Fast and sensitive aflatoxin B1 and total aflatoxins ELISAs for analysis of peanuts, maize and feed ingredients

Aflatoxins are a group of carcinogenic compounds produced by Aspergillus fungi that can grow on different agricultural crops. Both acute and chronic exposure to these mycotoxins can cause serious illness. Due to the high occurrence of aflatoxins in crops worldwide fast and cost-effective analytical methods are required for the identification of contaminated agricultural commodities before they are processed into final products and placed on the market. In order to provide new tools for aflatoxin screening two prototype fast ELISA methods: one for the detection of aflatoxin B₁ and the other for total aflatoxins were developed. Seven monoclonal antibodies with unique high sensitivity and at the same time good cross-reactivity profiles were produced. The monoclonal antibodies were characterized and two antibodies showing IC₅₀ of 0.037 ng/mL and 0.031 ng/mL for aflatoxin B₁ were applied in simple and fast direct competitive ELISA tests. The methods were validated for peanut matrix as this crop is one of the most affected by aflatoxin contamination. The detection capabilities of aflatoxin B₁ and total aflatoxins ELISAs were 0.4 μg/kg and 0.3 μg/kg for aflatoxin B₁, respectively, which are one of the lowest reported values. Total aflatoxins ELISA was also validated for the detection of aflatoxins B₂, G₁ and G₂. The application of the developed tests was demonstrated by screening 32 peanut samples collected from the UK retailers. Total aflatoxins ELISA was further applied to analyse naturally contaminated maize porridge and distiller's dried grain with solubles samples and the results were correlated with these obtained by UHPLC-MS/MS method.

Développement et applications d’un outil bio-informatique pour la détection de similarités de champs d’interaction moléculaire

Résumé : Les méthodes de détection de similarités de sites de liaison servent entre autres à la prédiction de fonction et à la prédiction de cibles croisées. Ces méthodes peuvent aider à prévenir les effets secondaires, suggérer le repositionnement de médicament existants, identifier des cibles polypharmacologiques et des remplacements bio-isostériques. La plupart des méthodes utilisent des représentations basées sur les atomes, même si les champs d’interaction moléculaire (MIFs) représentent plus directement ce qui cherche à être identifié. Nous avons développé une méthode bio-informatique, IsoMif, qui détecte les similarités de MIF entre différents sites de liaisons et qui ne nécessite aucun alignement de séquence ou de structure. Sa performance a été comparée à d’autres méthodes avec des bancs d’essais, ce qui n’a jamais été fait pour une méthode basée sur les MIFs. IsoMif performe mieux en moyenne et est plus robuste. Nous avons noté des limites intrinsèques à la méthodologie et d’autres qui proviennent de la nature. L’impact de choix de conception sur la performance est discuté. Nous avons développé une interface en ligne qui permet la détection de similarités entre une protéine et différents ensembles de MIFs précalculés ou à des MIFs choisis par l’utilisateur. Des sessions PyMOL peuvent être téléchargées afin de visualiser les similarités identifiées pour différentes interactions intermoléculaires. Nous avons appliqué IsoMif pour identifier des cibles croisées potentielles de drogues lors d’une analyse à large échelle (5,6 millions de comparaisons). Des simulations d’arrimage moléculaire ont également été effectuées pour les prédictions significatives. L’objectif est de générer des hypothèses de repositionnement et de mécanismes d’effets secondaires observés. Plusieurs exemples sont présentés à cet égard.

The analysis and detection of new psychoactive substances in biological matrices

New psychoactive substances (NPSs) have appeared on the recreational drug market at an unprecedented rate in recent years. Many are not new drugs but failed products of the pharmaceutical industry. The speed and variety of drugs entering the market poses a new complex challenge for the forensic toxicology community. The detection of these substances in biological matrices can be difficult as the exact compounds of interest may not be known. Many NPS are sold under the same brand name and therefore users themselves may not know what substances they have ingested. The majority of analytical methods for the detection of NPSs tend to focus on a specific class of compounds rather than a wide variety. In response to this, a robust and sensitive method was developed for the analysis of various NPS by solid phase extraction (SPE) with gas chromatography mass spectrometry (GCMS). Sample preparation and derivatisation were optimised testing a range of SPE cartridges and derivatising agents, as well as derivatisation incubation time and temperature. The final gas chromatography mass spectrometry method was validated in accordance with SWGTOX 2013 guidelines over a wide concentration range for both blood and urine for 23 and 25 analytes respectively. This included the validation of 8 NBOMe compounds in blood and 10 NBOMe compounds in urine. This GC-MS method was then applied to 8 authentic samples with concentrations compared to those originally identified by NMS laboratories. The rapid influx of NPSs has resulted in the re-analysis of samples and thus, the stability of these substances is crucial information. The stability of mephedrone was investigated, examining the effect that storage temperatures and preservatives had on analyte stability daily for 1 week and then weekly for 10 weeks. Several laboratories identified NPSs use through the cross-reactivity of these substances with existing screening protocols such as ELISA. The application of Immunalysis ketamine, methamphetamine and amphetamine ELISA kits for the detection of NPS was evaluated. The aim of this work was to determine if any cross-reactivity from NPS substances was observed, and to determine whether these existing kits would identify NPS use within biological samples. The cross- reactivity of methoxetamine, 3-MeO-PCE and 3-MeO-PCP for different commercially point of care test (POCT) was also assessed for urine. One of the newest groups of compounds to appear on the NPS market is the NBOMe series. These drugs pose a serious threat to public health due to their high potency, with fatalities already reported in the literature. These compounds are falsely marketed as LSD which increases the chance of adverse effects due to the potency differences between these 2 substances. A liquid chromatography tandem mass spectrometry (LC-MS/MS) method was validated in accordance with SWGTOX 2013 guidelines for the detection for 25B, 25C and 25I-NBOMe in urine and hair. Long-Evans rats were administered 25B-, 25C- and 25I-NBOMe at doses ranging from 30-300 µg/kg over a period of 10 days. Tail flick tests were then carried out on the rats in order to determine whether any analgesic effects were observed as a result of dosing. Rats were also shaved prior to their first dose and reshaved after the 10-day period. Hair was separated by colour (black and white) and analysed using the validated LC-MS/MS method, assessing the impact hair colour has on the incorporation of these drugs. Urine was collected from the rats, analysed using the validated LC-MS/MS method and screened for potential metabolites using both LC-MS/MS and quadrupole time of flight (QToF) instrumentation.

Uma Lectina D-lactose específica da esponja marinha Cinachyrella apion: purificação, caracterização e interação com Leishmania chagasi

Four different sponge species were screened using Ouchterlony agarose gel and immunodiffusion tests to identify cross-reactivity with the polyclonal antibody IgG anti-deglicosilated CvL, a lectin from Cliona varians. Crude extract from the sponge Cinachyrella apion showed cross-reactivity and also a strong haemmaglutinating activity towards human erythrocytes of all ABO groups. Thus, it was submitted to acetone fractionation, IgG anti-deglicosilated CvL Sepharose affinity chromatography, and Fast Protein Liquid Chromatography (FPLC-AKTA) gel filtration on a Superose 6 10 300 column to purify a novel lectin. C. apion lectin (CaL) agglutinated all types of human erythrocytes with preference for papainized type A and O erythrocytes. The haemagglutinating activity is independent of Ca2+, Mg2+ and Mn2+ ions, and it was strongly inhibited by the disaccharide D-lactose, up to a minimum concentration of 6.25 mM. CaL molecular mass determined by FPLC-AKTA gel filtration on a Superose 12 10 300 column and SDS gel electrophoresis was approximately 124 kDa, consisting of eight subunits of 15.5 kDa, assembled by hydrophobic interactions. The lectin was relatively heat- and pH-stable. Leishmania chagasi romastigotes were agglutinated by CaL, indicating that lactose receptors could be presented in this parasite stage. These findings are indicative of the physiological defense roles of CaL and its possible use in the antibiosis of pathogenic protozoa

Anticorpos anti-trypanosoma cruzi como preditivos de infecção por leishmania infantum

Leishmania infantum and Trypanosoma cruzi are trypanosomatids of medical importance and are, respectively, the etiologic agents of visceral leishmaniasis (VL) and Chagas disease (CD) in Brazil. People infected with L. infantum or T. cruzi may develop asymptomatically, enabling the transmission of pathogens through blood transfusion and / or organs. The assessment of the infection by T. cruzi is included among the tests performed for screening blood donors in Brazil, however, there is no availability of tests for Leishmania. Serological tests for T. cruzi are very sensitive, but not specific, and may have cross-reactions with other microorganisms. Thus, the aim of this study was to determine the prevalence of Leishmania infection in blood donors and assess whether the serological test for T. cruzi detect L. infantum. Among the 300 blood samples from donors, discarded in 2011, 61 were T. cruzi positive, 203 were from donors with other infections and 36 were from handbags with low blood volume, but without infection. We also assessed 144 samples from donors without infections and able to donate blood, totaling 444 subjects. DNA was extracted from blood samples of all to perform quantitative PCR (qPCR) to detect Leishmania DNA. The buffy coat obtained from all samples was grown in Schneider medium supplemented and NNN. All samples were evaluated for the presence of anti-Leishmania antibody. The serological results indicate a percentage of 22% of Leishmania infection in blood samples obtained from discarded bags. A total of 60% of samples positive in ELISA for T. cruzi were negative by IFI, used as confirmatory test, ie 60% false positive for Chagas. Among these samples false positive for Chagas, 72% were positive by ELISA for Leishmania characterizing the occurrence of cross reaction between serologic assays. Of the 300 cultures performed, 18 grew parasites that were typed by qPCR and specific isoenzymes, found the species Leishmania infantum crops. Among the 18 cultures, 4 were purged from scholarships for low volume and all negative serology blood bank, thus demonstrating that there is a real risk of Leishmania transmission via transfusion. It is concluded that in an area endemic for leishmaniasis in Brazil, serological diagnosis performed to detect infection by T. cruzi among blood donors can identify infection by L. infantum and although cause false positive for Chagas, this cross-reactivity reduces the risk of Leishmania infection via blood transfusion, since tests are not applied specific detection of the parasite. In this way, there remains the need to discuss the implementation of a specific serological screening test for Leishmania in endemic countries such as Brazil

Alergia à picada de Himenópteros em cães

Dissertação de Mestrado Integrado em Medicina Veterinária

Avaliação da sensibilidade ao latex em profissionais de saúde num hospital do norte alentejano

Nos profissionais de saúde a alergia ao látex representa um importante problema de saúde ocupacional. Em contexto hospitalar, a principal fonte de exposição alergénica é a utilização de luvas látex empoadas e a inalação dos bioaerossóis que se formam pela manipulação das mesmas. Mediante consentimento informado, os participantes preencheram um inquérito de resposta fechada e realizaram os testes cutâneos (TC). Aqueles que apresentaram reacção foram encaminhados para a consulta de imunoalergologia e realizaram TC para alimentos, doseamentos séricos de imunoglobulinas (lgE total e específica). Foram incluídos no estudo 44 dos 48 participantes (a=0.1). A sensibilidade ao látex foi posta em evidência em 15 dos elementos da amostra, seis dos quais reagem ao extracto de látex utilizado nos TC. A alergia ao látex foi diagnosticada em duas pessoas. A aplicação de questionários na avaliação sensibilização ocupacional ao látex pode vir a constituir ferramenta válida de auscultação deste problema nas instituições prestadoras de cuidados de saúde. ABSTRACT: Latex allergy is a major occupational disease among health care workers. ln hospitals, the main source of allergen exposure is the powdered latex gloves latex and the inhalation of the aerosols formed by its direct manipulation. By means of informed assent, the participants filled an inquiry and carried out the SPT. Those with cutaneous reaction to the latex extract went to an immunoallergology appointment. SPT for fruit and pollen with cross reactivity to latex allergens where performed, and blood levels of total and specific lgE where determined. The study group included 44 of the 48 participants (a=0.1). Fifteen elements showed latex sensitization and six of them had cutaneous reaction to the latex extract used in the SPT. Latex allergy was found in two health care professionals. Questionnaires may provide a reliability tool to access the sensitization due to natural rubber products and the symptoms more commonly associated with it, in health care facilities.

Study of the allergenic profile of pollen from Platanus hybrida in the city of Evora, Portugal

Background and Aim: Although grasses and olive are the most relevant allergenic species in the Alentejo region, aggravation of allergic symptoms in the early spring, unrelated with those species pollen seasons, has been reported, particularly in urban environment. Plane trees, hence pollen, are highly abundant in the city of Évora, nonetheless allergen pollen profile has not yet been evaluated. The aim of this work was to characterize the allergen profile of pollen from Platanus hybrida, one of the most representative species in Evora showing pollination prior to the main pollen season in Alentejo. Methods: Pollen from Platanus hybrida and Dactylis glomerata was extracted with ammonium bicarbonate buffer, lyophilized and stored at -80ºC until analysis. Protein content was determined by the Bradford method. SDS-PAGE followed by western blot, using allergic patient sera (obtained from the Hospital do Espírito Santo de Évora – HESE), were performed to evaluate the allergen profile of the pollen. Sensitization and cross-reactivity was assessed by solid phase immunoblot. Results: Half of the patient exhibited sensitization to pollen extracts of P. Hybrida. Western blot have shown several immunoreactive bands in the Mr 10-90 kDa range. Immunoreactive bands were also observed in the protein profile according to the pI in the pI range 4.0-6.1. Cross-reactivity of P. hybrida with D. glomerata was found. Although several bands are common to D. glomerata, a band with ~50kDa was observed in P. hybrida but not in D. glometata. Conclusion: These results evidenced allergens found in P. hybrida pollen. Moreover, cross–reactivity between P. hybrida and highly allergenic species such as D. glomerata was found which probably contributes for aggravation of pollinosis in the early spring. Acknowledgments: This work was supported by FEDER through the “Programa Operacional Fatores de Competitividade – COMPETE” (Strategic projects of ICAAM and ICT 2013-2015).

Allergenic profile of Quercus rotundifolia pollen in Alentejo, Portugal

Background and Aim: Grasses and olive are the most relevant allergenic species in the Alentejo region. However, aggravation of allergic symptoms has been reported in the early spring, before grass and olive pollen seasons. Quercus pollen is the most abundant pollen type in the early spring in Alentejo, nonetheless its allergen profile has not yet been evaluated. The aim of this work was to characterize the allergen profile of pollen from Quercus rotundifolia among the most representative species showing pollination in April, prior to the main pollen season in Alentejo. Methods: Pollen from Quercus rotundifolia, Olea europaea and Dactylis glomerata was extracted with ammonium bicarbonate buffer, lyophilized and stored at -80ºC until analysis. Extract from Quercus ilex pollen was kindly offered by Bial. Protein content was determined by the Bradford method. SDS-PAGE followed by western blot, using allergic patient sera (obtained from the Hospital do Espírito Santo de Évora – HESE), were performed to evaluate the allergen profile of the pollen. Sensitization and cross-reactivity was assessed by solid phase immunoblot. Results: Most of the patient evidenced sensitization to pollen extracts of Q. rotundifolia. Protein profile of Q. rotundifolia has shown several bands in the Mr 10-90 kDa, mostly overlapping with Q. ilex. Western blot have shown several immunoreactive bands. Immunoreactive bands were also observed in the protein profile according to the pI in the range 4.0-6.1. Cross-reactivity between Q. rotundifolia with O. europaea and D. glomerata was found. Conclusion: These results evidenced allergens found in Q. rotundifolia pollen. It also shows that protein profile of Q. rotundifolia and Q. ilex are mostly alike suggesting that similarities in allergen profile are expected. Moreover, cross–reactivity between Q. rotundifolia and highly allergenic species such as O. europaea and D. glomerata was found which probably contributes to the aggravation of pollinosis in the early spring. Acknowledgments: This work was supported by FEDER through the “Programa Operacional Fatores de Competitividade – COMPETE” (Strategic projects of ICAAM and ICT 2013-2015). We also aknowledge Bial-Aristegui for supplying pollen and extract samples of Q. ilex.

Perfil em alergénios do pólen de Platanus hybrida

Objetivos: O plátano (Platanus hybrida) é uma árvore frequentemente utilizada em ambiente urbano, com fins ornamentais. Sendo uma árvore de grande porte, produz pólen em grande quantidade. Embora seja responsável por níveis de exposição a pólen elevados no início da primavera, que são coincidentes com queixas da população, o seu potencial alergénico está pouco caracterizado. Este trabalho teve, assim, como objetivo caracterizar o perfil em alergénios do pólen de plátano na cidade de Évora, Alentejo. Métodos: Prepararam-se extratos de amostras de pólen de Platanus hybrida ou Dactylis glomerata utilizando tampão bicarbonato. Os extratos foram liofilizados e conservados a -80ºC. O conteúdo em proteínas foi determinado pelo método de Bradford. O perfil em alergénios foi avaliado por western blot utilizando soros humanos (obtidos mediante consentimento informado de doentes do Hospital do Espírito Santo de Évora – HESE). Resultados: Observou-se teste positivo a P. hybrida em metade dos soros testados. O perfil em proteínas de P. hybrida exibiu diversas bandas imunorreativas com massas moleculares compreendidas entre 10-90 kDa e com pI no intervalo 4,4-7,0. Foram encontradas imunorreativas comuns a Q. rotundifólia e/ou a D. glomerata. Duas bandas identificadas na gama de 50kDa e 60 kDa parecem específicas de P. hybrida. Também se registou reatividade cruzada com D. glomerata. Conclusões: Este trabalho evidencia alguns alergénios encontrados em pólen de P. hybrida. Para além disso mostra ainda a existência de reatividade cruzada com pólen de gramíneas. Estes resultados sugerem que o pólen de plátano, dada a sua grande abundância na cidade de Évora, poderá contribuir para o agravamento a sintomatologia da população que sofre de polinose, em particular no início da primavera. Agradecimentos: Este trabalho foi financiado por fundos do FEDER através do Programa Operacional Fatores de Competitividade – COMPETE”. Um agradecimento especial ao nosso colega, já falecido, Prof. Rui Brandão, pelo estímulo que deu a este trabalho e pela sua dedicação para a implementação e desenvolvimento da Aerobiologia na Universidade de Évora. Temos a honra de dedicar este trabalho à sua memória. Background and Aim: Although grasses and olive are the most relevant allergenic species in the Alentejo region, aggravation of allergic symptoms in the early spring, unrelated with those species pollen seasons, has been reported, particularly in urban environment. Plane trees, hence pollen, are highly abundant in the city of Évora, nonetheless allergen pollen profile has not yet been evaluated. The aim of this work was to characterize the allergen profile of pollen from Platanus hybrida, one of the most representative species in Evora showing pollination prior to the main pollen season in Alentejo. Methods: Pollen from Platanus hybrida, Quercus rotundifolia or Dactylis glomerata was extracted with ammonium bicarbonate buffer, lyophilized and stored at -80ºC until analysis. Protein content was determined by the Bradford method. SDS-PAGE followed by western blot, using allergic patient sera (obtained from the Hospital do Espírito Santo de Évora – HESE), were performed to evaluate the allergen profile of the pollen. Results: Protein profile of P. Hybrida has shown several bands in the Mr 10-90 kDa. Western blot have shown several immunoreactive bands. Protein profile according to the pI showed immunoreactive bands in the pI range 4.0-6.1. Cross-reactivity of P. hybrida with Q. rotundifolia and D. glomerata was found. Conclusion: These results evidenced allergens found in P. hybrida pollen. Moreover, cross–reactivity between P. hybrida and highly allergenic species such as D. glomerata was found which probably contributes for aggravation of pollinosis in the early spring. Acknowledgments: This work was supported by “FEDER - Programa Operacional Factores de Competitividade – COMPETE”. A special acknowledgment to our colleague Prof. Rui Brandão, deceased, for his dedication to the present work, to the implantation and development of Aerobiology in the University of Évora. We have the honour of dedicating this work to the memory of Prof. Rui Brandão.

Perfil em alergénios do pólen de Quercus rotundifólia

Background and Aim: Grasses and olive are the most relevant allergenic species in the Alentejo region. However, aggravation of allergic symptoms has been reported in the early spring, before grass and olive pollen seasons. Quercus pollen is the most abundant pollen type in the early spring in Alentejo, nonetheless its allergen profile has not yet been evaluated. The aim of this work was to characterize the allergen profile of pollen from Quercus rotundifolia the most representative species showing pollination in April, prior to the main pollen season in Alentejo. Methods: Pollen from Quercus rotundifolia, Olea europaea and Dactylis glomerata was extracted with ammonium bicarbonate buffer, lyophilized and stored at -80ºC until analysis. Extract from Quercus ilex pollen was kindly offered by Bial. Protein content was determined by the Bradford method. SDS-PAGE followed by western blot, using allergic patient sera (obtained from the Hospital do Espírito Santo de Évora – HESE), were performed to evaluate the allergen profile of the pollen. Results: Protein profile of Q. rotundifolia has shown several bands in the Mr 10-90 kDa, mostly overlapping with Q. ilex. Western blot have shown several immunoreactive bands. Protein profile according to the pI showed immunoreactive bands in the pI range 4.0-6.1. Cross-reactivity of Q. rotundifolia with O. europaea and D. glomerata was found. Conclusion: These results evidenced allergens found in Q. rotundifolia pollen. It also shows that protein profile of Q. rotundifolia and Q. ilex are mostly alike suggesting that similarities in allergen profile are expected. Moreover, cross–reactivity between Q. rotundifolia and highly allergenic species such as O. europaea and D. glomerata was found which probably contributes for aggravation of pollinosis in the early spring. Acknowledgments: This work was supported by “FEDER - Programa Operacional Factores de Competitividade – COMPETE”. A special acknowledgment to our colleague Prof. Rui Brandão, deceased, for his dedication to the present work, to the implantation and development of Aerobiology in the University of Évora. We have the honour of dedicating this work to the memory of Prof. Rui Brandão.

Quantitative Analysis of Immunoglobulin E Reactivity Profiles in Patients Allergic or Sensitized to Natural Rubber Latex (Hevea Brasiliensis)

Characterized native and recombinant Hevea brasiliensis (rHev b) natural rubber latex (NRL) allergens are available to assess patient allergen sensitization profiles. OBJECTIVE: Quantification of individual IgE responses to the spectrum of documented NRL allergens and evaluation of cross-reactive carbohydrate determinants (CCDs) for more definitive diagnosis. METHODS: Sera of 104 healthcare workers (HCW; 51 German, 21 Portuguese, 32 American), 31 spina bifida patients (SB; 11 German, 20 Portuguese) and 10 Portuguese with multiple surgeries (MS) were analysed for allergen-specific IgE antibody (sIgE) to NRL, single Hev b allergens and CCDs with ImmunoCAP technology. RESULTS: In all patient groups rHev b 5-sIgE concentrations were the most pronounced. Hev b 2, 5, 6.01 and 13 were identified as the major allergens in HCW and combined with Hev b 1 and Hev b 3 in SB. In MS Hev b 1 displayed an intermediate relevance. Different sIgE antibody levels to native Hevea brasiliensis (nHev b) 2 and rHev b 6.01 allowed discrimination of SB with clinical relevant latex allergy vs. those with latex sensitization. Sensitization profiles of German, Portuguese and American patients were equivalent. rHev b 5, 6.01 and nHev b 13 combined detected 100% of the latex-allergic HCW and 80.1% of the SB. Only 8.3% of the sera showed sIgE response to CCDs. CONCLUSIONS: Hev b 1, 2, 5, 6.01 and 13 were identified as the major Hev b allergens and they should be present in standardized latex extracts and in vitro allergosorbents. CCDs are only of minor relevance in patients with clinical relevant latex allergy. Component-resolved diagnostic analyses for latex allergy set the stage for an allergen-directed immunotherapy strategy

Cross-reactions vs co-sensitization evaluated by in silico motifs and in vitro IgE microarray testing

Using an in silico allergen clustering method, we have recently shown that allergen extracts are highly cross-reactive. Here we used serological data from a multi-array IgE test based on recombinant or highly purified natural allergens to evaluate whether co-reactions are true cross-reactions or co-sensitizations by allergens with the same motifs.

Rheumatic Fever and Rheumatic Heart Disease: Cellular Mechanisms Leading Autoimmune Reactivity and Disease

Rheumatic fever (RF) is an autoimmune disease caused by the gram-positive bacteria Streptococcus pyogenes that follows a nontreated throat infection in susceptible children. The disease manifests as polyarthritis, carditis, chorea, erythema marginatum, and/or subcutaneous nodules. Carditis, the most serious complication, occurs in 30% to 45% of RF patients and leads to chronic rheumatic heart disease (RHD), which is characterized by progressive and permanent valvular lesions. In this review, we will focus on the genes that confer susceptibility for developing the disease, as well as the innate and adaptive immune responses against S. pyogenes during the acute rheumatic fever episode that leads to RHD autoimmune reactions. The disease is genetically determined, and some human leukocyte antigen class II alleles are involved with susceptibility. Other single nucleotide polymorphisms for TNF-alpha and mannan-binding lectin genes were reported as associated with RF/RHD. T cells play an important role in RHD heart lesions. Several autoantigens were already identified, including cardiac myosin epitopes, vimentin, and other intracellular proteins. In the heart tissue, antigen-driven oligoclonal T cell expansions were probably the effectors of the rheumatic heart lesions. These cells are CD4(+) and produced inflammatory cytokines (TNF alpha and IFN gamma). Molecular mimicry is the mechanism that mediated the cross-reactions between streptococcal antigens and human proteins. The elucidation of chemokines and their receptors involved with the recruitment of Th1, Th2, and Th17 cells, as well as the function of T regulatory cells in situ will certainly contribute to the delineation of the real picture of the heart lesion process that leads to RHD.