Abstracts of Congress for Papers and Posters
PI 12
CORRELAÇÃO ENTRE BCG INTRADÉRMICO E LINFOPROLIFERAÇÃO E PRODUÇÃO DAS CITOCINAS IFN-γ. IL-12. IL-10 E IL-4 EM PACIENTES COM HANSENÍASE E EM SEUS COMUNICANTES
Mirian Lane de O Rodrigues Castilho, Maria Aparecida Nunes Ferreira,Norma T Foss
Disciplina de Dermatologia do Departamento de Clínica Médica da Faculdade de Medicina de Ribeirão Preto- USP.
O BCGid é preconizado para a imunoprofilaxia da hanseníase. Apesar de freqüentemente utilizado, especialmente em comunicantes de doentes de áreas endêmicas, os seus mecanismos imunológicos de estimulação protetora são ainda pouco conhecidos para a doença. Como o desenvolvimento da hanseníase correlaciona-se diretamente com a resposta imunecelular, avaliou-se a relação entre a aplicação de uma dose de BCGid em doentes e comunicantes, associando com alterações imunecelulares. A avaliação foi através da linfoproliferação e da quantificação das citocinas IFN-γ. IL.-12. IL-10 e IL-4. Verificou-se ainda se houve diferença entre a resposta imunecelular induzida pelo BCGid em doentes e comunicantes. Foram avaliados 34 indivíduos, antes e após o BCGid. sendo 15 doentes e 19 comunicantes sadios. A linfoproliferação foi desenvolvida na presença de Con-A, BCG e HSP-65. durante 96 horas. Os sobrenadantes foram coletados e estocados a - 70C para dosagem de citocinas, e a proliferação foi avaliada pela incorporação de 3H-timidina. Linfócitos de doentes e comunicantes apresentaram maior proliferação na presença de Con-A e BCG. Comparando-se os resultados antes e após a aplicação de BCGid. foram notadas maiores respostas nos indivíduos submetidos ao BCGid (p<0,05). Foi observado também que as células dos comunicantes pós BCGid apresentaram maior capacidade de estimulação na presença dos antígenos do BCG, quando comparadas às dos doentes (p=0,004). Os resultados da quantificação das citocinas (através do Elisa) mostraram que a aplicação de BCGid leva á maior produção de IFN-γ (p<0,05), sendo essa produção significativamente maior na presença do BCG em células de comunicantes que de doentes TT (p=0,004). IL-12. pós BCGid. apresentou níveis equivalentes em células de doentes e comunicantes (p=0.1713), frente ao estímulo BCG. Entretanto, a produção de IL-12 em comunicantes foi significativamente maior pré BCGid, na presença do BCG (p=0,0029), o que não se observou entre os doentes (p=0.4648). Além de IFN-γ e IL-12. BCG induziu a produção de IL-10, detectada em sobrenadantes de comunicantes em níveis significativamente maiores após o BCGid (p=0.0098), frente ao BCG. Esses resultados sugerem que a resposta imune predominante induzida pelo BCGid foi do tipo protetora, associando-se à detecção de IFN-γ. Assim, foi possível concluir que a aplicação do BCGid é útil em áreas endêmicas, pois pode induzir a resposta imunecelular específica das células do hospedeiro. A capacidade do BCGid induzir a ativação da resposta imunológica de doentes e comunicantes está associada à maior produção IFN-7 em ambos os grupos.
PI 13
CORRELAÇÃO ENTRE BCG INTRADÉRMICO E NÍVEIS DE ANTI-PGL-1 EM PACIENTES COM HANSENÍASE E EM SEUS COMUNICANTES
Mirian Lane de O Rodrigues Castilho, Maria Aparecida Nunes Ferreira, Norma T Foss
Disciplina de Dermatologia do Departamento de Clínica Médica da Faculdade de Medicina de Ribeirão Preto- USP.
O BCG intradérmico é preconizado para a imunoprofilaxia da hanseníase. Apesar de freqüentemente utilizado, especialmente em contatos de doentes de áreas endêmicas, os seus mecanismos imunológicos de estimulação protetora são ainda pouco conhecidos para a doença. Como o desenvolvimento da hanseníase correlaciona-se diretamente com a resposta imunecelular, avaliou-se a relação entre a aplicação de uma dose de BCGid em doentes e comunicantes, associando com alterações imunecelulares. Um dos métodos usados para a avaliação foi através da produção de anticorpos específicos do M. leprae (Anti-PGL-I). Verificou-se ainda se houve diferença entre a resposta imunecelular induzida pelo BCGid em doentes e comunicantes. Foram avaliados 34 indivíduos, antes e após o BCGid, sendo 15 doentes e 19 comunicantes sadios.
Foi utilizado o ensaio enzimático para detecção de anticorpos Anti-PGL-1 (Elisa Anti-PGL-1). Os níveis dos anticorpos Anti-PGL-1 no soro de pacientes e comunicantes foram avaliados antes e após uma dose de BCGid, sendo observados baixos níveis de anticorpos Anti-PGL-1 em tuberculóides (= 1,86), médios em borderlines (= 4,56) e elevados em virchovianos (= 15,75), correlackwando-se com as baciloscopias. Os níveis dos anticorpos Anti-PGLl no soro dos comunicantes foram menores do que aqueles encontrados nos doentes (p<0,05). Após a aplicação do BCGid, houve diminuição significante dos níveis de Anti-PGL-1 em doentes e comunicantes (p<0,000l), o que pode sugerir que o BCG induz a ativação da resposta imunecelular (tipo Th1), potencializando a destruição dos bacilos pelos macrófagos e capacitando a defesa específica dos doentes. Os resultados tornam-se relevantes, porque até a avaliação pós BCGid, os doentes permaneceram sem tratamento específico. A redução dos níveis dos anticorpos específicos ocorreu nos pacientes, independentemente de nível baixo ou mais elevado e de forma clínica da doença. Portanto, a capacidade do BCGid induzir a ativação da resposta imunológica de doentes e comunicantes pode estar associada à queda dos níveis dos anticorpos Anti-PGL-1, em ambos os grupos.
PI 14
CRIPTOCOCOSE EM PACIENTE DE HANSENÍASE: RELATO DE CASO
Joel Carlos Lastória, Maria Regina C. Silvares, Ana Paula Delgado. Maria Stella M. A. Putinatti
Depto de Dermatologia F. M. de Botucatu - UNESP
Introdução: Doença infecciosa causada por levedura de distribuição universal, o Cryptococus neoformans, disseminada através de dejetos de pássaros e adquirida através de inalação. A infecção cutânea em indivíduos sadios é rara. No entanto, sua freqüência aumenta em adultos com doenças sistêmicas como lúpus erilematoso, linfomas, em estados de imunossupressão de origem infecciosa ou medicamentosa.
Relato do caso: Os autores apresentam caso clínico ocorrendo em paciente de 39 anos de idade, pedreiro, portador de hanseníase virchoviana tratada, mas apresentando surtos freqüentes de eritema nodoso hansênico controlados há cerca de 6 anos com doses variadas de corticosteróides sistêmicos que há 6 meses apresentou lesão cutânea ulcerada diagnosticada como criptococose e tratada com Huoconazol, tendo evoluído com cicatrização da lesão. Os autores discutem e chamam a atenção sobre a ocorrência de imunossupressão iatrogênica na tentativa de se controlar complicação importante da hanseníase que é o ENH.
Considerações finais: Trata-se de patologia cutânea incomum ocorrida por imunossupressão iatrogênica.
PI 15
CYTOKINE LEVELS CORRELATE WITH MULTIDRUG-RESISTANT PULMONARY TUBERCULOSIS IN RIO DE JANEIRO
Fortes A., Antas P.R.Z., Daleolmo M.*, Milagres A.***, Oliveira E., Hijjar M.A.*. Pereira K. Kritski A.**. Ottenhoff T.H.M.#. Sampaio E.P.
Leprosy Laboratory, Oswaldo Cruz Institute, FIOCRUZ, Rio de Janeiro; *Reference Center Hélio Fraga, Ministry of Health - RJ; ***Departament of Pneumology, Hospital Raphael de Paula e Souza -RJ**; Department of Pneumology, Federal University of Rio de Janeiro; #Department of Blood Transfusion, Leiden University, The Netherlands.
Introduction: Resistance of M. tuberculosis to antimycobacterial agents has recently received increased attention worldwide. The participation of T cell response in multidrug resistant TB is not yet clearly understood. In addition, progression to fatal outcome in these patients might be related to enhanced inflammatory response in vivo.
Aim: To evaluate T cell and inflammatory response in MDR patients in comparison toTB patients not MDR.
Methods: MDR TB cases were defined as resistant to at least INH e RMP. The immune response was evaluated in 12 MDR patients who were tested negative (ELISA) for HIV. Peripheral blood was collected before the initial specific MDR treatment. For detection of cytokines. IFNγ. sTNF-RII (p75), and 1L-6 were measured in PBMC cultures stimulated or not with PPD and the recombinant antigens FSAT-6 and 85B. Supernatants were harvested either after 20 or 72h (IL-6, TNF-RII) and after 5 days (IFNγ) of culture and were assayed by specific ELISA.
Results: Preliminary immunological analysis showed lower IFN levels in response 10 ESAT-6 in MDR patients (mean SEM = 590 223pg/ml) when compared to pulmonary TB patients (n = 50; mean = 1553 420pg/ml). and similar to the response of PPD negative healthy donors (491 74pg/ml). More interestingly. IFNy in response to PPD in the MDR group (mean = 431 260pg/ml) was also lower when compared lo both groups. TB patients (1564 1 l(lpg/ml) and controls (1332+411 pg/ml). Evaluation of the inflammatory response in MDR was showed to be up-regulated, since values of soluble TNF-R in these cultures were 753 384pg/ml (72h culture) vs. 2067 923pg/ml in the TB group. In addition, for IL-6, constitutive cytokine levels were 4744 832pg/ml in the TB patient's vs. 1468 878pg/ml in the MDR.
Conclusion: The data indicate that T cells from MDR patients respond to mycobacterial antigens in vitro at a lower extent when compared to TB patients and that inllammatory responses are also exacerbated. Follow-up studies are still necessary to determine whether worsening of clinical conditions in MDR are related to such immunological parameters.
PI 16
CYTOKINE mRNA EXPRESSION IN THE EPIDERMIS OF LEPROSY PATIENTS: DIFFERENTIAL TNFα mRNA REGULATION DURING INFLAMMATION
Teles R.M.B ., Moraes M.O., Geraldo N.T.R., Salles A.M., Sarno E.N., Sampaio E.P.
Laboratorio de Hanseníase FIOCRUZ Rio de-Janeiro.
Introduction: The epidermis can represent an important site of immuno-inflammatory response in the skin. In leprosy, histopathological alterations are described both in the dermis and epidermis, mainly during the reactional states (reversal reaction. RR and erythema nodosum leprosum, ENL).
Objective: To evaluated the expression of cytokinesand ICAM-1 genes by RT-PCR in the epidermis of reactional leprosy patients.
Methodology: Skin biopsies were collected of the 25 reactional leprosy patients. RNA was extracted from the dermis and epidermis and RT-PCR was performed to â-aclin. TNFa, IL-6, IL-8. IL-12 and ICAM-1. The amplified products were analyzed through electrophoresis in agarose gel and the radioactive hybridization was performed with specific probes to all molecules.
Results: Detection of TNFα and IL-6 mRNA in the epidermis was evidenced in all individuals during ENL and RR. IL-8 message was delected in 66.6 and 62.5% of the patients, IL-12 mRNA was present in 91.6 and 62.5% and ICAM-I in 100 and 71.4%, respectively. In addition, when skin biopsies were obtained from the same patients before and during the reactional episode, an enhancement in cytokine mRN,. but not of ICAM-1, was noted. Seven patients were also evaluated at the onset of reaction and during anti-inflammatory treatment. In contrast to a preferential decrease in the TNFα gene detected in the dermis, during the treatment phase, a persistent/enhanced TNFa mRNA expression was detected in the epidermis in 6 out of the 7 patients assessed.
Conclusion: The present data indicate that the epidermis has an important participation in the local inflammatory response in leprosy and it seems to parallel the histological changes observed in situ.
PI 17
CYTOKINES QUANTIFICATION IN SUPERNATANT OF MONONUCLEAR CELL CULTURE OF PATIENTS WITH LEPROSY: PRELIMINARY RESULTS
Fátima Vilani-Moreno 1; Esther Nogueira1; Eliane Silva1; Elaine Marcos1; Somei Ura1; Diltor Opromolla1; Pran Das2; Ben Naafs3
1Equipe Técnica de Imunologia - Instituto Lauro de Souza Lima. Bauru - SP. CP 3021. www.ilsl.br
2Department of Pathology. Amsterdan Medical Centre, The Netherlands
3Department of Dermatology. Leiden University Medical Centre. The Netherlands
Objective: to evaluate the cytokine profile in the supernatant of a mononuclear cell culture of patients with leprosy, at the moment of diagnosis and six months after multidrugtherapy.
Methods: mononuclear cells from peripheral blood from 15 patients (5 LL. 7 B. 3 TT) were cultivated, in 37ºC and 5% CO2, for 24 and 48 hours in the presence and absence of PHA (8g/ml), LPS ( 10g/ml) and integral M. leprae ( 10 bacilli/cell). The supernalants were collected and the cytokines IL-2 and IFNγ (Th1,) IL-4 and IL-10 (Th2), IL-1 and TNFα were quantified by the ELISA technique (R&D Systems).
Results: multibacillary patients (12) produced greater levels of IL-4 before treatment (PI1A= 80 ± 72 pg/ml; M. leprae= 4 ± 7 pg/ml; spontaneously= 10 ± 18 pg/ml) than six months after the treatment (PHA= 37 ± 55 pg/ml; M. leprae= 2 ± 2 pg/ml; spontaneously= 3 ± 2 pg/ml) and high levels of IL-10 before and after multidrugtherapy (PHA= 2392 ± 1673 pg/ml; M. leprae= 430 ± 623 pg/ml; spontaneously- 790 ± 1030 pg/ml: PHA= 2489 ± 1332 pg/ml: M. leprae= 564 ± 33 I pg/ml: spontaneously = SI I ±613 pg/ml. respectively).
Conclusion: the results obtained suggest that multibacillary patients produce high levels of cytokines of the Th2 pattern (IL-4 and IL-10) at the moment of disease diagnosis.
PI 18
DETECTION of TNFα mRNA EXPRESSION BY DIRECT IN SITU RT-PCR IN THE PBMC OF PATIENTS WITH LEPROSY
Teles. R.M.B. , Aarestrup. F.M., Sarno. E.N., Pinto. G.S., Sampaio. E.P.
Laboratorio de Hanseníase FIOCRUZ Rio de Janeiro
Introduction: TNFα is an important cytokine in leprosy pathogenesis and it has been shown to be involved in the immuno-inflammatory processes during this infectious disease.
Objective: To standardize the method of the direct in situ RT-PCR for evaluation of TNFα mRNA expression induced by M. leprae in vitro.
Methodology: PBMC of 7 leprosy patients were isolated by Ficoll-hypaque density centrifugal ion, plated on Teflon beakers and stimulated or not with LPS (lOng/ml) or M. leprae (Ig/nil) for 1 and 3 hours. Amplification for TNFa was performed by direct in situ RT-PCR, using digoxigenin-dUTP.
Results: Detection of TNFα mRNA positive cells (blue-black staining) was higher in the LPS-stimulated cultures when compared with the unstimulated cells in all experiments. Similar results were found using M. leprae stimulated cells. TNFα mRNA expression in the PBMC from one BT patient was detected by direct in situ RT-PCR in the unstimulated. M. leprae- and LPS- stimulated cultures. It was possible to detect a qualitative difference between unstimulated and stimulated cells, which contain a higher number of positive cells (blue-black staining) as compared to the former one. Moreover, after 1 hour of stimulation, the number of positive cells was higher than after 3 hours. The same kinetic response For TNFα mRNA expression was obtained in both, solution RT-PCR and in situ RT-PCR. M. lepra estimulated PBMC showed higher amount of TNF mRNA 1 hour after the stimulus.
Conclusion: The in situ RT-PCR method will allow the more precise determination of the amount of cells that actively express cytokine message. Current experiments are being developed to determine the differential profile of TNFα synthesis by monocytes and T-lymphocytes in vitro.
PI 19
DIFFERENTIAL SERODIAGNOSIS OF LEPROSY AND TUBERCULOSIS BY IMMUNOBLOT BAR READING AND ELISA USING SHARED MYCOBACTERIAL ANTIGENS
Burggraaf, J.D.1, Verhagen, C.E.1,2, Baas, J.C.3, van den Bos. I.C.1, Faber, W.R2, Naafs, B.1,4,5, Fleury, R.5 and Das. P.K. 1,2,3
Departments of 1Pathology and 2Dermatology, Academic Medical Center. University of Amsterdam:
3Instituteof Public Health Bilthoven;
4Department of Dermatology of Leiden University, Medical Center Leiden, The Netherlands and
5Institute "Lauro De Suza Lima", Bauru, Brazil.
Mycobacteria are composed of complex mixture of antigens and many of them are shared by all mycobacterial species. Owing to the ubiquitous nature of mycobacteria, human sera always show serum antibody activities to shared mycobacterial (Smyc) antigens. Studies on the sero-antibody responses to Smyc antigens by immunoblot assay showed that hosts recognise different antigenic bands in the fashion of "bar" as disease specific manner. This is an important basis for discriminating leprosy patients from tuberculosis patients and patients with other types of inflammation. The group of leprosy patients were histopathologically and clinically classified. The present report is the analysis of a total of 200 patients (130 leprosy. 75 tuberculosis and 75 patients with non-mybacterial imflanimation by immunoblot 'BAR' reading (ImBBR) method). The results show that 99% of LL and 77% of BL patients'sera reacted with a doublet 29/33 KD antigens whereas TT and BT sera (94% and 63% respectively) reacted to a 64/65 KD singlet Smyc antigen. Interestingly, sera of BB patients did not show any clear cut reactivity pattern rather a smeartype pattern. In contrast sera from tuberculosis patients, that were clinically and micro-biologically defined, reacted with a group of Smyc antigenic bands, e.g. 58-60 KD. 38-40 KD. 18-22 KD. 14-16 KD regions. In parallel, enzyme linked immunosorbent assay (ELISA) for measuring IgG. IgM and IgA antibodies using gel purified 29/33 KD doublet, 64/65 KD singlet and a subcellular pellet fraction (P90) Smyc antigens was used. The ELISA results show that different types of leprosy can be discriminated distinctly from each other and from tuberculosis as well as from control patients. Moreover, since the immunoblots and antigen coated microtitre plates are stable at room temperature such combined assays can be used for screening population in the countries endemic for both of these pathogenic mycobacterial infection. In conclusion it appears that the combination of ImHBR and ELISA using either antigen mixtures or a panel of isolated Smyc antigens will be a valuable tool for identification of potential leprosy and tuberculosis patients in endemic population.
PI 20
DIFFERENTIAL TRAFFICKING OF Mycobacterium tuberculosis AND Mycobacterium leprae IN HUMAN MONOCYTIC THP-1 CELLS
Indira Nath *, N. Vemuri* P. Malik*, Y.J. Murali Mohan*, T. Das**, M.J. Colston# and J.L. Krahenbuhl##
*Immunology Laboratory. Biotechnology Department, **Electron Microscopy Division.
Anatomy Department, All India Institute of Medical Sciences, New Delhi, India;
# National Institute of Medical Research. Mill Hill, London; ## National Hansen's Disease Program, Baton Rouge, Lousiana, U.S.A.
Supra vital dye -labelled Mycobacterium tuberculosis and Mycobacterium leprae and vacuolar markers were used to study the phagosomal biogenesis in host cells (THP-1. a human monocytic cell line) by Laser Scanning Confocal microscopy (LSCM) and Electron Microscopy. Fluorescein (green), or PKH26 (red)-labeled mycobacteria and two acidotropic probes Lysolracker Red DND-99 and Lysotracker Green-26 were used to monitor the events by LSCM while Rab5 and Cathepsin D were used to identify phagosomes and lysosomes by immunoelectron microscopy. Lysotracker probes localised preferentially within lysosomal compartments whereas phagosomes were identified by transferrin receptors. Live M. tuberculosis co-localised with transferrin-labeled organelles up to 48hrs. Whereas M. leprae co-localisation with transferrin was restricted to 6 hours only. The phagolysosomal fusion event also differed with both organisms, with viability of the organisms being the pre-disposing factor during this phenomenon. Interestingly, M. leprae co-localised with acidic organelles up to 48 hours while M. tuberculosis containing phagosomes resisted fusion with lysosomes. Results indicate that although cultivable and non-cultivable mycobacteria have evolved ways to circumvent the hostile environment of the macrophage, the mechanisms employed by them are varied.
PI 21
DISTRIBUTION OF HLA CLASS II ALLELES IN LEPROSY PATIENTS OF KAZAKH POPULATION
L.V. Saroyants , M.N. Boldyreva. I. A. Guskova, A.A. Juscenko, L.P. Aleksejev
Leprosy Research Institute, Astrakhan, Russian Federation
Institute of Immunology, Moscow, Russian Federation
Genetic factors play a significant role in susceptibility to infectious diseases. A great number of investigations to study HLA-antigenic distribution in leprosy were carried out mainly on Orients and Hindu. In Russia leprosy patients of Kazakh nationality are next to Russian patients. Meanwhile. HLA-genetic profile in leprosy patients of Kazakh population has not been studied. Our work is aimed at studying distribution of class II HLA-antigens in Kazakh population. Distribution of class II HLA-antigens of DRB1, DQA1 and DQB1 loci was defined in 52 leprosy patients and 60 healthy non-relatives of Kazakh nationality. HLA-genotyping was carried out by means of PCR. It was stated that in leprosy patients DRB1-01 and DRB1-17 antigens occurred more frequently (P<0,05). Frequency of DRB1-10. -09 and DQA1-601-alleles was considerably low as compared with healthy persons. Relative risk (RR) of the disease was 2.8 and 3,6 for DRB1-01 and -17. correspondingly.. The data obtained permit lo consider HLA-DRB1-01 and DRB1-17 antigens as genetic markers of susceptibility to leprosy in Kazakh population. Having regard to the fact that HLA-DRB1-17 allele enters into serologically defined HLA-DR3-specificity that is defined as a marker of leprosy susceptibility in different ethnic populations, one might suggest that the highest risk of leprosy is associated with increased frequency of haplotypes with above allele. Thus, the observed peculiarities of distribution of alleles of HLA-loci strongly necessitate investigations on HLA markers of diseases in different ethnic populations. It will permit a more accurate identification of risk groups, on the one hand, and target searches for universal markers of leprosy susceptibility, on the other.
PI 22
DISTRIBUTION OF II CLASS HLA ALLELES IN RUSSIAN LEPROSY PATIENTS
L.V. Sarovants , M.N. Boldyreva, I.A. Guskova, A.A. Juscenko, L.P. Aleksejev
Leprosy Research Institute, Astrakhan, Russian Federation
Institute of Immunology, Moscow, Russian Federation
Distribution of DRB1, DQA1 and DQB1 -antigens of II class HLA was studied in 55 leprosy patients and 50 healthy non-relatives of Russian nationality. HLA-genotyping was carried out in PCR. Leprosy patients showed antigens DRBI-15, DRB1-16, DQAI-102, DQB1-602/8 and DQBI-502/4 with more high frequency (P<0,05). Frequency of HLADQA1-301 allele was significantly low in leprosy patients as compared with healthy subjects. With correction for the number of test-antigens significant differences maintained for alleles of HLA-DQA1-102 and -DRBI-15 genes. A study of distribution of haplotypes in leprosy patients showed increased frequency of DRBI -15-DQAI -102-DQB1 -602/8 and DRBI-16-DQAI-102-DQBI-502/4 as compared with control. At the same time frequency of DRB1-11-DQA1-501-DQB1-301-haplotype was low in leprosy patients. Taking into account that HLA-DR1-15 and 16 alleles are a part of serologically detectable specificity of HLA-DR2 which was earlier defined by us as indicating leprosy susceptibility in Russian population, one might suggest that the highest risk of leprosy disease in Russian population should be associated with high frequency of haplotypes of the above alleles. The results obtained permit to consider HLA-DRB1 -15 and DQA1 -102 molecules as genetic markers of susceptibility to leprosy in Russian population. Thus, investigation of distribution of allelic loci of HLA-system when identifying specific haplotypes significantly increase the effectiveness of defining their associations with leprosy and, hence, permit a more accurate identification of risk groups based on genetic analysis.
PI 23
ESTUDO COMPARATIVO ENTRE REAÇÃO DE MITSUDA E FENOTIPAGEM HLA EM PACIENTES HANSENIANOS: RESULTADOS PRELIMINARES
Fabiana Covolo de Souza; Elaine Valim Camarinha Marcos; Somei Ura; Maria Esther Sàlles Nogueira
Instituto Lauro de Souza Lima, Bauru - SP Equipe Técnica de Imunologia E.mail: imunologia@ilsl.br
Sabe-se que na hanseníase as respostas imunológicas do hospedeiro determinam as formas clínicas da doença, verificadas pela reação de Mitsuda (teste incorporado como auxílio diagnóstico, principalmente, dos grupos indeterminado e dimorfo). Do ponto de vista genético, ha evidências de que o complexo HLA seja o responsável pelas diferentes formas da doença, mas não existem relatos de trabalhos que descrevam tal comparação.
Desta forma, temos por objetivo verificar se existe relação entre os resultados da reação de Mitsuda, formas clínicas da doença e fenótipos HLA encontrados nos pacientes que compõe o estudo.
Realizamos, até o momento, tipagem HLA classe II por PCR-SSP, em 75 hansenianos caucasóides (21HT; 26 HV; 28 HD) e comparamos os dados (freqüência HLA) com amostra da população caucasóide do estado de São Paulo (n=142).
Dos 21 pacientes HT, 20 são Mitsuda positivo e apresentam freqüência elevada do HLA-DR2 (52,4% x 19%); dos 26 pacientes HV, 24 são Mitsuda negativo e apresentam freqüência elevada do HLA-DQ1 (73% x 50%); e dos 28 HD, 11 são Mitsuda positivo e 17 negativo, contudo, não observamos freqüência elevada de qualquer fenótipo HLA.
Outro dado verificado nos pacientes hansenianos, independentemente das formas clínicas, é a diminuição do HLA-DR5 e DQ7. Acreditamos que se aumentando o tamanho da amostra, poderemos confirmar as associações observadas nesses pacientes.
PI 24
EVALUATION OF ADRENAL AND GONADAL FUNCTIONS IN LEPROSY: COMPARISON TO IMMUNE RESPONSE
A.M. Leal, P.K. Magalhães N.T. Foss , A.C. Moreira
Faculty of Medicine of Ribeirão Preto of São Paulo University.
Leprosy is a chronic inflammatory disease which not only involves skin and peripheryc nerves but also endocrine organs. This disease has a clinical and pathologic spectral nature associated with distinct immunologic response. In the present study an attempt has been made to assess the functional integrity of the hypothalamic-pituitary-adrenal (HPA) and gonadal (HPG) axis and their relatonships with the immune systems in leprosy. Ten multibacillary (MB, 40 3yr) and 8 paucibacillary (PB, 44 3yr) male untreated patients and 10 healthy controls (31 2yr) were evaluated. Day 1 at 9:00am: baseline plasma samples were taken for Cortisol, DHEA-S, LH, FSH, testosterone, TNFα, ILβ, IL6 and human CRH test (lg/Kg iv) was then performed. Day 2-9am, synthetic ACTH (124,250g) was given IV and plasma samples were collected at 60 minutes. After stimulation with hCRH the plasma ACTH and Cortisol area under the curve (AUC, 15-120min) did not differ between controls and patients. Compared to controls, total and net ACTH (1-24)- stimulated Cortisol levels were not different. TNF and IL6 were significantly elevated in MB and PB patients compared to controls (p0.01). IL1 beta was not different between controls and patients. Plasma DHEA-S levels were significantly lower in patients than in controls, but there was no difference between MB and PB patients. A negative correlation between DHEA-S and IL6 was observed(r-0.48: pO.01). Although plasma testosterone levels did not differ between controls and patients, LH and FSH were significantly higher in MB than in controls and PB patients. It was observed no correlation between plasma ACTH or Cortisol AUC and Interleukins. Regarding the HPG axis. LH and FSH levels were significantly correlated with IL6 0-0.46 and r=0.64, respectively; p0.01) and TNF 0-0.49 and 1-0.67, respectively; p0.01).These data suggest that DHEA-S, LH and FSH are best indicators of HPA and HPG axis function in leprosy and may be influenced by IL6 and TNFα.
PI 25
EVALUATION OF T CELL IMMUNE RESPONSE TO THE ESAT-6 HOMOLOGUE OF Mycobacterium leprae IN LEPROSY
Elizabeth P. Sampaio, Annemieke Geluk*, Paulo R.Z. Antas, Kees L.M.C. Franken*, Eliane B. Oliveira, Kelly C. Pereira, Andreia M. Fortes, Euzenir N. Sarno and Tom H. M. Ottenhoff*
§ Leprosy Laboratory IOC Fiocruz, Rio de Janeiro, Brasil; * Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, The Netherlands
In contrast to the highly homologous mycobacterial heat shock proteins, the ESAT-6 of M. leprae (L-ESAT) only shows 35% identity with its homologue in M. tuberculosis (T-ESAT). Based on the high specificity of the T-ESAT for immunodiagnosis of tuberculosis (TB), even in BCG-vaccinated individuals, it is argued whether the ESAT homologue in M. leprae could provide such a diagnostic reagent for the detection of leprosy. Thus, the T cell response against the recombinant protein L-ESAT was analyzed in Brazilian leprosy patients (n=23). TB patients (n=22), and healthy controls (n=15). Leprosy patients were 5LL, 6BL. 6 BT, and 6 reactional (RR). PBMC derived from most M. leprae responding patients produced IFN following in vitro stimulation with l0g/ml of L-ESAT (mean SEM = 384+ 125). A total of 40% lepromatous patients did respond to L-ESAT as compared to 83.3% tuberculoid, and 66.6% of the RR. Concordant responses between L-ESAT and whole M. leprae was found in 80% of the cases. However, TB patients (57.1 %) and healthy controls either positive (62.5%) or negative (71.4%) tuberculin skin test responded equally well to L-ESAT. Among the untreated TB patients (n=9), 66.6% responded to this antigen as did 50% of the treated TB (n=12). In addition, no striking differences in IFN levels induced by L-ESAT were found between patients and healthy controls derived from a tuberculosis/leprosy-endemic area, which excludes the use of L-ESAT as a diagnostic tool for leprosy.
PI 26
EVALUATION OF THE 10 MINUTES ML FLOW ASSAY USING WHOLE BLOOD
S. Amador2, G.C. Gussenhoven1, Edvaldo C.B. Loureiro2, S. Bührer-Sékula1
1KIT Biomedical Research, Meibergdreef 39, 1105 AZ Amsterdam, The Netherlands
2IEC, Av. Almte Barroso. 492, Belem, Pará.
We describe a further simplification of the ML Flow assay for the detection of antibodies to phenolic glycolipid I (PGL-1) of Mycobacterium leprae by using whole blood and evaluated the assay performance in the leprosy endemic area of Belem in Brazil. The agreement between results of the test performed using whole blood and sera was 85.9% (value= 0.7, SE=0.042). This simple assay is proposed for classification of leprosy patients after clinical diagnosis and identification of high-risk contacts of leprosy patients. Identifying and monitoring the contacts of leprosy patients with higher risk of developing leprosy may be a tool for the interruption of transmission of leprosy, one of the main challenges for leprosy control.
The ML Flow assay is a fast and easy-to-perform method for the detection of IgM antibodies to PGL-1 of M. leprae; it does not require any special equipment and the highly stable reagents make the test robust and suitable for use in tropical countries.
PI 27
EXPRESSION OF ANTI-INFLAMMATORY AND INFLAMMATORY CYTOKINES IN THE LESIONS OF T1R PATIENTS DURING PREDNISOLONE TREATMENT
Sara Atkinson, Anna K. Andersson, David Little,Saroj Khanolkar-Young , Sujai Suneetha and Diana N.J. Lockwood
Department of Infectious and Tropical Diseases, London School of Hygiene & Tropical Medicine, Keppel Street. London, WCIE 7HT, United Kingdom
BPRC, Hyderabad, India
This study investigates effect of prednisolone (30mg daily) on the expression of the cytokines in the lesions of patients with Type I reactions (T1R). The hypothesis that the inflammatory cytokines observed in T1R patients decrease with treatment and that the anti-inflammatory cytokines are concurrently increased has been tested.
Study: Skin biopsies were taken from 15 patients (6 BL and 9 BT) at time points (weeks 0, 1, 4 and 24) during depleting prednisolone treatment. Immunohistochemical analysis of the expression of the cytokines IFN-γ, TNF-α, TGF-1, IL-6, IL-12, IL-13, IL-10 and iNOS were determined.
Results: The inflammatory cytokines (TNF-α. IFN-γ, IL-12) levels were found to decrease with treatment (significantly by 4 weeks). This study also demonstrates that the levels of the anti-inflammatory cytokines IL-10 and IL-13 decrease (significantly by 4 weeks).
Conclusion: This work suggests that prednisolone non-specifically down regulates the whole spectrum of inflammatory cytokines rather than altering the pro-/anti-inffammatory cytokine balance and that this effect is not observed in skin lesions until 4 weeks after the start of treatment..
PI 28
EXPRESSION OF PERFORIN MRNA AT THE LESION SITE IN LEPROSY REACTION
Geraldo, N.T.R.; Teles, R.M.B.; Nery, J.C.A.; Sarno, E.N.; Sampaio, E.P.
Introduction: Perforin is a cytolytic pore-forming protein that colocalizes with granulysin in cytotoxic granules, and is responsible for the cytolytic activity of CD8+T cells. Perforin mRNA was detected in leprosy lesions and enhanced expression of perforin in the blood of patients with reaction (erythema nodosum leprosum, ENL and reversal reaction, RR) was also described.
Objective: To investigate the expression of perforin mRNA at the site of the leprosy lesion in patients with reaction and its induction following M. leprae stimulation in vitro.
Methods: Skin biopsies of 12 leprosy patients (10 BL and 2 LL) were collected and total RNA was extracted. For the in vitro experiments, PBMC was obtained from 5 patients, and kinectic cultures were established. Following RNA isolation, RT-PCR for perforin was performed, and the amplified products analyzed through electrophoresis in agarose gel.
Results: When comparing patients with reaction, ENL patients are likely to show higher relative amounts of perforin mRNA in the lesion than patients with RR. In addition, around 50% of the RR patients (n=7) expressed perforin message in the dermis, whereas, 100% of the ENL (n=3) were positive for this cytolytic mediator. Four of these patients were also evaluated during treatment for reaction and down-regulation of perforin mRNA was noted in 2 individuals. Kinectic evaluation of perforin mRNA following M. leprae stimulation showed to be similar for that of TNF-α as it peaks around 3h after adding the stimulus.
Conclusions: Perforin mRNA is expressed in the dermis of the reactional leprosy lesion. The present data suggest that cytotoxic mechanisms may play a role in the pathogenesis of reaction in leprosy.
PI 29
GANGLIOSIDE IMMUNO ASSAY EVALUATION FOR LEPROSY PATHOLOGICAL DAMAGE
Sardella, I.G., Schwerer, B., Saad, M.H.F.
Leprosy Laboratory, FIOCRUZ, RJ, Brazil), Neurology Institut, Wien University, Austria
In some forms of motor neuron diseases and peripheral neuropathy the occurrence of high titers of antibody against Gm1 gangliosyde has been described. Gm1 is an acid glycolipids composed of lipid and carbohydrate moieties. The carbohydrate portion of ganglioside contains sugars (gal and glue) and sialic acid. The carbohydrate portion of the gangliosyde could be the epitope in autoimmune reactions caused after nervous damage. Leprous neuritis caused by Mycobacterium leprosy is the most common peripheral neuropathy in developing countries. Since lipids such as gangliosydes, cerebrosides and sulfatides are known to be immunogenic and are present in peripheral nerve and perhaps the nerves are the first system to be attacked by M. leprae presenting along of the time irreversible damage, we investigate the existence of an auto antibody response to ganglioside in leprosy patients using Gm1 antigen (monosialoganglioside). Elisa Anti-Gm1 antibodies (IgG, IgM and IgA) were measured in sera from leprosy patients, household contacts and healthy individuals. Comparison of anti-Gm1 IgG, IgM and IgA rates between leprosy patients and healthy individuals did not show significant statistical difference (p>0,05). Antibodies levels were very low, 84% (85/101) of leprosy sera showed ▲ E<0.1 and only 3% (03/101) presented ▲ E>0.1. With Household contacts and Normal results were similar: 43% (21/49) showed ▲ E<0.1 and 8% presented ▲ E>0.1. Most of the sera presented high background: this could be, perhaps, by the use of detergent tween 20 in the block and washing solution. In fact Anti-Gm1 antibodies do not have value for evaluate pathological damage in leprosy.
Support: CNPq, FAPERJ
PI 30
HEPATITIS B AND C INFECTION AMONG LEPROSY PATIENTS ATTENDING THE SANATORIUM OF FONTILLES (SPAIN)
P. Torres1, J.R. Gomez1, J.J. Camarena2, J.M. Nogueira2, J.C. Navarro2
1Sanatorium San Francisco de Borja, Fontilles;2 Servicio de Microbiología, Hospital Universitario Dr Peset.
A possible association between infection by hepatitis viruses B (HBV) and C (HCV) and leprosy has been proposed. Hepatitis B (HBV) and hepatitis C (HCV) viruses are transmitted by blood (transfusions, parenteral injections) possibly sexual contacts and probably other unknown routes. They can cause chronic liver disease. Populations with increased risk of these viral infections, specially patients with hemophilia and on hemodialysis have been identified. Patients with leprosy possibly also form a high risk group because of skin lesions, blood transfusions and confinement in institutions during prolonged periods of time. Some consider that the 2 polar forms of leprosy (tuberculoid and lepromatous) provide a model of interaction between cellular immunity and the hepatitis viruses.
In this study, the distribution of HBV and HCV virus markers were evaluated in 214 leprosy patients mostly long term institutionalised in the Sanatorium of Fontilles and compared with matched controls, using the same protocols required for screening of blood donors. Initially, two third generation microparticle enzyme immunoassays and positive results were confirmed by PCR methods.
The HBsAg and HCV positivity rates were 6% and 35% respectively, significantly higher than in the corresponding control groups (2% and 3.5%). The influence of possible risk factors (blood transfusion, confinement in leprosaria during prolonged periods of time, open skin lesions etc.,) on this group of patients is discussed.
PI 31
HUMAN T CELL RESPONSES TO PEPTIDES OF THE Mycobacterium leprae 45-KDA SERINERICH ANTIGEN
S. Brahmbhatt1, R. Hussain2, S. Zafar3, G. Dawood4, T.H.M. Ottenhoff5, J. W. Drijfhout5, G. Bothamley6,
S. Smith1, F. Vega Lopez7, H.M. Dockrell1
1London School of Hygiene & Tropical Medicine, London WC1E 7HT, UK; 2Aga Khan University, PO Box 3500, Karachi 74800, Pakistan; 3Marie Adelaide Leprosy Centre, PO Box 8666, Karachi 74400, Pakistan; 4Masoomeen Hospital, Karachi 53, Pakistan; 5Leiden University Medical Centre, Postbus 9600. 2300 RC Leiden, The Netherlands: 6Homerton Hospital, London E96 SR. UK; 7The Middlesex Hospital. London, W1N 8AA. UK.1
In order to identify T cell epitopes within the Mycobacterium leprae 45-kDa serine-rich antigen, T cell responses to overlapping 17-mer peptides encompassing the whole antigen were analysed in non-exposed UK controls, Pakistani leprosy patients and tuberculosis patients in both the UK and Pakistan. This antigen has been described as M. leprae-specific, although it has a hypothetical homologue in Mycobacterium tuberculosis. Peripheral blood mononuclear cells were stimulated with peptide for 5 days and IFN-γ measured in supernatants by ELISA. Some peptides were more frequently recognised by T cells from tuberculoid leprosy patients than those from UK controls, suggesting that such T cell epitopes might have diagnostic potential. Short-term cell lines and How cytometry confirmed specific T cell recognition of these peptides. However, T cells from many tuberculosis patients also recognised these potentially specific peptides suggesting that there could be a true 45-kDa homologue present in M. tuberculosis, or that tuberculosis patients living in a leprosy-endemic area have also been exposed to M. leprae.
PI 32
IDENTIFICATION AND CHARACTERIZATION OF THE ESAT-6 HOMOLOGUE OF M. leprae AND T CELL CROSSREACTIVITY WITH M. tuberculosis
Annemieke Geluk*. Krista E. van Meijgaarden*, Kees L.M.C. Franken*, Yanri W. Subronto*, Brigittc Wieles*, Sandra M. Arend∇*, Elizabeth P. Sampaio∆, Tjitske de Boer*, William R. Fabef¶, Ben Naafs#, and Tom H. M. Ottenhoff*
*Department of Immunohematology and Blood Transfusion. #Department of Dermatology and ∇Department of Infectious Diseases, Leiden University Medical Center, The Netherlands, ¶Department of Dermatology, Amsterdam Medical Center, The Netherlands, ∆Leprosy Laboratory IOC Fiocruz, Rio de Janeiro, Brazil.
The present study describes the identification and characterization of M. leprae ESAT-6 (L-ESAT). the homologue of M. tuberculosis ESAT-6 (T-ESAT). TESAT-6 is expressed by all pathogenic strains belonging to the M. tuberculosis complex, but absent from virtually all other mycobacterial species, and is a promising antigen for immunodiagnosis of TB. Therefore, we have analyzed whether L-ESAT-6 represents a similarly powerful tool in leprosy, by examining T cell responses against L-ESAT-6 in leprosy patients, TB patients and exposed or non-exposed healthy controls from leprosy/TB endemic and nonendemic areas. L-ESAT-6 was recognized by T cells from leprosy patients, TB patients. TB patients' contacts and healthy individuals from a TB/leprosy endemic area, but not by non-M. tuberculosis, non-M. leprae-exposed individuals. Moreover, M. leprae-unresponsive leprosy patients failed to respond to LESAT-6. A very similar pattern was seen in case of TESAT-6. These results show that L-ESAT is a potent M. leprae antigen that stimulates T cell-dependent IFN-γ production in a large proportion of M. leprae exposed individuals. Moreover, our results suggest the existence of significant cross reactivity between T- and L-ESAT-6. which has implications for the use of ESAT-6 as diagnostic tool for diagnosis of leprosy and TB in areas endemic for both diseases.
PI 33
IMMUNE SERIC PARAMETERS IN LEPROSY REVERSAL REACTION
Sales. A.M .; Nery, J.A.C; Oliveira. R.B.; Machado. P.; Scheinberg. M.; Sampaio, E.P.; Sarno, E.N.
Leprosy Laboratory, Oswaldo Cruz Foundation (FIOCRUZ), Rio de Janeiro. R.J.. Brazil
Introduction: It is well known that reactions are commonplace occurrences during the course of leprosy disease representing a clinical challenge in view of the immune response involved. In terms of immunology, the acute clinical episodes taking place in some chronic diseases are attributed to an exacerbation of the immune-inflammatory response expressed through seric and cellular markers, which are clinically evaluated, for example, by way of the erythrocyte sedimentation rate (ESR), reactive protein C, and nuclear activity factor. Other markers, like Neopterin, β2-microglobulin, and tumor necrosis factor (TNF) have recently been shown to also be significant in this context. In leprosy, however, while the Lepromin test and the linfoproliferative assay are capable of confirming exacerbation of the immune response, no clinical or laboratory evidence has yet been reported to uphold this claim.
Objective: To evaluate the serum markers used to assess immunological activity during and after reversal reaction (RR).
Material and Methods: The first reactional episodes of 21 multibacillary (MB) leprosy patients who developed RR during specific MDT treatment were studied. The patients were classified according to the Ridley and Jopling criteria, having been submitted to routine clinical, histopathological, bacteriological, and immunological tests at diagnosis and then at the onset of a reactional episode. Neopterin and β2-microglobulin levels, tumor necrosis factor, and soluble TNFα receptors 1 and 2 were likewise assessed at the beginning of the RR episode and after treatment.
Results: Slightly over 90% (90.5%) of the first reactional episodes occurred during the first year of treatment; and the great majority of the patients (71,4%) experienced only one. During reaction, increased levels of the studied markers, which declined after treatment, were observed. Increased values of neopterine (66,6%) were seen more frequently during RR than were other markers, such as β2-microglobulin (62%). The levels of these two markers showed a statistically significant (p= 0.007 and p=0,01. respectively) regression pattern subsequent to RR treatment.
PI 34
IMMUNOEPIDEMIOLOGICAL MONITORING OF LEPROSY
A.A. Juscenko, M.N. Dyachina, V.V. Duiko, O.V. Degtyarev, VP. Tsemba
Leprosy Research Institute, Astrakhan, Russian Federation
Identification of risk groups for leprosy disease among contacts and population of regions with sporadic cases of leprosy is rather topical. Survey was carried out of 316 household contacts with leprosy cases and 516 inhabitants of Astrakhan region where sporadic cases of leprosy are registered. Epidemiological monitoring involves specific serological diagnosis (ELISA) and skin tests. In DIS-BSA-based ELISA anti-PGL-1 antibodies and antibodies against M. leprae sonicates were determined. DTH response to lepronin (Leprosy Research Institute, Astrakhan, Russia) and leprosin A (WHO Bank) was determined. As controls 150 volunteers out of inhabitants of non-endemic for leprosy regions of Russia were used. Among 316 contacts 39 subjects showed positive serological results (12.3%), and among 516 inhabitants anti-M. leprae antibodies were observed in 9 cases (1,7%). Levels of anti-PGL-1 antibodies (0,23+0,05) and anti-M. leprae antibodies (0,27 ± 0,12) in contacts significantly differed from indices in control subjects (0,08 ± 0,04 and 0.13 ±0,01. respectively. Serologically positive contacts were investigated each 6 months during 2 - 2,5 years. Contacts with permanently high titers of antibodies against M. leprae were given additional 6-months' course of preventive therapy resulting in serological conversion in some of them. Among 138 contacts 28% gave a positive reaction to lepronin and 35% to leprosin A (coincidence for the two antigens 81%). Whereas among 419 inhabitants positive reactions to lepronin were observed in 21% and to leprosin A - in 18% (coincidence for the two antigens 88%. Thus, the data of serological monitoring and skin testing of household contacts and general population of leprosy endemic regions favor for identification of risk groups and might serve as additional characteristic of epidemiological situation in a region.
PI 35
IMMUNOLOGICAL PROPERTIES OF THE Mycobacterium leprae HLP PROTEIN
Rodrigues. L.S., Antunes, S.L.G., Lima. M.C.B.S., Martins, M.V.B.S., Sarno, E.N., Pessolani, M.C.V.
Leprosy Laboratory, Oswaldo Cruz Institute, FIOCRUZ, Rio de Janeiro, Brazil
Mycobacterium leprae Hlp (histone-like protein) is a cationic protein that is able to bind the laminin α2 and other extracellular matriz proteins present on the surface of Schwann cells (Marques et al., Microbes and Infection. 2:1407, 2000). Besides its potential role as a M. leprae adhesin, it has been reported that a Hlp found in a number of Streploccocus species may play a role in the pathogenesis of bacterial-induced tissue inflammation (Choi et at., Clin. Immunopathol. 76:68. 1995). In this context, it has been speculated that Hlp can accumulate within the basal lamina of infected nerves inducing tissue damage in leprosy. To elucidate its potential role in the immunopathogenesis of leprosy, the present study investigated the immunological properties of Hlp and its expression in infected tissues. Hlp was able to elicit high levels of IFN-γ secretion in mononuclear cells isolated from bordeline tuberculoid leprosy patients. Experiments are under way to determine the capacity of Hlp to induce TNF-α secretion in the same cultures. Additionally, the presence of antibodies specific to Hlp was detected in leprosy patient's sera, indicating that this protein is able to induce both cellular and humoral immune responses in M. leprae infected individuals. Immunohistochemical analysis using a specific antibody anti-Hlp showed that the protein is expressed in the cutaneous infiltrates of leprosy lesions as Hlp-positive phagocyled materials inside macrophages. These data indicate that Hlp is immunogenic and could contribute to tissue inflammation in leprosy. Currently, we are analysing the presence of Hlp in nerve biopsies to define its potential role on the persistent inflammation and delayed sequelae observed following M. leprae endoneural infection.
Supported by CNPq and NIH.
PI 36
IMPORTÂNCIA DAS PROVAS INFLAMATORIAS INESPECÍFICAS NA EVOLUÇÃO DO TRATAMENTO DA HANSENÍASE MULTIBACILAR
Augusto S. Monteiro Jr, Fabiana M. Silva, Sarah Saul, Vania S. Manso, Sidney de Souza Lima
Hospital Infantil Darcy Vargas. Rua Seraphico de Assis Carvalho, 34- Morumbi cep 05614-040 Fone 37233753
O sistema imune responde geralmente a agentes lesivos animados, microorganismos, com a produção de anticorpos e células sensíveis. Anticorpos são proteínas originadas na progênie dos linfócitos B, os plasmócitos,caracterizados como globulinas,e portanto pela alteração dos seus níveis plasmáticos podem caracterizar a presença de infecção, embora se compreenda de forma inespecífica.
Uma infecção pode ser rastreada quanto à sua evolução através da repetição destes exames que podem evidenciar a melhora ou não do processo infeccioso,no caso a hanseníase multibacilar.
Nosso serviço pela simplicidade do armamentário diagnóstico laboratorial disponível tem lançado mão daquilo que chamamos de "provas funcionais de proteínas inflamatórias", acrescidas do leucograma para evidenciar leucocitose.
Os resultados obtidos mostram que de uma forma mais simples, em serviços mais periféricos descontrole da Hanseníase, este rastreamento pode ser uma forma a mais de consolidar a alta clínica.
O estudo foi realizado na Unidade Básica de Saúde de Itapevi, estado de São Paulo, onde foram acompanhados 23 pacientes multibacilares. Foram solicitadas provas inflamatorias durante o curso de tratamento. De acordo com o estudo pudemos evidenciar que os únicos exames que parecem estar relacionados com critérios para alta de multibacilares são proteína C reativa (positiva em 40% dos casos) eVHS (positiva em 60%)
PI 37
INDUCTION OF CELL DEATH (APOPTOSIS) BY TNFα AND TGFβ IN A HUMAN SCHWANN CELL LINE IN VITRO
Oliveira R.B ., Sampaio E.P., Sanio E.N.
Leprosy Laboratory, Oswaldo Cruz Insitute, FIOCRUZ, Avenida Brasil 4365, Manguinhos, Cep 21045-900, Rio de Janeiro, R.J. Brasil
A major complication in leprosy is the development of deformities along the chronic course of disease. In the present study, we used a human malignant Schwann cell line to characterize mechanisms of cell death in vitro. The ST88-14 tumor cell line was established from malignant schwannoma of neurofibromatosis (NF1) patient. The cells (SC) were grown in complete RPMI medium. The purity of Schwann cells was assessed by morphologic examination through Wright Giemsa, toluidine blue staining and S-100 protein. In the present study, we investigated physiological and morphological characteristics of ST8814. Cytokine gene expression and secretion was assessed by RT-PCR and ELISA respectively. Constitutive mRNA (for TNFα, TNF-R1. TNF-R2. IL-8, ICAM-1 and c-fos) was present in these cultured cells. Albeit TNFa gene expression was detected, no TNFα protein was observed in culture supernatants. However, soluble TNF-R1 and TNF-R2 were released in the culture medium. FACS analysis demonstrate, for the first time, expression of both TNF-R on the human SC surface. When SC were cultured in the presence of TNFα (10ng/ml) and TGF↓ (40ng/ml), around 30% of cell death was detected in vitro, a 3 fold enhancement when compared to the unstimulated cultures. No significant effect was noted when either TNF or TGF↓ were used as the stimulus. In order to determine whether the sineraistic effect of TNF and TGF↓ leads to apoptosis in vitro, adherent and free cells in the culture medium were stained with propidium iodide and analyzed by How cytometry. Preliminary results demonstrate an increase in the subdiploid peak in cells cultured in the presence of TNF/TGF as compared to the controls. The present data indicate that expression of the TNF and TNF-R genes in the SC may have implications in the pathogenesis of nerve damage in leprosy and induction of cytokine-mediatted SC death in vivo.
PI 38
INHIBITION OF M. leprae-INDUCED APOPTOSIS AND CYTOKINES PRODUCTION BY THALIDOMIDE AND ANALOGUES
Elizabeth P. Sampaio,Daniel S. Carvalho, Jorgenilce S. Sales, Eliane B. Oliveira, Tatiana O. Fulco, Rodrigo C. Peres, Paulo R. Z. Antas, Shannon E. J. e Euzenir N. Sarno
Leprosy Laboratory, IOC/FIOCRUZ, Av. Brasil 4365, Manguinhos, Rio de Janeiro, Brasil. E-mail: esampaio@gene.dbbm.tiocruz.br
One of the clinical manifestations of leprosy is the erythema nodosum leprosum (ENL) characterized by enhanced TNF levels. IFN and IL-12 were shown to be up-regulated during the reactions and are likely to be involved in the high TNF production in response to M. leprae in vivo. The use of thalidomide (THAL) in the treatment of several pathologies has been described by several authors. The search for new stable analogues with increased effectiveness and lower side effects has been requested. In order to conceive the mechanisms of THAL action, the M. leprae-'mduced production of IL-12 and TNF (secretion and mRNA synthesis), and its impact on cell death was investigated in cells culture. PBMC was stimulated with irradiated M. leprae (10g/ml) in the presence of THAL and analogues (25g/ml). RT-PCR performed of cultured cells 3h after stimulation displayed a decreased TNF and IL-12 mRNA by effects of THAL and N-hydroxyphtalimide (NH). After 20h of culture, TNFot production (2306 190pg/ml, n=5) was decreased by THAL (64 ± 12%), NH (81 ± 8.7%) and N-butylphtalimide (NB, 48 ± 4.4%). N-methylethylketonephtalimide (NK) showed no effect so far. Analysis of intracellular cytokine staining demonstrated an enhancement in TNF positive cells for both CD4+ and CD8+ T subsets induced by M. leprae, which was higher in the former group (3.7 ± 0.8% Vs. 1.3 ± 0.3%). In the presence of THAL, CD8+ T cells seem to be more affected than CD4+ T cells. When incubated with THAL, analysis of the rate of apoptosis induced by M. leprae in monocytes showed a reduction of 58,3%. Our preliminary data indicate that THAL, NB and NH lead to inhibition of pro-inflammatory cytokines on both monocytes and T cells, and the potential role to modulate M. lepraeinduced features is evidenced.
PI 39
INVOLVEMENT OF B CELLS IN LOCAL IMMUNITY OF LEPROSY PATHOLOGY
Sengupta U.l,2, Moens A.2,3, Mohanty K.1, Katoch K.1, Faber W.R.3, Bellone A.4, Fleury R.4, Naafs B.2,4,5 and Das P.K.2,3
1Central JALMA Leprosy Institute, Agra. India;
Departments of 2Pathology and 3Dermatology Academic Medical Center UvA, Amsterdam, The Netherlands;
4Instituto "Lauro De Suza Lima", Bauru, Brazil;
5Department of Dermatology Leiden University Medical Center (LUMC), Leiden, The Netherlands.
Mycobacterium leprae responsive T cell subsets are regarded to control the clinical and immunological spectrum in leprosy. On the otherhand. the longlasting m.leprae specific antibodies are diagnostically important, despite approximately 60% of paucibacillary (PB) patients can not be diagnosed by sero antibody assays. Low level of systemic antibody in PB patients can be due to the absence of optimal antigenic load in circulation that is needed for stimulating circulating B cells. Moreover, it is not understood why the PB lesions show continued clinical activity long after stopping the treatment. Since it is believed that appropriate quantum of immunologically defined both Th1 and Th2 cells exist in these PB patients, it is not known whether subsets of B cells are locally present. Further it remains unknown whether these also present B cells can be activated by the in situ persistence of in.leprae antigens and thus causing reactivation of the disease. Interestingly role of specific T cells in skin inflammation is studied exclusively but the participation of B cells in skin has not been studied.
In this study, we report the presence of B cell subsets as identified immunohistochemically by means of monoclonal antibodies e.g. CD20, CD79 and Syndican-1(CD138), that are involved in local interaction with residual in.leprae antigens and other immune cells e.g. T cells and subsets of antigen presenting cells (APC). We analysed the immune infiltrates and antigen expressions in both skin and nerve biopsies of leprosy patients originating from Brazil, India and The Netherlands. Our results show that all the subsets of B cells e.g. CD20+/CD79+/CD138-, CD20-/CD79+/CD 138-, CD20+/CD79-/CD138- and CD20-/CD79-/CD138+ cells are present in varying proportion and distribution in both skin and nerve lesions. The often presence of these B cells and persistent presence of m.leprae antigens are seen microscopically interacting with T cells and APC in both nerve and skin. We hypothesise that locally produced antibodies by these B cells may play an important effector role together with T cells and APC in the dynamicity of leprosy pathology.
PI 40
M. leprae INDUCED APOPTOSIS PARALLELS A DOWN REGULATION OF CD14
Hernandez, M.O., Shannon, E.J.*, Sarno, E.N., Sampaio, E.P.
Leprosy Laboratory, Oswaldo Cruz Foundation-FIOCRUZ, Rio de Janeiro - Brazil.
*Veterinary Medical School, Baton Rouge, Louisiana - USA.
Until very recently, the function of CDI4 was thought to be limited to innate immunity, as the major endotoxin receptor. Nowadays, a role for CD 14 in the regulation of monocyte apoptosis is being reported. It has been shown that down regulation of CD 14 or its removal triggers apoptosis, whereas up-regulation promotes survival. Since our previous results indicate that M. leprae induces monocyte-derived macrophages (MDM) apoptosis in a dose dependent manner, the expression of CD 14 in MDM stimulated with increasing concentrations of M. leprae or LPS was monitored by flow cytometry. When dead M. leprae (1g/ml) or LPS were added to cultures for 2 days an up-regulation of CD 14 and an increase in cell viability was observed. However, when higher amounts of the bacteria (10 or 20g/ml). reported to induce apoptosis, where used, a down regulation of CD 14 expression was noted after the same period of culture. When live M. leprae was used a similar profile in CD14 expression was detected. Our results indicate, a selective and progressive CD 14 down regulation, which parallels apoptosis induced by dead M. leprae in MDM, suggesting that CD 14 down regulation is an early signal of cell death, as previously reported for MTB.
PI 41
MATURE DENDRITIC CELLS INFLUENCE TH1/TH2 CYTOKINE PROFILES IN STABLE LEPROMATOUS LEPROSY PATIENTS
Nalini Vemuri *, R.K. Jain**, M.J. Colston# and I. Nath*
*Immunology Laboratory, Biotechnology Department, Anatomy Department, All India Institute of Medical Sciences, New Delhi, India; **Dermatology Department, Safdarjung Hospital, New Delhi, India; #National Institute of Medical Research, Mill Hill, London.
The role of dendritic cells (DC) in immune response to M. leprae antigen was investigated in lepromatous leprosy patients. Dendritic cells (DC) are unique antigen presenting cells specialised in antigen capture and triggering adaptive immune responses. Immature DCs act mainly to capture antigens whereas mature DCs acquire high lenvels of MHC class I/II and co-stimulatory molecules, present antigens and initiate T cell and B cell responses. The role of both populations Of DCs in Th cell differentiation in leprosy was evaluated. Co-expression of Th cytokines IFNy and IL4 and regulatory cytokines IL10 and IL12p40 was compared in antigen stimulated peripheral blood mononuclear cells (PBMCs). T-cells reconstituted with autologous monocytes (Mo). T-cells reconstituted with imniarure DC (high intracellular MHC II, low CD83 and p55) and T-cells reconstituted with mature DC (CD 11C+. high surface MHC II, high CD83 and p55) by conventional and Real Time lluorogenic based RT-PCR (Reverse Transcription Polymerase Chain Reaction) and by ELISA. Reconstitution of purified T-cells with autologous Mo and immature DC resulted in down regulation of IL4 and ILK). On the other hand reconstitution of purified T cells with mature DC resulted in upregulation of IFNy and dysregulation of IL4. The fact thai stimulation of different populations of DCs could alter the cytokine profile in reconstituted cultures, suggests that they may have a varied influence on the imuunological stablily of this disease.
PI 42
MODULATION OF CYTOKINE RESPONSE TO POLYCLONAL AND MYCOBACTERIAL STIMULI BY THE PHENOLIC GLYCOLIPID-I(PGLI)
Esquenazi D.A .1,2, Alvim I.M.P.1,2, Guimarães M. da S.M.1, Santos S.L.C.1, Nery J.A.C.1, Sarno E.N.1, Pessolani M.C.V.1, and Pereira G.M.B.1,2
1Leprosy Lab, IOC-FIOCRUZ;
2Lab of Immunopathology, FCM-UERJ; Rio de Janeiro, Brazil
Previously. PGLI has been shown to inhibit Tcell activation parameters and to enhance TNF-a production by M. Ieprae-engulfing mononuclear phagocytes. In order to further assess the role of PGLI in cytokine production, peripheral blood leukocytes from healthy volunteers, tuberculoid and lepromatous leprosy patients were stimulated in vitro (ConA, M. Leprae, PPD), in the presence of PGLI, and the levels of 6 cytokines (IFN-α, TNF-α, IL-2, IL-4, II.5 and IL-10) were evaluated in the culture supernatants, using the cytokine bead array flow cytometric method and ELISA. In the presence of PGLI, IFN-γ levels were reduced/absent in ConA and M. Leprae-stimulated wells, but unafected or increased with PPD. PGLI reduced TNF-α in response to ConA, but markedly enhanced this cytokine concentration when added to M. Leprae-stimulated wells. PGLI inhibitory actions were not associated to increase in IL-10 levels. The observed effects of PGLI were seen in the patients and healthy volunteers. Taken together these observations show that PGLI effects change with different stimuli, perhaps reflecting a different sensitivity to PGLI by the leukocyte subsets and/or pathways involved in cytokine production induced by ConA and these mycobacterial stimuli.
Supported by PROATEC-UER.I. PADCT/CNPq. and FAPERJ.
PI 43
NÍVEIS DE ÓXIDO NÍTRICO EM PLASMAS DE PACIENTES HANSENIANOS
Luiz Cosme Cotta Malaquias 1, Fabiana Magalhães Coelho1, Daniela Aquino Dusi de Nazareth1, Francisco Carlos Félix Lana2, Simone Teixeira3, Francisco Carlos Pereira4, Regina Lúcia Barbosa Cypriano4 e Alexandre Castelo Branco4
1Faculdade de Ciências, Educação e Letras/UNIVALE, Gov. Valadares, MG, Brasil;
2Escola de Enfermagem/UFMG, Belo Horizonte, MG, Brasil.
3Secretaria Municipal de Saúde, Gov. Valadares, MG, Brasil.
4Policlínica Central Municipal de Saúde, Gov. Valadares, MG, Brasil
As infecções causadas por parasitos intracelulares obrigatórios tais como o Mycobacterium leprae são contidas pela imunidade celular. Os pacientes portadores desta infecção apresentam um variado espectro de manifestações clínicas, o que reflete o estado imunológico em que se encontram. Pacientes multibacilares podem apresentar alta bacteremia em virtude de uma resposta imune celular deficiente. Por sua vez, pacientes paucibacilares apresentam baixa bacteremia associado a uma resposta imune celular eficiente. A produção de reativos intermediários do oxigênio e do nitrogênio por macrófagos ativados é crucial para o desenvolvimento da imunidade contra microrganismos intracelulares. Este estudo tem por objetivo quantificar os níveis de óxido nítrico (NO) em plasmas de pacientes portadores das diferentes formas clínicas da Hanseníase. A quantificação de NO será realizada indiretamente através da medida de nitritos e nitrados. Metodologia: 50 L das amostras de plasmas diluídos em água foram incubados overnight com uma mistura contendo FAD, NADPH e nitrato redutase. As amostras foram desproteinadas por ZnS04. A seguir, 100 L das amostras em duplicatas foram misturadas com 100 L do reagente de Griess e a absorbância determinada em leitor de ELISA. Os resultados expressos em M/mL foram obtidos pela extrapolação de uma curva padrão com NaN02. Resultados: os níveis de NO encontrados nos plasmas dos pacientes foram: paucibacilares: 218,8 M/mL(n=3), multibacilares: 183,4 M/mL (n=10) e controles sadios: 217.7 M/mL (ii=10) respectivamente. Conclusão: os resultados sugerem não haver diferenças significativas nos níveis séricos de NO entre pacientes hansenianos e controles sadios.
Apoio Financeiro: FAPEMIG
PI 44
PARACOCCIDIOIDOMICOSE E HANSENÍASE: RELATO DE CASO
Ana Regina Alencar Santos, Clarisse Zaitz, Clarice Marie Kobata, Juliana Rogério Prado
Clínica De Dermatologia Da Santa Casa De Misericórdia De São Paulo
Rua Cesário Motta Júnior. 112 Cep:01221-020 Vila Buarque, São Paulo
clakohata@yahoo.com
Paciente masculino, negro, 44 anos, natural de Teófilo Otoni- MG, procedente de São Paulo.
Procurou nosso serviço em Dezembro de 1999 apresentando dermatose localizada em ângulo esquerdo da boca e língua caracterizada por exulceração com cerca de 2 cm de diâmetro, de limites irregulares, mal delimitadas em cuja superfície se observava exsudato seropurulento e crostas hemáticas.
O diagnóstico de Paraeoccidioidomicose foi confirmado pelo exame micológico direto e cultura para fungos.
Foi introduzido o tratamento com cetoconazol, porém o paciente abandonou o tratamento.
Em Julho de 2001 procurou o serviço de Otorrinolaringologia com queixa de rouquidão progressiva e perda ponderal de 5 kg. Na mucosa nasal foi observado lesão ulcerosa infiltrativa e exsudativa.
Na mesma época retornou ao nosso serviço.
Ao exame dermatológico observou-se: dermatose disseminada caracterizada por nódulos e placas que variavam de 2 a 7 cm de diâmetro.Face infiltrada, nódulos em pavilhões auriculares e madarose.
Na região de sulco nasogeniano e axila direita nódulos ulcerados com cerca de 2 cm de diâmetro.
Baciloscopia com bacilos álcool ácido resistentes isolados e em globias.
Exame micológico direto e cultura para fungos positivos para Paraeoccidioidomicose.
Exame anti-HIV negativo.
Iniciou-se tratamento com poliquimioterapia esquema multibacilar para Hanseníase Virchowiana e Itraconazol 100 mg ao dia para Paraeoccidioidomicose.
Trata-se de uma associação rara de duas doenças infecto contagiosas, com mecanismos imunológicos distintos, onde o paciente apresenta a forma crônica multifocal da Paraeoccidioidomicose com resposta Th 1 e Hanseníase Virchowiana com resposta Th2.