Double-blind trial of the efficacy of pentoxifylline vs thalidomide for the treatment of type II reaction in leprosy

Type II reaction in leprosy, or erythema nodosum leprosum (ENL), is often characterized by severe clinical symptoms together with nerve function impairment leading to permanent disabilities. Thalidomide has been shown to be a highly effective drug for the treatment of ENL. It is, however, contraindicated for women of childbearing age due to its teratogenicity. On the other hand, pentoxifylline, used to treat hypercoagulable states, is not teratogenic and, like thalidomide, can inhibit the synthesis of tumor necrosis factor-a and other cytokines. In the present randomized double-blind clinical study we compared the effectiveness of orally administered pentoxifylline vs thalidomide in treating type II reaction in 44 patients. Daily doses of 300 mg thalidomide or 1.2 g pentoxifylline were administered for 30 days to multibacillary leprosy patients undergoing type II reaction. Randomly chosen patients were included in the study before, during, and after specific multidrug therapy. Clinical evaluations were performed on the 1st, 7th, 14th, 21st, and 30th days of treatment and laboratory tests were carried out on the 1st and 30th days. As expected, overall, thalidomide proved to be more effective in the treatment of type II leprosy reaction. Nevertheless, continuous treatment with pentoxifylline was effective in relieving the clinical signs of ENL, especially limb edema and systemic symptoms, in 62.5 percent of the patients.

An immunohistochemical, clinical and electroneuromyographic correlative study of the neural markers in the neuritic form of leprosy

The nerve biopsies of 11 patients with pure neuritic leprosy were submitted to routine diagnostic procedures and immunoperoxidase staining with antibodies against axonal (neurofilament, nerve growth factor receptor (NGFr), and protein gene product (PGP) 9.5) and Schwann cell (myelin basic protein, S-100 protein, and NGFr) markers. Two pairs of non-adjacent histological cross-sections of the peripheral nerve were removed for quantification. All the fascicles of the nerve were examined with a 10X-ocular and 40X-objective lens. The immunohistochemistry results were compared to the results of semithin section analysis and clinical and electroneuromyographic data. Neurofilament staining was reduced in 100 percent of the neuritic biopsies. NGFr positivity was also reduced in 81.8 percent, PGP staining in 100 percent of the affected nerves, S100 positivity in 90.9 percent, and myelin basic protein immunoreactivity in 90.9 percent. Hypoesthesia was associated with decreased NGFr (81.8 percent) and PGP staining (90.9 percent). Reduced potential amplitudes (electroneuromyographic data) were found to be associated with reduced PGP 9.5 (63.6 percent) and nerve fiber neurofilament staining (45.4 percent) by immunohistochemistry and with loss of myelinated fibers (100 percent) by semithin section analysis. On the other hand, the small fibers (immunoreactive dots) seen amid inflammatory cells continued to be present even after 40 percent of the larger myelinated fibers had disappeared. The present study shows an in-depth view of the destructive effects of leprosy upon the expression of neural markers and the integrity of nerve fiber. The association of these structural changes with the clinical and electroneuromyographic manifestations of leprosy peripheral neuropathy was also discussed.

Patterns of intracellular cytokines in CD4 and CD8 T cells from patients with mycobacterial infections

Using a short-term bulk culture protocol designed for an intracellular-staining method based on a flow cytometry approach to the frequencies of cytokine-producing cells from tuberculosis and leprosy patients, we found distinct patterns of T cell subset expression. The method also reveals the profile of peak cytokine production and can provide simultaneous information about the phenotype of cytokine-producing cells, providing a reliable assay for monitoring the immunity of these patients. The immune response of Mycobacterium leprae and purified protein derivative (PPD) in vitro to a panel of mycobacteria-infected patients from an endemic area was assessed in primary mononuclear cell cultures. The kinetics and source of the cytokine pattern were measured at the single-cell level. IFN-gamma-, TNF-alpha-, IL-4- and IL-10-secreting T cells were intracytoplasmic evaluated in an attempt to identify M. leprae- and PPD-specific cells directly from the peripheral blood. The analysis by this approach indicated that TNF-alpha was the first (8 h) to be produced, followed by IFN-gamma (16 h), IL-10 (20 h) and IL-4 (24 h), and double-staining experiments confirmed that CD4+ were a greater source of TNF-alpha than of CD8+ T cells (P < 0.05). Both T cell subsets secreted similar amounts of IFN-gamma. We conclude that the protocol permits rapid evaluation of cytokine production by different T cell populations. The method can also be used to define immune status in non-infected and contact individuals.

Normal skin of HIV-infected individuals contains increased numbers of dermal CD8 T cells and normal numbers of Langerhans cells

Dysregulation of the skin immune system (SIS) could explain the high prevalence of skin disorders in HIV+ individuals. The present study was carried out to determine whether alterations in the cell population of SIS and epidermal immunoactivation occur in the normal skin of HIV+ individuals. Forty-five biopsies were taken from the normal upper arm skin of 45 HIV+ patients and of 15 healthy controls. HIV+ individuals were divided into three categories according to their CD4 cell blood count (<200, 200-499 and 500/æl). Hematoxylin-eosin was used to stain tissue sections for morphological analysis and immunohistochemistry was used for the evaluation of the frequency of macrophages, Langerhans cells, and CD lymphocyte subsets. In addition, semiquantitative analysis of LFA-1, ICAM-1 and HLA-DR was determined in epidermal cells. Macrophages, Langerhans cells, and CD lymphocyte subsets did not differ significantly between any of the patient categories and the control group. When all HIV+ individuals were compared as a group to the control group, a significant increase in dermal CD8+ T lymphocytes (P < 0.01) and lower CD4-CD8 ratios (P < 0.01) were observed in the HIV+ individuals. Epidermal ICAM-1 and HLA-DR expression was negative in both HIV+ and normal skin biopsies. No evidence of a depletion of the SIS population or of epidermal immunoactivation in normal skin from HIV+ individuals was demonstrable, suggesting that alterations in the central immune system are not necessarily reflected in the SIS of HIV-infected patients.

Hansens disease in the laboratory / ?

Médica, doutora em patologia, Euzenir Sarno é estudiosa da imunopatologia da hanseníase, infecção crônica das mais antigas que constitui uma doença exclusivamente humana. Integrante de um dos ambulatórios de referência sobre a doença no Brasil, no qual são diagnosticados de 220 a 250 pacientes novos/ano, ressalta que uma das consequências da impossibilidade de se cultivar o Mycobacterium Leprae é a permanência das mesmas questões seculares a respeito da transmissão e a suscetibilidade à doença. Há, também, mais interrogações no terreno epidemiológico que permanecem sem resposta. Estimá-se que entre as pessoas que mantêm contato com pacientes multibacilares, 90% são infectados mas apenas 8% mais ou menos ficam doentes. O índice elevado de infecção de quem convive com doentes multibacilares, sem que a doença se manifeste, indica que apenas um pequeno número de indivíduos não tem resistência ao Mycobacterium leprae. Essa é uma das questões que a imunologia não consegue responder: por que algumas pessoas têm resistência e outras não. A proporção é menor se o contato ocorrer com pacientes paucibacilares, uma forma de manifestação com poucos bacilos. A hanseníase é conhecida como a doença dermatológica, mas a especialista desta que a primeira lesão é anestésica: o nervo é atingido. Além dos nervos sensitivos da pele, há danos que determinam lesões motoras e deformidades irreversíveis, que levam à amputação de extremidades. O Mycobacterium leprae foi uma das primeiras bactérias patogênicas que tiveram o genoma completamente seqüenciado, em 2000. Agora é que se está começando a ter realmente condições para obter testes mais precisos. A doença não é hereditária e apenas em 1986 os serviços de saúde no Brasil passaram a se organizar para combatê-la, pois durante os vinte anos de ditadura militar o sistema foi desmantelado. Em 1991, o tratamento de um ano que inclui três drogas-Dapsona, Rifanpicina e Clofazimina-foi introduzido em nosso país. Apenas 30% dos casos são negativados. Segundo a entrevistada, enquanto a tuberculose é doença altamente bacilar e virulenta, o bacilo da lepra não é virulento, é "preguiçoso"; é um germe que está no fim de seu processo evolutivo; 1/3 de seu genoma não funciona

Further biochemical characterization of Mycobacterium leprae laminin-binding proteins

It has been demonstrated that the alpha2 chain of laminin-2 present on the surface of Schwann cells is involved in the process of attachment of Mycobacterium leprae to these cells. Searching for M. leprae laminin-binding molecules, in a previous study we isolated and characterized the cationic proteins histone-like protein (Hlp) and ribosomal proteins S4 and S5 as potential adhesins involved in M. leprae-Schwann cell interaction. Hlp was shown to bind alpha2-laminins and to greatly enhance the attachment of mycobacteria to ST88-14 Schwann cells. In the present study, we investigated the laminin-binding capacity of the ribosomal proteins S4 and S5. The genes coding for these proteins were PCR amplified and their recombinant products were shown to bind alpha2-laminins in overlay assays. However, when tested in ELISA-based assays and in adhesion assays with ST88-14 cells, in contrast to Hlp, S4 and S5 failed to bind laminin and act as adhesins. The laminin-binding property and adhesin capacity of two basic host-derived proteins were also tested, and only histones, but not cytochrome c, were able to increase bacterial attachment to ST88-14 cells. Our data suggest that the alanine/lysine-rich sequences shared by Hlp and eukaryotic H1 histones might be involved in the binding of these cationic proteins to laminin

The use of pentoxifylline in the treatment of type 2 reactional episodes in leprosy.

It has been suggested that erythma nodosum leprosum (ENL) is associated with enhanced production of TNF-alpha resulting in increased inflammation of the skin and nerve function impairment. Thalidomide and steroids are the major drugs used in the treatment of ENL, but due to the serious problems associated with their use, alternative therapeutic interventions are being considered. In the present retrospective study, the authors report their clinical observations on the effect of pentoxifylline (PTX) in the treatment of ENL. Parameters, such as the clinical involution of reactional lesions, the regression of the inflammatory symptoms associated with the lesions, and the impact on the systemic symptoms common to ENL were assessed at regular intervals during PTX therapy. It was found that PTX therapy led to total elimination of systemic symptoms within the first week of treatment. This improvement was maintained until the end of the study (60 days of treatment). Moreover, the evolution of nodular lesions showed a 100% improvement within the first 14 days of treatment. However, by the 60th day, worsening of the lesions was noted in 20% of the cases. The impression is that PTX is well tolerated, and it may be used for improving patient's clinical condition during ENL reaction. Nevertheless, a randomized, double blind, controlled trial to compare the effects of the widely-accepted thalidomide and the yet untested pentoxifylline for treatment of type 2 reaction is still necessary.

Expression and cytokine secretion in the states of immune reactivation in leprosy

Leprosy is a chronic inflammatory disease caused by Mycobacterium leprae. The human response to this pathogen exhibits intriguing aspects which are up to now not well understood. The present study discusses the probable mechanisms involved in T cell-specific unresponsiveness observed in lepromatous patients. Analysis of the cytokine profile either in blood leukocytes or in skin specimens taken from leprosy lesions indicates that some parameters of Th1 immune response are present in lepromatous patients under reactional states.

Thalidomide protects mice against LPS-induced shock

Thalidomide has been shown to selectively inhibit TNF-alpha production in vitro by lipopolysaccharide (LPS)-stimulated monocytes. TNF-alpha has been shown to play a pivotal role in the pathophysiology of endototoxic shock. Using a mouse model of LPS-induced shock, we investigated the effects of thalidomide on the introduction of TNF-alpha and other cytokines and on animal survival. After injecton of 100-350 mug LPS into mice, cytokines including TNF-alpha, IL-6, IL-10, IL-1beta, GM-CSF and IFN-gamma were measured in the serum. Administration of 200 mg/kg thalidomide to mice before LPS challenge modified the profile of LPS-induced cytokine secretion. Serum TNF-alpha levels were reduced by 93 percent, in a dose-dependent manner, and TNF-alpha mRNA expression in the spleens of mice was reduced by 70 percent. Serum IL-6 levels were also inhibited by 50 percent. Thalidomide induced a two-fold increase in serum IL-10 levels. Thalidomide treatment did not interfere with the production of GM-CSF, IL-1beta or IFN-gamma. The LD50 of LPS in this model was increased by thalidomide pre-treatment from 150 mug to 300 mug in 72 h. Thus, at otherwise lethal doses of LPS, thalidomide treatment was found to protect animals from death.

Two multidrug fixed-dosage treatment regimens with multibacillary leprosy patients.

This study compares the clinical, bacilloscopic, and histopathological evolution of 140 patients classified as having multibacillary leprosy with no previous specific treatment who were submitted to two multidrug treatment regimens with a fixed dose. Regimen I-Group 1: 70 cases received 600 mg rifampicin (RMP) + 100 mg dapsone (DDS) daily for three consecutive months followed by 100 mg DDS daily, self-administered doses for 21 months. Regimen II-Group II: 70 cases received 600 mg RMP + 300 mg clofazimine (CLO) once a month under supervision plus self-administered doses of 50 mg CLO + 100 mg DDS daily for 24 months. The bacilloscopic, histopathological and neuromotor evaluation parameters showed no statistically meaningful differences (P > 0.05) between the two groups except for reaction frequency (P < 0.05) in that group II patients presented the least number of reactional episodes during the treatment and in the dermatological examination at discharge. Follow-up after treatment was carried out for a consecutive four year period. During routine clinical examination one case submitted to regimen I developed nodular skin lesion over the right arm. Skin biopsy was done for histopathological examination and mouse foot-pad experiment by Shepard technique. The drug susceptibility test with DDS and RPM showed that M. leprae strain isolated was susceptible to both the drugs.

The effect of thalidomide on BCG-induced granulomas in mice

Granuloma proliferation is the result of a series of complex biological events in wich a variety of cell types and cytokines are involved. Tumor necrosis factor alpha (TNF-Ó) plays a central role. In the present study, we investigated the effect of thalidomide (Ó-N-pthalimidoglutarimide), a sective inhibitor of TNF-Ó synthesis, on granuloma formation during BCG infection in Oncins France 1 (OF-1) mice. Subcutaneous injections of 30 mg/kg body weight of thalidomide daily for 14,21 or 28 days into the mice resulted in the reduction of the size and total number of liver granulomas. The most strikinf effect of thalidomide was observed after 28 days, when the total number of liver granulomas was reduced by as much as 40 percent (P<0.05). Serum TNF-Ó levels of thalidomide-treatment mice were significantly lower (85 percent) than control mice on day 14 and remained lower...

Hanseníase e gravidez / Leprosy and pregnancy

Foram estudadas vinte grávidas hansenianas do ponto de vista clínico [oito lepromatosas (L), sete borderline, quatro tuberculóide (T) e uma indeterminada (I)] e avaliados seus recém-nascidos. Três pacientes apresentaram os primeiros sintomas da doença durante uma das gestaçöes e outra no puerpério. Uma paciente teve a doença durante 16 anos e outra recebeu tratamento durante 14. O exame bacterioscópico foi positivo em 13 e negativo em sete. O teste de Mitsuda foi negativo em 16, positivo em duas, e em outras duas näo foi realizado. O esquema terapêutico em 14 pacientes foi poliquimioterapia (diaminodifenilsufona, rifampicina e clofazimina), a monoterapia (diaminodifenilsufona) foi realizada em três e nenhuma delas mostrou resistência a droga. Outras três só iniciaram o tratamento após o parto. Os estados reacionais foram tratados com corticosteróide e ácido acetilsalicítico. O uso de drogas foi realizado de modo irregular em nove pacientes e em oito regularmente. Em dez pacientes, observaram-se surtos reacionais: oito durante a gestaçäo, uma no puerpério e outra na lactaçäo. Oito apresentaram quadro de eritema nodoso leproso e duas reaçäo reversa, sendo que uma delas desenvolveu neurite ulnar no 2§ mês de gestaçäo. Uma paciente mostrou sorologia positiva para sífilis sem manifestaçöes clínicas da doença. Outra apresentou baciloscopia positiva durante a lactaçäo. Um recém-nato apresentou quadro de dermatite esfoliativa nas primeiras horas de vida e sua mäe tinha recebido diaminodifenilsufona durante a gravidez. Entre os 20 recém-natos, cinco tinham peso inferior a 2.500g e quatro eram prematuros. Nós concluímos que os estados reacionais das pacientes, o baixo peso e a prematuridade dos recém-natos ocorreram nas grávidas do grupo de lepromatosas e borderline

Imunidade celular e humoral na hanseníase bordeline tuberculoid (BT) / Humoral and cellular immunity in bordeline tuberculoid (BT) leprosy

Foram estudados 20 pacientes com diagnóstico de hanseníase bordeline tuberculoid (BT), classificados segundo os critérios de Ridley & Jopling, bem como dois pacientes com forma Tuberculoid tuberculoid (TT), todos com baciloscopia negativa, exceto um, que apresentou índice baciloscópico 1+. Todos os pacientes foram avaliados quanto a sua capacidade de resposta imune humoral ao DBSA (antígeno sintético semelhante ao glicolipídeo fenólico I, específico do M, leprae) e 18 pacientes foram submetidos a testes de avaliaçåo da resposta imunocelular in vivo (teste Mitsuda) e in vitro (linfoproliferaçåo e produçåo de interferon-gama) frente ao Mycobacterium leprae. Observamos que 90 por cento dos pacientess apresentaram resultados negativos quanto à pesquisa de IgM anti-DBSA pelo método imunoenzimático ELISA (densidade óptica , 0,27), o que demonstra ser este teste inadequado para a detecçåo de pacientes paucibacilares. Quanto aos testes de imunidade celular, oito pacientes (44,4 por cento) apresentaram teste de Mitsuda positivo (>= 5mm), sendo os demais considerados negativos. Cerca de 89 por cento dos pacientes tiveram teste de Mitsuda maior ou igual a 3mm. Doze pacientes (66,7 por cento) tiveram resposta linfoproliferativa positiva (índice estimulatório >= 3,0) para o M. leprae. Vinte e dois por cento dos pacientes apresentaram níveis de interferon-gama acima do limite de positividade (40 U/ml). Houve 66,7 por cento de correlaçåo entre os testes de Mitsuda e interferon-gama; 55,6 por cento de correlaçåo entre os testes in vitro (linfoproliferaçåo e interferon-gama). Quando estes três testes foram considerados em conjunto, uma correlaçåo de 38,9 por cento foi observada. Este estudo demonstra a heterogeneidade do comportamento imunológico mediado por células e anticorpos em pacientes com hanseníase BT, apesar de todos histologicamente serem capazes de conter a multiplicaçåo bacilar e de formar granulomas epitelióides

Effect of cutaneous cell-mediated immune response to rIFNy on mycobacterium leprae viability in the lesions of lepromatous leprosy / Efeito da célula cutanea - resposta imune intermediária para rIFNy sobre viabilidade de Mycobacterium leprae nas lesöes de lepra lepromatosa

Studies were caried out to determine the effect of intra-dermal injections of recombinant human interferon-gamma (rIFNy) on the viability of Mycobacterium leprae. Twenty-three untreated and 4 treated multibacillary patients, 12 with lepromatous leprosy (LL) and 15 with bordeline lepromatous leprosy (BL), were selected for intradermal administration of rIFNy or PPD. Treated patients (LL and BL) had received multi-drug therapy according to the recommendations of the World Health Organization, i. e., rifampicxin (600 mg/month), dapsone (100 mg/day) and clofazimine (50 mg/day and 300 mg/month) for 1-4 months. Three daily doses of 10 or 30 ug rIFNy induced local induration and mononuclear leucocyte accumulation. Bacteria isolated from a punch biopsy of the site 21 days after lymphokine administration were injected into mouse foot pads and evaluated for viability and growth. The local response to rIFN (specific activity 2 x 10 7 units/mg protein) induced a delay or total inhibition of M. leprae growth in the mouse foot pad, ,indicating that the cellular response to the antigen reduced local M. leprae viability. The extent of reduction in viability depended on the dose of rIFNy injected and the extent of local induration induced by the lymphokine. With a vigorous cell-mediated immune response growth was fully inhibited. A similar but less extensive effect on M. leprae viability was observed in resapo0nse to the local injection of 5 units in 0.1 ml of purified protein derivative of tuberculin (PPD)

Metodos histoquimicos para a identificacao de leucocitos e macrofagos. / Histochemical methods for the identification of leukocytes and macrophages

Os autores descrevem a padronizacao de tecnicas histoquimicas, fosfatase acida, alfa-naftil-acetato esterase e peroxidase para identificacao de linfocitos e macrofagos em tecido. Recomenda-se a fixacao em formol sacarose tamponado e inclusao em goma de Holt como a mais eficaz ja que nao so conserva a atividade enzimatica, como tambem e de realizacao acessivel

Identificacao de antigenos da hepatite B em tecido hepatico nas diversas formas de hepatite aguda. / Identification of hepatitis B antigens in hepatic tissue in the various forms of acute hepatitis

Os autores pesquisaram os antigenos da Hepatite B (antigenos de superficie e antigeno do nucleocapsideo), em fragmentos de biopsia hepatica de 223 casos de hepatite viral aguda (203 hepatite viral aguda classica, 16 hepatite aguda com necrose em ponte, 4 necrose hepatica macica); 85 casos apresentavam antigenos de superficie no soro. Foi utilizada a tecnica imunohistoquimica de PAP (complexo peroxidase-antiperoxidase) em blocos de tecido fixados em formol e incluidos em parafina.Sete casos apresenteram um ou ambos os antigenos: Antigeno do nucleocapsideo (AgHBc) isolado foi encontrado em cinco casos. Antigeno de superficie (AgHBs) isolado em um caso e concomitante com antigeno do nucleocapsideo em outro. Com excecao de um caso, todos os que exibiam antigenos em tecido tinham antigeno de superficie no soro. Tres casos evoluiram para hepatite viral cronica ativa, um destes veio a falecer com cirrose. Conclui-se ser a fase aguda da hepatite viral concomitante com clearence viral, dai a pobreza de sua deteccao. A observacao de antigenos em biopsia nesta fase clinica teria algum potencial evolutivo

Heterogeneidade de resposta do hospedeiro ao virus da hepatite B. / Heterogeneity of host response to hepatitis B virus

O comportamento de 36 pacientes de uma Unidade de depuracao e transplante renal, cuja taxa de ataque pelo virus da hepatite B era 35% ao ano, foi analisado pelo periodo de 3 anos.Pesquisaram-se o antigeno de superficie da hepatite B (AgHBs) e seu anticorpo (anti-HsB) e o anticorpo contra o antigeno central do virus (anti-HBc) no soro e ambos os antigenos AgHBs e AgHBc no tecido hepatico, correlacionando-se estes dados com os aspectos histologicos das biopsias hepaticas. Vinte e dois pacientes mostraram um ou mais dos marcadores imunologicos da hepatite B e sao objeto do presente trabalho. Dezesseis apresentaram AgHBs no soro em algum momento do acompanhamento,sendo que em 11 pacientes houve permanente persistencia da positividade. A biopsia hepatica demonstrou hepatite cronica em 6 destes casos e alteracoes inespecificas ou ausencia de alteracoes nos demais. A concomitancia de AgHBs e AgHBc em tecido so foi observada em casos de Hepatite cronica com uma excecao. AgHBs isolado foi encontrado em lesoes hepaticas e em um caso de hepatite cronica persistente. Hepatite viral aguda, B, clinicamente diagnosticada ocorreu em 4 casos tendo 3 evoluido para cura clinica ou histologica e um para hepatite cronica