Abstracts of Congress for Papers and Posters
PI 45
PREVALÊNCIA DE ANTICORPO ANTICARDIOLIPINA NAS DIFERENTES FORMAS CLÍNICAS DE HANSENÍASE
Rosa Maria Cordeiro Soubhia ; Denise Rodrigues; Geysa Camirim; Prof. Dr. José Maria Godoy; Prof. Dr. João Roberto Antonio
Faculdade Estadual de Medicina de São José do Rio Preto. Ambulatório de Dermatologia do Hospital de Base. Av. Brigadeiro Faria Lima no. 5416 São José do Rio Preto-SP Brasil.
Os Anticorpos Anticardiolipina (AA) são autoanticorpos associados com trombose vascular e aborto de repetição, porém vários relatos têm associado com doenças infecciosas como a Hanseníase. O objetivo deste estudo foi avaliar a prevalência dos AA nas diferentes formas clínicas de Hanseníase. Foram estudados 42 pacientes, sendo 26 do sexo masculino e 16 do sexo feminino, com idades entre 17 e 77 anos com média de 48 anos .Todos pacientes eram portadores de Hanseníase confirmados pela clínica, baciloscopia e biópsia de pele. As manifestações clínicas da Síndrome do Anticorpo Antifosfolípede (SAF) não foram encontradas. Os pacientes foram submetidos a classificação de Ridley-Jopling: 9,5% indeterminado(I), 16,6% tuberculóide(T), 16,6%dimorfo tuberculóide(DT), 7,1 %dimorfo dimorlb(DD), 4,7%dimorfo virchowiano(DV) e 45,2% virchowiano (V). A avaliação dos níveis de AA no soro foi realizada pelo método ELISA (Enzyme-linked Imunnosororbbent Assay).Foi utilizado na análise estatística o teste exato de Fisher com p< 0,05 e IC 95%.Os dados obtidos foram comparados com grupo controle formado por 100 doadores do banco de sangue de estudo prévio da instituição.A prevalência global dos AA foi de 47%, sendo significante p< 0,0001 em relação ao grupo controle, sendo encontrado positividade na forma I (25%), T (28,5%), DT (28,5%), DD (33,3%), DV (50%) e na forma V (68,4%). Os pacientes com baciloscopia positiva apresentaram significância estatística quando comparados com os negativos p< 0,01. Conclui-se que os pacientes com Hanseníase apresentam alta prevalência de AA, sendo as formas com baciloscopia positiva mais prevalente que as formas com baciloscopia negativa.
PI 46
REAÇÃO DO GRANULOMA IN VITRO COM ANTIGENOS DO Mycobacterium leprae E CÉLULAS MONONUCLEARES DO SANGUE PERIFÉRICO DE DOENTES COM HANSENÍASE
Souza. CS .1; Foss, N.T.1; Cunha, F.Q.2
1Divisão de Dermatologia e 2Departamento de Farmacologia Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo.
As razões para diversidade da resposta imune nas formas polares da doença frente ao mesmo agente agressor, Mycobacterium leprae, não estão completamente elucidadas. Buscamos desenvolver um modelo de granuloma in vitro com antígenos do M. leprae e células mononucleares do sangue periférico de doentes com as formas polares da hanseníase e avaliar as diferenças na produção de citocinas e de óxido nítrico em culturas de células. Foram selecionados, 10 doentes com hanseníase sem tratamento, atendidos no HC-FMRPUSP: Grupo V (virchowianos polares e borderline-lepromatosos) e Grupo T (tuberculóides polares e borderline-tuberculóides). Procedeu-se o isolamento de células mononucleares do sangue periférico utilizando-se centrifugação sob gradiente de Ficoll-Hypaque. Foram realizadas culturas duplicatas (2 x 106 linfócitos/ml): controles, com meio de cultura ou beads isolados; com antígenos de 28kD ou de 36kD do M. leprae conjugados a beads de poliacrilamida e com a presença ou ausência de aminoguanidina. As placas foram incubadas a 37º C em atmosfera úmida contendo 5% de CO2, durante 21 dias e, colhidas amostras do sobrenadante no 7º e 21º dias. A pesquisa das citocinas ILL IL6, IL10, IL8, TNF, IFN no sobrenadante foi feita pelo método ELISA e a atividade da enzima NO simase foi avaliada pelo ensaio modificado da citrulina. Ao 7º dia, observou-se aumento significativo da produção de IL-6 e IL10 nas culturas controles e com antígenos no grupo V, comparada à do grupo T. Não se detectou produção de TNF em nenhum grupo neste período. Ao 21º, os grupos V e T produziram IL6 de modo similar, na presença ou ausência de antígenos nas culturas. Entretanto, no grupo V. observou-se redução da produção de IL10 nas culturas com antígenos. A produção de TNF tende a ser mais acentuada no grupo T e reduzida com a presença de qualquer um dos antígenos testados, em ambos os grupos. Com a presença de antígenos, a produção de ILI foi reduzida no 21" dia. Não se detectou a atividade da enzima NO sintase nas culturas. A IL6 atua preferencialmente em células B. proporcionando a produção de anticorpos, e a IL10 exerceria efeitos imunossupressores. No modelo desenvolvido, o aumento destas citocinas no grupo V e a inibição da produção de TNF e IL1, na presença de antígenos, são indicadores da imunomodulação. Vale ressaltar, que o modelo de granuloma in vitro mostrou-se útil no estudo da imunomodulação na hanseníase com amplas perspectivas de aplicação experimental
PI 47
REGULATION OF MACROPHAGE BCL-2 GENE FAMILY EXPRESSION DURING M. leprae-INDUCED APOPTOSIS: INVOLVEMENT OF BAX AND BAK
Hernandez, M.O.,Salles, J.S., Sarno, E.N., Sampaio, E.P.
Leprosy Laboratory, Oswaldo Cruz Foundation - FIOCRUZ, Rio de Janeiro-Brazil.
Bcl-2 protein family regulates cell death. This family has antiapoptotic (Bcl-2 and Bcl-xL) and proapoptotic (Bad, Bax and Bak) proteins. These proteins form homo and heterocomplexes that can, among other functions, regulate mitochondria release of the cytochrome C. Mycobacterias were described to induce apoptosis in mononuclear phagocytes in vivo and in vitro. Our previous results demonstrate that M. leprae can also induce apoptosis in leprosy patients monocytes in a dose dependent manner in vitro. In order to investigate the molecular mechanisms underlying the apoptotic pathway in this model, RT-PCR was used to evaluate the expression of some members of the Bcl-2 family, namely Bax-a and Bak. Initial experiments demonstrated that M. leprae could modulate the mRNA expression of some proapoptotic genes. Cells stimulated with low concentration of M. leprae (lg/ml), not able to induce macrophages apoptosis, also did not induce expression of Bak or Bax. However, cells stimulated with 10 or 20g/ml of M. leprae showed increased expression of both genes and exhibited a significant enhancement in the rate of apoptosis. LPS, an inhibitor of macrophage cell death, did not induce large amounts of these genes. Bad expression was not detected in either culture. These results suggest that Bcl-2 family proteins may be involved in M. leprae-induced cell death in vitro.
PI 48
SELECTIVE INCREASE IN THE CD4+ T CELL ACTIVATION THRESHOLD AS A POTENTIAL MECHANISM FOR THE BIOLOGICAL ROLE OF THE PHENOLIC GLYCOLIPID-I (PGL-I) IN LEPROSY
Alvim, IMP1,2, Guimarães. M da S.M.1, Esquenazi, D..A1,2. Santos. S.L.C.1. Nery. J.A.C.1, Sarno, E.N.1, Pessolani, M.C.V and Pereira, G.M.B.1,2
1Leprosy Lab, IOC-FIOCRUZ;
2Lab of Immunopathology, FCM-UERJ: Rio de Janeiro, Brazil
Pathogens frequently synthesize immune response modifiers to enhance their survival in the host microenvironment, In order to evaluate the role of PGL-I, a M. leprae (ML) glycolipid, as a T cell function modifier, PBLs from healthy volunteers and leprosy patients were stimulated in vitro in the presence of PGL-I. Flow cytometry of the cultured PBLs demonstrated PGL-I inhibition of CD28 expression in CD4+. but not CD8+T cells. The induction of CD69 and CD25 in ConA or anti-CD3 stimulated T cells was markedly reduced by PGL-I. The proliferative responses, IL-2/TCGF and TNF bioactivities, as well as IFN-γ levels (ELISA) were also reduced by PGL-I. All the effects of PGL-I were seen in healthy volunteers and in patients across the spectrum of leprosy. PGL-I actions occurred only at suboptimal levels of stimulation, being reversed by increasing the stimulus. This observation suggests that this glycolipid increases the threshold for T cell activation. The inhibition of CD28 expression/function in CD4+ T cells and, as a consequence, less effective sorting of activation-associated molecules in the immunological synapses is a potential mechanism for PGL-I action on T cell activation. These effects of PGL-I can lead to pathogen-specific anergy and defective effector function against ML. if associated to conditions allowing the initial survival of ML in an infected individual.
Supported by PROATEC-UERJ. PADCT/CNPq. FAPERJ.
PI 49
SERODIAGNOS1S OF Mycobacterium leprae INFECTED INDIVIDUALS IN HIGHLY ENDEMIC AREAS OF MYANMAR
Khin Nwe Oo .1 Namisato M.,2 Kashiwabara Y.,3 Fujiwara T.,4 Nwe Nwe Yin.1 Kyaw Nyunt Sein,5 Kyaw Myints
1Immunology Research Division, Department of Medical Research, Yangon, Myanmar
2National Sanatorium Kuryu-Rakusenen. Japan
3Leprosy Research Center. National Institute of Infectious Diseases, Japan
4Nara University, Japan
5Department of Health. Yangon. Myanmar
This study was carried out on serodiagnosis of M. leprae-infection in highly endemic areas of Myanmar. It was studied on the residents of Kyanbokone (A-village) and Konethandin (B-village) in Bago Division and NTP-BSA ELISA was done on their sera. The IgM and IgG seropositives in A-villages were 59/263 (22.43%) and 34/263 (12.93%) in the first year, 41/223 (19.21%) and 46/223 (20.63%;) in the second year. The same in B-village is 41/115 (35.65%) and 9/115 (7.82%) in the 1st year. The age group of 10-19 year-old was the commonest one in age specific distribution of anti IgM and IgG antibodies to NTP-BSA antigen. Some villagers who presented with high litre of antibodies should be followed up considering the indication of chemoprophylaxis. Through the follow up study of M. lepraeinfection in particular populations, some valuable information about the process from infection to development of disease can be expected.
PI 50
SERUM CYTOKINES AND MONOKINES IN LEPROSY AS A MARKER FOR MONITORING REVERSAL REACTIONS
Faber. WR 1, Iyer, AM1,2, van der Pol, T3, Dekker, T1,2, Walsh. GP4+, Fijardo4 and Das. PK1,2
1Departments of Dermatology. 2 Pathology and 3Internal Medicine, Academic Medical Center. University of Amsterdam, The Netherlands
4Leonard Wood Memorial Leprosy Foundation, Phillipines.
During the course of leprosy episodes, two types of "reactions", classified either type-1 (reversal reaction: RR) or type II (erythema nodosum leprosum: ENL) occur among 30-40% of the patients. The immunopathology of leprosy is primarily due to immune interaction between subsets of T cells, macrophages/relevant antigen presenting cells and M.leprae antigens. Such interactions produce Th1/Th2 cytokines, activated macrophage products and monokines, which act as molecular signals for communication between immune cells and the organ specific cells, which play a pivotal role in the dynamics of host immune response and tissue damage. The latter, in case of leprosy, is the destruction or dysfunction of peripheral nerves and deformity. In vitro and in vivo studies have been used to delineate the immunologic aspects of leprosy and the reactional states. In this respects serum cytokine and monokine levels had been considered as useful markers for monitoring the leprosy patients during the course of the disease. In this study we measured, by either EL1SA or radioimmunoassay, serum levels of T cell cytokines: IFN-γ, TNF-α, TNF-αR (p75. p55), IL-4. IL-5 and neopterine in seven leprosy patients from Phillipines before treatment and thereafter for 1-6 months when patients developed clinical signs of RR with the expectation that serum cytokine and monokine profile at the onset of reaction may provide a clue for early prediction of reactions to be experienced by the patients. The results show that at the onset of RR and/or one month after, 6/7 patients showed significantly increased levels of neopterine and in some cases remained high even after being treated with immunosuppressive drugs. On the other hand serum IFN-, IL-4, IL-5 showed inconsistent increase in not more than 2/7 patients. Interestingly, levels of TNF-γ. TNF-R were increased in 6/7 patients either at the onset or after 1 month of the onset and remained high above the normal value. It appears from this pilot study that measurement of serum neopterine and TNF-α, TNF-αR could be valuable in monitoring the RR patients during the treatment.
PI 51
SPECIFICITY OF PGL-I SEMI- SYNTHETIC ANTIGENS IN LEPROSY SEROLOGY
S. Bührer-Sékula1, P. Jansen, M. Hatta2, L. Oskaml, W.R. Faber and PR. Klatser1
1KIT Biomedical Research, Meibergdreef 39, 1105 AZ, Amsterdam, The Netherlands
2Department of Microbiology, Faculty of Medicine, Hasanuddin University, Makassar, Indonésia
Several serological tests to detect M. leprae infection have been developed using PGL-I. a cell wall component specific for M.leprae. Semi-synthetic derivatives were produced by either directly linking a disaccharide group to BSA, leading to disaccharide BSA (DBSA). or by linking the synthesised sugar groups (natural di- or tri-saccharides [ND or NT]) to BSA with an oclyl (O) or a phenolic linker (P).
We evaluated whether the antibody response to PGLI was specific for M.leprae and determined whether scrum samples gave similar results with different PGL-I-based semi-synthetic antigens.
143 serum samples from different groups were tested using the native PGL-I and three semi-synthetic antigens (ND-O-BS A, DBSA and NT-P-BSA). ND-OBSA was more often positive than the others. All antigens were compared with ND-O-BSA. DBSA gave the highest agreemenl and PGL-I the lowest. There was no disagreement in the patients group with OD values above 1.5 and the groups formed by contacts and non contacts of leprosy patients with negative ND-O-BSA. The group composed of non-contacts with positive ND-O-BSA results gave the lowest agreemenl between antigens, namely 62, 83 and 94% when compared with DBSA, NT-P-BSA and PGL-I, respectively.
Comparison between different batches of ND-OBSA gave a strong suggestion that non-specific binding in a particular batch could be occurring. This resuits indicate that a rigorous quality control of the antigen should be performed.
PI 52
SPINDLE CELLS AND MACROPHAGES IN HISTOID LEPROSY
Tomimori-Yamashita J .1, Dos Santos M.J.CP.2, De Seixas M.T.2, Michalany N.2
Department of Dermatology1 and Pathology2, Federal University of São Paulo (UNIFESP), Rua Botucatu 740, CEP: 04023-900, São Paulo/SP, Brazil.
Purpose: Histoid form of leprosy is a rare manifestation of lepromatous form. The histological aspect is the presence of a pseudocapsule, with fusiform cells surrounding this structure. The aim of this study was to demonstrate that this spindle cells express the same antigen as the foam cell and that this form are related to high positivity of acid fast bacilli.
Methods: We have performed an histological (HE and Fite-Faraco stain) and immunohistochemical study (anti-CD4. anti-CD8. anti-CD68 and anti-BCG) in 8 patients, presenting clinical criteria for histoid leprosy.
Results: In HE stain, the infiltrate was mainly constituted by Virchow's cells. The spindle cells were present in four patients. In only two cases, we could distinguish pseudocapsule in peripheral localization of the macrophagic granuloma. We observed a relationship between globi and presence of spindle cells, by Fite-Faraco stain. Virchow's cells were related to isolated and granular bacilli. The expression for BCG antigen was also strong in every patients. CD4+ and CD8+ cells were diffusely distributed in the infiltrate, without any typical pattern. CD68 antigen expression was strong in Virchow's and spindle cells. The expression for BCG and CD68 antigen was positive in the cytoplasm of fusiform cells, constituting the pseudocapsule.
Conclusion: Spindle cells are typical of this resistant form of leprosy. Expressing the same antigen (CD68+) in Virchow's and spindle cells, we could consider that these spindle cells have macrophagic function.
PI 53
STUDIES ON REGIONAL IMMUNITY USIN EX-VIVO EXPLANT CULTURES OF SKIN LESIONS IN RELATION TO LEPROSY PATHOLOGY
Mohanty, K.P.1, Lehé, C.2,3, Dekker, T.2,3, Katoch, K.1, Das. P.K. 2,3 and Sengupta. U.1,2
1Central JALMA Leperosy Institute, ICMR, Agra, India;
Departments of 2Pathology and 3Dermatology, Academic Medical Center, University of Amsterdam, The Netherlands.
Prevention of acute reactions in leprosy is the principal goal to be tackled for the containment of leprosy in foreseeing future. Understanding the local immunity on skin lesions is therefore, a pre-requisite so that a more direct approach for early diagnosis of leprosy lesions can be developed. At present, several sérodiagnostic tests using antibody titres are used for leprosy diagnosis. Although these tests can diagnose 90-100% of multibacillary (MB) leprosy, they failed to detect 40-60% cases of paucibacillary (PB) patients, but 40% of these PB patients show serum antibody to m.leprae antigens. In addition, several investigators used serum cytokine levels as the marker for disease activity but with limited value, particularly in the context of discriminating leprosy from other inflammatory skin diseases.
We established organotypic cultures of full thickness skin to study the local production of anti-m.leprae antibodies and cytokines in the lesions of paucibacillary (PB), multibacillary (MB) patients with and without leprosy reaction. These presently studied PB and MB patients were histopathologically classified as BT/TT and BL/LL respectively. Kinetics of antibody production and cytokines in the culture supernatants were analysed.
Results: show that production of antibody peaked at 48 hours in all leprosy lesions but negative in all control specimens. Such antibody production could not be seen when the biopsies were autoclaved before culturing. On the otherhand kinetics of IFN-, TNF- in the same supernatants that peaked at 24 hours were significantly more pronounced in PB lesions and lesions with reversal reactions than those in MB and control specimens. However, there was no difference in the local production of IL-4 and IL-10 among the specimens. Interestingly. IL-6 production peaked at 48 hours that was variable but pronounced in the lesions of PB patients although statistically not different from those in MB lesions.
Conclusion: The present data taken together with the phenotypes of in situ immune-infiltrates in PB lesions suggest that a combined role of locally produced antibody and T cell response is important in leprosy pathology.
PI 54
T- AND B-CELL RESPONSES TO Mycobacterium leprae HOMOLOGUE OF ESAT-6 IN LEPROSY PATIENTS AND CONTROL INDIVIDUALS
Mônica C.B.S. Lima 1,2,3*, Márcia V.B.S. Martins2*, John S. Spencer1, Maria A.M. Marques1, Heejin Kim1, Bruce C. Gregory1, Nadia C. Duppre1, José A.C. Nery1, Geraldo M.B. Pereira2,3, Maria C.V. Pessolani2, Euzenir N. Sarno2, and Patrick J. Brennan1
1Department of Microbiology, Colorado Stale University, CO, USA;
2Leprosy Laboratory, Oswaldo Cruz Institute, Oswaldo Cruz Foundation and
3Laboratory of lmmunopathology, School of Medical Sciences, State University of Rio de Janeiro, RJ, Brazil.
ESAT-6 (early secreted antigen target 6 kDa protein) has been described as an immunodominant antigen in the context of Mycobacterium tuberculosis infection in animal models and humans. ESAT-6 generates CD4+ Till cells and antibody production, thus showing promise as a specific diagnostic tool for active tuberculosis. It was recently demonstrated that M. leprae expresses in vivo an ESAT-6 homologue sharing only 36% identity with its M. tuberculosis counterpart. The present study investigated the specific T- (IFN-γ secretion) and B- (IgG antibody) cell responses to M. leprae ESAT-6 in the context of leprosy. T-cell response to M. leprae ESAT-6 was measured in peripheral blood mononuclear cells (PBMC) from leprosy patients. A7. leprae-exposed individuals, tuberculosis patients, and healthy individuals faun an endemic area. Cells were stimulated with the antigen and secreted IFN-? levels were quantified by standard ELISA in cultured supernatanls. Initial results show that M. leprae ESAT-6 was recognized by cells from leprosy patients (6/9), exposed individuals (8/12). tuberculosis patients (1/2), and healthy individuals (2/2) from an endemic area. IgG antibody response was observed in lepromatous leprosy patients ( 11/13), tuberculoid leprosy palients (6/8), TB patients (1/10), and exposed individuals (14/18). Currently, M.leprae ESAT-6 peptides are being analyzed for IFN-γ induction in PBMC. These preliminary results indicate that ESAT-6 induces T- and B-cell responses in most leprosy patients and healthy exposed individuals. The potential cross-reactivity with M. tuberculosis ESAT-6 together with the positive responses observed in individuals from an endemic area suggest limitations on the use of M. leprae ESAT-6 in a specific diagnostic test for leprosy.
(Research supported by the NIAID. NIH).
PI 55
THALIDOMIDE CAN CO-STIMULATE OR SUPPRESS CD4+ CELLS' ABILITY TO INCORPORATE [H3]-THYMIDINE - A DEPENDENCE ON THE PRIMARY STIMULANT
Shannon, E.J ., Sandoval, F.
Hansen's Disease Programs, Laboratory Research Branch at L.S.U. Baton Rouge, La.
Thalidomide is the treatment of choice for erythema nodosum leprosum and has immunomodulatory properties. To assess if the stimulant and/or thalidomide could modify the synthesis of IL-2, IFN-? and incorporation of [H3]-thymidine, peripheral blood mononuclear cells (PBMC) were incubated for three days in the presence or absence of thalidomide and Staphylococcal enterotoxin A (SEA), anti-CD3, Con-A or PHA.
Regardless of the mitogen used to stimulate the PBMC. the thalidomide-treated-PBMC produced more IL-2 than controls. Thalidomide enhanced IFN-? synthesis in the Con-A and anti-CD3-simulated PBMC. It suppressed the ability of SEA and PHA stimulated PBMC to incorporate [H3]-thymidine; whereas it enhanced incorporation of [H3]-thymidine in PBMC's stimulated with anti-CD3.
When the PBMC were enriched for CD4+ or CD8+ cells. the SEA-stimulaled CD4+ cells responded far better than the CD8+ cells in the synthesis of IL-2 and incorporation of [H3]-thymidine. In CD4+ cells thalidomide acted as a co-stimulant with SEA to enhance the synthesis of IL-2. but it suppressed incorporation of [H3]-thymidine.
In the anti-CD3-stimulated-thalidomide treated cultures of PBMC enriched for CD4+ or CD8+ cells, thalidomide acted as a costimulant to enhance the synthesis of IL-2 and incorporation of [H3]-thymidine. Thalidomide cooperated with all of the mitogens to enhance T-cell synthesis of IL-2: however, depending on the stimulant, thalidomide could suppress or enhance cellular incorporation of [H3]-thymidine. The SEA-slimulated cell targeted by thalidomide to suppress incorporation of ju'l-thyniidine was CD4+. CD4+ and CD8+ cells stimulated with anti-CD3 were enhanced by thalidomide in their ability to synthesize IL-2 and to incorporate [H3]-thymidine.
PI 56
THALIDOMIDE DID NOT MODIFY LEPROSY PATIENTS CELLS' ABILITY TO PROLIFERATE IN RESPONSE TO M.leprae ANTIGENS
Azeb Tadesse1,2,3, Engeda Taye4, F. Sandoval3, E.J. Shannon 3
1Armauer Hansen Research Institute, Addis Ababa, Ethiopia.
2Dept. of Radiobiological Science, School of Veterinary Medicine, L.S.U., Baton Rouge, LA.
3Gillis W. Long Hansen's Disease Center, Lab Research Branch, L.S.U., Baton Rouge, LA.
4All Africa Leprosy and Rehabilitation Training Center (ALERT), Addis Ababa, Ethiopia.
The immune response in reversal reaction. (RR) and in erythema nodosum leprosum (ENL) is characterized in vitro by an enhancement in lymphocyte blast transformation to M. leprae. As thalidomide is effective treatment for ENL, this study assessed the effect of thalidomide on these phenomena. Mononuclear cells from patients attending the clinic at ALERT and healthy staff were exposed for 5 days to integral M. leprae, or a modified Dharmendra antigen, or a preparation of PPD from M. tuberculosis. The cultures were treated with thalidomide. In one set, thalidomide was added once at the initiation of the culture, and in another set it was added for a second time (2x), 24 hr prior to harvesting the cells.
The mononuclear cells, in the absence of thalidomide, from the healthy staff (N=11), borderline tuberculoid patients (BT, N= 14) and the BT patients in RR (BT/RR, N=11) responded best to PPD >Dharmendra > M. leprae. The cells from patients who were being treated with prednisone to suppress ENL (N=7) did not respond well to the M. leprae antigens. Thalidomide (2x) enhanced proliferation to PPD in the ENL group (paired t-test, p=0.02). No significant changes occurred for the other groups. Comparing PPD-stimulated cells treated with thalidomide once to those treated with thalidomide twice, thalidomide (2x) suppressed incorporation of [H3]-thymidine by the PPD-stimulated cells in the healthy staff group (p= 0.04). In the Dharmendra-stimulated cells from the healthy staff thalidomide significantly suppressed TNF-a (p=0.01). A mixed effect was seen within and between the other groups, but there was a trend for thalidomide to suppress of TNF- induce by the M. leprae and PPD antigens.
PI 57
THE EFFECT OF THE ACTIVITY OF MICROSOMAL ENZYMES AND ACETILATION ON METHEMOGLOBIN RATE IN LEPROSY PATIENTS
V.Z. Naumov, VP. Tsemba, E.A. Zadneprovskaya
Leprosy Research Institute, Astrakhan, Russian Federation
As it is known, dapsone at certain doses may induce hemolysis, especially in persons with glucosesphosphate dehydrogenase (G6PDH) deficiency, occurring in about 10% of leprosy patients. However. DDS-induced hemolysis might be due to other factors among which peculiarities and intensity of drug metabolism, including rate of sulphone acetylating and hydroxylation, play an important role. Patients with lepromatous leprosy were given various schemes of MDT with dapsone 100 mg daily as a main component. Activity of microsomal enzymes by the time of antipyrine half-secretion (T1/2) and acetylation rate of sulfadimizine was studied. All the patients studied had no G6PDH-deficiency. It was observed that in patients showing rather high activity of microsomal enzymes (T1/2 =12,5 h in average) blood methemoglobin rate was significantly higher (P<0,05) than in those with low activity of these enzymes (T1/2=23.5 h in average). Though methemoglobin rate in the most patients did not exceed 1,5%, it approached 2.5-3,9% in persons with a combination of low acetylating rate and high activity of microsomal enzymes. It might be a consequence of increase in derivatives of N-hydroxylation of dapsone with methemoglobin-forming properties in persons with predominance of oxydative phenotype of xenobiotic biological transfor.
PI 58
THE REPORT FOR THE SKIN SMEARS QUALITY CONTROL ON LEPRESY IN SICHUAL PROVINCE IN THE PAST 15 YEARS
Wang Rongmao , Liu Xueming. Zheng Yiqiang. Yu Linchong
Sichuan Institute of Dermatology, Chengdu, 610031, China
The skin smears quality control on leprosy was implemented in the leprosy epidemic counties in Sichuan Province, in order to improve the quality of skin smears and implementation of MDT. 10% of skin smears, came from the leprosy epidemic counties, were selected randomly with double-blind method and evaluated in smears, stain and diagnosis in Sichuan Leprosy Laboratory on the basic of the criterion of the skin smears quality on leprosy in the Handbook of MDT on Leprosy. Meanwhile, the skin smears came from Sichuan Leprosy Laboratory were also checked and contrasted by the paramedical workers. In the past 15 years, the skin smears quality control was implemented and the quality of skin smears was improved between 17 and 97 leprosy epidemic counties in Sichuan. 4529 pieced of skin smears were checked. The average qualified rate of smears, stain and diagnosis was 96.88%, which was 86.97% in 1986. The implementation of skin smears quality control could improve professional level of paramedical workers and the quality of leprosy control
PI 59
TOLL-LIKE RECEPTOR 2 ON HUMAN SCHWANN CELLS.
1,2Oliveira. R.B., 2Sarno, E.N., 1Modlin. R.L.
1Division of Dermatology, Department of Microbiology and Immunology, University of California (Los Angeles) School of Medicine, 90095, Los Angeles, California, USA; :Leprosy Laboratory, Oswaldo Cruz Insitute, FIOCRUZ. Avenida Brasil 4365, Manguinhos, Cep 21045-900, Rio de Janeiro, RJ, Brasil.
Nerve damage is a characteristic clinical feature of leprosy. We investigated the ability of human Schwann cells to participate in microbial recognition according to their expression of Toll-like receptor 2 (TLR2). In this paper, FACS analysis of a human Schwann cell line ST88-14 and immunohistochemistry of leprosy skin lesions demonstrate expression of TLR2 on the surface of human SC (double-fluorescence labeling showed colocalization of a Schwann cell marker, neural cell adhesion molecule (NCAM) and TLR2). Given that TLR2 mediates recognition of microbial lipopeplides, we engineered a synthetic lipopeptide comprising the first six amino acids of the putative M. leprae 19 kD antigen. Activation of the human Schwann cell line with the the M. leprae lipopeptide triggered an increase in the number of cells with condensed nuclei and evidence of DNA fragmentation, characteristics consistent with cell death. Hoescht stain and 7-AAD showed a 2 or 3 fold enhancement in the cell death when compared to the unstimulated cultures. The ability of M. leprae components to induce apoptosis of Schwann cells through Toll receptors might provide a mechanism for nerve damage in leprosy in the absence of inflammation.
MICROBIOLOGY & MOLECULAR BIOLOGY