre as cÃlulas mononucleadas, principalmente linfÃcitos e nÃo induziu resposta inflamatÃria aguda, quando administrada pela via oral. Efeitos alergÃnicos puderam ser detectados, por via subcutÃnea, com as fraÃÃes PL e BL, e nÃo foram observados por via oral. EvidÃncias para uma possÃvel tolerÃncia sistÃmica Ãs proteÃnas do lÃtex por via oral foram fornecidas. As proteÃnas do lÃtex podem atuar como imunoestimulantes. As proteÃnas do lÃtex permanecem uma fonte interessante de molÃculas biologicamente ativas que devem ser estudadas em detalhe quanto Ãs suas propriedades estruturais, funcionais e aplicativas. / The latex of the lactiferous plant Calotropis procera (Ait.) R. Br. is an important biologically active compound that displays relevant properties like antiinflammatory and antidiarrhea activities, although the latex has previously been shown to produce considerable toxic effects on animals. Another question is that it is not known yet whether the latex proteins from C. procera induce allergenic effects, just like the latex proteins from Hevea brasiliensis. The potential of the latex as a pharmaceutical, therefore, depends on separating the curative properties from the toxic and allergenic properties, but there has been a lack of scientific investigation in this area. The objective of the present study was to investigate biochemical, toxicological and allergenic aspects of the latex from the plant C. procera, through the study of in vitro and in vivo digestibility, evaluation of acute and subchronic toxicity by oral route, and analysis of immune response induction by subcutaneous and oral route of its fractions. The latex was fractionated into three distinct fractions according to their water solubility and molecular size. The fractions were named as: latex proteins (LP), corresponding to the major latex proteins; dialysis proteins (DP), representing low molecular size substances; and rubber latex (RL), which was highly insoluble in water. LP fraction was investigated in some biochemical aspects, like its protein profile by SDS-PAGE analysis and its amino acid composition determination. Latex proteins were also subjected to in vitro digestion with trypsin, chemotrypsin, pepsin and Streptomyces griseus protease and in vitro digestibility was evaluated by gel filtration and SDS-PAGE analysis. The in vivo digestibility of LP was analyzed when experimental animals ingested this fraction for 35 days. The volume uptake of LP was recorded daily and samples of fecal material from animals were collected, treated and submitted to SDS-PAGE analysis and radial double immunodifusion assays, with polyclonal antibodies raised against LP. In relation to toxicological aspects, LP fraction was orally administered to experimental animals for 35 days, to allow subchronic toxicity evaluation. Increases in body mass were recorded and blood samples were analyzed weekly. After the test period, the animals were sacrificed and a number of biochemical and physiological parameters determined. These included sera determinations of blood glucose, total cholesterol, HDL-cholesterol, triglycerides, hepatic function tests (total proteins, albumin, alanine aminotransferase â ALT and aspartate aminotransferase â AST), renal function tests (urea and creatinin), total and differential leukocyte counts in blood and peritoneal fluid and analysis of sera proteins by SDS-PAGE. Animals had internal key organs (liver, kidneys, spleen, small intestine, large intestine, pancreas and stomach) dissected and the relative fresh masses were determined. In the acute toxicity evaluation, LP fraction was administered by oral route to experimental animals and behavioral changes in animals were also monitored. With reference to allergenic aspects, immunological responses of latex from C. procera were investigated by oral and subcutaneous routes in mice. Anti-sera against LP, DP and RL fractions were assayed for IgG and IgA titration by ELISA, while IgE and IgG1 were accessed by passive cutaneous anaphylaxis (PCA) in rats and mice, respectively. Protein profiles of LP and RL fractions were analyzed by SDS-PAGE. Concerning biochemical aspects results, LP fraction possesses appreciable amount of protein, low content of sulphurated amino acids (methionine and cysteine) and a considerable number of amino acids (arginine, lysine, phenylalanine and tyrosine) that represent cleavage sites to animal proteolytic enzymes studied. LP fraction was digested by the action of pepsin, trypsin and chemotrypsin as revealed by gel filtration and SDS-PAGE analyses. The full LP digestion was easily achieved by S. griseus protease treatment. Rabbit polyclonal antibodies raised against LP failed to detect cross-reactive molecules in faeces of experimental rats. Similar patterns of electrophoresis were observed for the negligible amounts of protein observed in the fecal materials of control and test animals. With relation to subchronic toxicity evaluation results, no death was observed during the experiment. Experimental and control animals presented similar growth rate and key organs exhibited similar relative fresh masses. Hepatic and renal functions, sera glycemic and lipidemic parameters were determined to be at normal levels. Likewise, the electrophoretic protein patterns of sera from untreated and treated animals exhibited quite similar profiles. Uptake of latex proteins did not induce acute inflammation into peritoneal cavity of rats as determined by the total and differential leukocytes counts. The most relevant result discovered was an apparent proliferative effect of the latex proteins upon blood lymphocytes that tended to increase, whilst neutrophils remained at normal level. It should be emphasized that the experimental rats were normal in their behavior, morphological and biochemical aspects, indicating that it is unlikely that they were under any pathological or infectious condition. Therefore, the increment of lymphocytes population in the blood serum should be attributed to the staking effect of the latex proteins rather than any harmful event. LP fraction was unable of presenting acute toxicity in experimental animals, because no behavioral changes in animals were observed. Concerning allergenic aspects results, none of the fractions induced antibodies level increases when mice received latex fractions by oral route and thus, did not develop allergy. Nonetheless, anti-sera of mice sensitized with LP and RL by subcutaneous administration displayed considerable immunological response, while DP did not induce antibodies synthesis. IgG level augmented consistently against LP and RL, while IgA response was detected to LP solely. LP and RL induced very strong PCA reactions suggesting that both fractions would contain latex substances involved in allergenicity. Furthermore, protein analysis of LP and RL suggests that RL still retain residual proteins, co-precipitated with rubber, abundantly found in LP, that could explain its similar allergenicity. No IgG1 reaction was detected in any of the anti-sera tested. It can be concluded that latex proteins were partially susceptible to digestive proteolysis when accessed by in vitro assays, and were digested and absorbed, or were absorbed in its intact form, when ingested and analyzed by in vivo tests. Toxic events or lethality associated with latex, as described in the literature, are not related to LP fraction. Latex proteins produced partial proliferative effect upon mononuclear cells, mainly lymphocytes and did not induce an acute inflammatory response, when administered by oral route. Allergenic effects could be detected, by subcutaneous route, with LP and RL fractions, and were not observed by oral route. Evidences for an inducible tolerance acquired to latex proteins by oral route were provided. Latex proteins may act as immune stimulants. The proteins from the latex remain an interesting source of biologically active molecules that should be studied in detail for their structural, functional and applicative properties.