Método imunocitoquímico para a identificação de amastigotas do Trypanosoma cruzi em cortes histológicos de rotina

A técnica da peroxidase antiperoxidase foi aplicada para a identificação de amastigotas do T. cruzi em cortes histológicos de rotina. Os tecidos foram obtidos de pacientes chagásicos crônicos e de animais na fase aguda da infecção chagásica. Anti-soros específicos produzidos em coelho contra as cepas CL, Y e Emane do T. cruzi foram utilizados como reagentes primários na técnica imunocitoquímica. Soro de coelho normal foi utilizado como controle negativo e culturas de macrófagos peritoniais de camundongos infectados com tripomastigotas e apresentando abundantes amastigotas intracelulares foram utilizadas como controles positivos. Coloração positiva ocorreu especificamente nos amastigotas intra e extra-celulares em todos os tecidos testados com os anti-soros contra as três diferentes cepas do T. cruzi. Os amastigotas isolados ou formando ninhos intracelulares tornaram-se facilmente identificáveis nas preparações histológicas utilizando-se o pequeno ou médio aumento do microscópio. O presente método aumenta a probabilidade do diagnóstico do parasitismo na doença de Chagas, e evita confundir-se amastigotas com outros microrganismos morfologicamente semelhantes.

Pesquisa de anticorpos IgM anti-Toxoplasma gondii por meio de técnica imunoenzimática reversa

Um teste imunoenzimático reverso foi padronizado utilizando-se como fase sólida, microplacas de polivinil sensibilizadas com anticorpos anti-IgM.7 Estas foram incubadas seqüencialmente com alíquotas de soros de pacientes com suspeita de toxoplasmose aguda, antígeno solúvel de Toxoplasma gondii, conjugado peroxidase F (ab')2 anti-toxoplasma e substrato enzimático. A atividade enzimática foi determinada por leitura espectrofotométrica, considerando-se como títulos dos soros a máxima diluição fornecendo valores de absorbância maiores que os obtidos com a menor diluição do soro padrão não-reativo. Em 69 amostras de soros de pacientes com toxoplasmose aguda, a média geométrica dos títulos no teste ELISA-Reverso IgM foi superior à de todos os outros testes para anticorpos IgM, não se observando resultados negativos falsos devidos a altos títulos de IgG específica. Não foi encontrada, também, reatividade cruzada em nenhuma das 104 amostras de soros de pacientes com outras patologias, inclusive em amostras contendo fator reumatóide IgM.

An immunohistochemic assay to localize leptospires in tissue specimens

An Immunoperoxidase technique for identification of leptospires in formalin fixed, paraffin embedded kidney sections is presented, using peroxidase-antiperoxidase complex. The anti-leptospiral antibody was raised in rabbit. Possible applications of this technique are discussed.

Immunoperoxidase technique in experimental chronic chagasic myocarditis

Chagas'disease has been described as the commonest form of chronic myocarditis. An immunologic pathogenesis has been discribed for this form of the disease. So far, no immunoperoxidase technique has been used for the detection of immunological deposits in chronic experimental Chagas'myocardiopathy. Forty-one Swiss mice, three months old were inoculated intraperitoneally with doses between 10 and 10(5) Tulahuen trypomastigotes. Mice were reinoculated one month after with doses between 10² and 10(5) and sacrificed at 6 (n=21) and 9 months (n=9) after the first inoculation. ECGs were recorded before sacrifice. Immunoperoxidase technique (peroxidase-antiperoxidase method), immunofluorescence (direct and indirect) as well as histological studies were performed in myocardiums and skeletal muscles of the surviving animals. The most sensitive methods for detecting chronic chagasic infection were the routine histologic studies (73%) and the ECGs 83% and 89% on 6 and 9 mo. post-infected mice, respectively. Myocardial involvement varied from interstitial mild focal lymphocyte infiltrates up to replacement of myocytes by loose connective tissue. Atrial myocardiums (21/23, 91%) were more affected than ventricles (9/23, 39%). Typical chagasic nests were rarely found. Skeletal muscle involvement (11/18 and 7/9) varied from mild to extensive lymphocyte and plasmacell infiltrates, and necrotic fibers. The involved antigen were shown in skeletal muscles by the immunoperoxidase technique as diffusely arranged granular intracytoplasmatic deposit for both IgC and total immunoglobulins. The coincidence between this technique and histologic muscle lesions was 11/18 (61(%) in 6 mo. and 6/8 (75%) at 9 mo. post-infection. In heart, delicate granular deposits of total immunoglobulins were seen diffusely arranged within the ventricular myocytes; coincidence between immunoperoxidase technique anl histologic involvement increased from 36 to 66% in animals sacrifeced 6 and 9 mo. post-infection. This strongly stressed the increase of immunologic phenomena with the chronification of infection. Concerning sensitivity, immunoperoxidase and direct immunofluorescence were highly sensitive in skeletal muscle (100%, p < 0.01). Conversely, direct immunofluorescence technique showed poor results in heart while immunoperoxidase increased its sensitivity from 21.4% (at 6 mo.) to 66.6% (at 9 mo.) post-infection (p < 0.001). Considering the necessity of obtaining an adequate vaccine in order to prevent this disease an experimental model like this, rendering immunological reactions as revealed by the immunoperoxidase technique, would be useful.

Methemoglobinemia associated with loxoscelism

In twenty five patients who presented the cutaneous form of loxoscelism, serum haptoglobin and lactic dehydrogenase, erythrocyte glucose-6-phosphate dehydrogenase, glutathione reductase, glutathione peroxidase, methemoglobin, bilirubin and reticulocytes were investigated after bite. No hemolysis was detected but an increase in methemoglobin was found in 54% of the cases; in 7% it was between 1.1% and 2%, in 27% it ranged from 2.1% to 4%, and in 20% from 4.1% to 8%. Blood samples of a normal, blood group 0 individual and of a patient who exhibited methemoglobinemia after Loxosceles bite were incubated separately with antisera against Loxosceles gaucho, Crotalus terrificus, Bothrops jararaca, with Loxosceles gaucho venom and 0.3% phenol. No methemoglobin was found after 1, 4,8 and 15 days in both sets of samples. At the 25th day all the samples, including the controls, exhibited similar methemoglobin reductase decrease. The data suggest that the methemoglobinemia which occurs in 50% of the patients probably arises from in vivo venom metabolism, inasmuch as the crude venom does not induce methemoglobinemia.

Diagnóstico precoce da leptopirose por demonstração de antígenos através de exame imuno-histoquímico em músculo da panturrilha

As dificuldades para estabelecer diagnóstico precoce e de certeza da leptospirose levaram-nos a analisar as alterações histopatológicas do músculo gastrocnêmio e avaliar, originalmente, a utilidade de método imuno-histoquímico, da peroxidase-antiperoxidase, quando desejada a demonstração do espiroquetídeo e de seus produtos no referido tecido. O estudo histopatológico de biópsias da panturrilha configurou quadro de miosite, caracterizado pela correlação entre o infiltrado inflamatório intersticial e as anormalidades degenerativo-necróticas das fibras musculares. As lesões foram consideradas mínimas em 69,45% dos pacientes, moderadas em 19,45% e intensas em 5,55%, estando ausentes nos demais. Por seu turno, o método imuno-histoquímico enzimático identificou antígeno leptospirótico em 94,45%, configurando resultado muito expressivo.

Dot-ELISA-IgM in saliva for the diagnosis of human leptospirosis using polyester fabric-resin as support (Preliminary Report)

In order to improve the diagnosis of human leptospirosis, we standardized the dot-ELISA for the search of specific IgM antibodies in saliva. Saliva and serum samples were collected simultaneously from 20 patients with the icterohemorrhagic form of the disease, from 10 patients with other pathologies and from 5 negative controls. Leptospires of serovars icterohaemorrhagiae, canicola, hebdomadis, brasiliensis and cynopteri grown in EMJH medium and mixed together in equal volumes, were used as antigen at individual protein concentration of 0.2 µg/µl. In the solid phase of the test we used polyester fabric impregnated with N-methylolacrylamide resin. The antigen volume for each test was 1µl, the saliva volume was 8 µl, and the volume of peroxidase-labelled anti-human IgM conjugate was 30 µl. A visual reading was taken after development in freshly prepared chromogen solution. In contrast to the classic nitrocellulose membrane support, the fabric support is easy to obtain and to handle. Saliva can be collected directly onto the support, a fact that facilitates the method and reduces the expenses and risks related to blood processing.

Pattern of mycobacterial antigen detection in leprosy

Immunohistochemistry reaction (Peroxidase anti-peroxidase - PAP) was carried out on fifty-two skin biopsies from leprosy patients with the purpose to identify the antigenic pattern in mycobacteria and to study the sensitivity of this method. Five different patterns were found: bacillar, granular, vesicular, cytoplasmatic and deposits, classified according to the antigenic material characteristics. Deposits (thinely particulate material) appeared more frequently, confirming the immunohistochemistry sensitivity to detect small amounts of antigens even when this material is not detected by histochemical stainings.

Immunoperoxidase for the detection for the detection of antibodies in cerebrospinal fluid in neurocysticercosis: use of Cysticercus cellulosae and Cysticercus longicollis particles fixed on microscopy slides

The ORF strain of Cysticercus longicollis represents an important model for the study of heterologous antigens in the immunodiagnosis of neurocysticercosis (NC). The immunoperoxidase (IP) technique was standardized using a particulate antigen suspension of Cysticercus longicollis (Cl) and Cysticercus cellulosae (Cc). Cerebrospinal fluid (CSF) samples were incubated on the antigen fixed to microscopy slides; the conjugate employed was anti-IgG-peroxidase and the enzymatic reaction was started by covering the slides with chromogen solution (diaminobenzidine/H2O2). After washing with distilled water, the slide was stained with 2% malachite green in water. Of the CSF samples from 21 patients with NC, 19 (90.5%) were positive, whereas the 8 CSF samples from the control group (100%) were negative. The results of the IP-Cl test applied to 127 CSF samples from patients with suspected NC showed 28.3% reactivity as opposed to 29.1 % for the IP-Cc test. The agreement index for the IP test (Cl x Cc) was 94.2%, with no significant difference between the two antigens.

AN ELISA SUITABLE FOR THE DETECTION OF RABIES VIRUS ANTIBODIES IN SERUM SAMPLES FROM HUMAN VACCINATED WITH EITHER CELL-CULTURE VACCINE OR SUCKLING-MOUSE-BRAIN VACCINE

An indirect ELISA for determination of post-vaccination rabies antibody was applied. Purified rabies virus was used as antigen to coat plates, and staphylococcal protein A linked with horseradish peroxidase was used for detecting IgG antibody in human sera. Sera from humans, vaccinated with cell-culture vaccine or suckling-mouse-brain vaccine, were examined. ELISA results were compared to those obtained from the virus neutralization test. The mean and standard deviation of OD were determined for 126 negative sera (pre-vaccination) and for 73 sera from vaccinated persons showing antibody titers lower than 0.5 IU/ml. Results were defined as ELISA -positive, -negative or -doubtful. Establishment of a doubtful region reduced the number of sera otherwise classified as positive (false-positive sera). In this way, the sensitivity, specificity and agreement values were respectively 87.5%, 92.4% and 88.5%. No significant differences were observed in these values when the group vaccinated with cell-culture vaccine and the group vaccinated with suckling-mouse-brain vaccine were compared. It was shown that much of the disagreement between the values obtained by neutralization test and ELISA occurred in sera obtained at the beginning of the immunization process, and was probably due to the presence of IgM in the serum samples, detected only by the former test. This ELISA method can be used as a screening test in rabies laboratories regardless of the kind of vaccine used for immunization.

Immunohistochemical changes in kidney glomerular and tubular proteins caused by rattlesnake (Crotalus vegrandis) venom

Renal damage is an important cause of death in patients who have survived the early effects of severe crotalid envenomation. Extracellular matrix of renal tissue is altered by Crotalus toxin activities. The aim of this study was to describe how cytoskeletal proteins and basal membrane components undergo substantial alterations under the action of Crotalus vegrandis crude venom and its hemorrhagic fraction (Uracoina-1) in mice. To detect the proteins in question, the immunoperoxidase method with monoclonal and polyclonal antibodies was used. Cell types within renal lesions were characterized by phenotypic identification, by means of immunohistologic analysis of marker proteins using different primary antibodies against mesangial cells, endothelial cells, cytoskeletal proteins (intermediate filament), extracellular matrix and basal membranes. Samples for morphological study by standard procedures (biotin-streptavidin-peroxidase technique) using light microscopy were processed. Positive and negative controls for each antigen tested in the staining assay were included. After crude venom and hemorrhagic fraction inoculation of mice, the disappearance of cytoskeletal vimentin and desmin and collagen proteins in the kidney was observed. In extracellular matrix and basal membranes, collagen type IV from envenomed animals tends to disappear from 24 h to 120 h after venom injection.

Astrocytic and microglial response and histopathological changes in the brain of horses with experimental chronic Trypanosoma evansi infection

This study aimed to characterize astrocytic and microglial response in the central nervous system (CNS) of equines experimentally infected with T. evansi. The experimental group comprised males and females with various degrees of crossbreeding, ages between four and seven years. The animals were inoculated intravenously with 10(6) trypomastigotes of T. evansi originally isolated from a naturally infected dog. All equines inoculated with T. evansi were observed until they presented symptoms of CNS disturbance, characterized by motor incoordination of the pelvic limbs, which occurred 67 days after inoculation (DAI) and 124 DAI. The animals in the control group did not present any clinical symptom and were observed up to the 125th DAI. For this purpose the HE histochemical stain and the avidin biotin peroxidase method was used. Lesions in the CNS of experimentally infected horses were those of a wide spread non suppurative meningoencephalomyelitis.The severity of lesions varied in different parts of the nervous system, reflecting an irregular distribution of inflammatory vascular changes. The infiltration of mononuclear cells was associated with anisomorphic gliosis and reactive microglia was identified. The intensity of the astrocytic response in the CNS of the equines infected by T. evansi characterizes the importance of the performance of these cells in this trypanosomiasis. The characteristic gliosis observed in the animals in this experiment suggests the ability of these cells as mediators of immune response. The parasite, T. evansi, was not identified in the nervous tissues.

Therapeutic Effects of Allium sativum and Allium cepa in Schistosoma mansoni experimental infection

The effects of both garlic (Allium sativum) and onion (Allium cepa) on some biochemical parameters in Schistosoma mansoni infected mice individually and mixed either with or without the currently used drug, praziquantel (PZQ) were investigated. These involved some immunological parameters, namely IgM, IgG, interleukins 2 and 6 (IL-2 and 6) and tumor necrosis factor (TNF-α), some antioxidant enzymes [catalase, superoxide dismutase (SOD) and glutathione peroxidase (GPX)]. In addition, parasitological and histopathological investigations were performed. No changes were observed in the normal control mice treated with dry extract of onion or garlic, individually or mixed, with or without PZQ, compared to the normal healthy control group. Infection with S. mansoni showed an increase in IgG, IgM, IL-2, IL-6, TNF-α and catalase enzyme, accompanied with a decrease in GPX and SOD antioxidant enzyme activities. Remarkable amelioration was noticed in the levels of all the measured parameters in S. mansoni infected mice after administration of the studied extracts. Moreover a significant reduction in worm burden, hepatic and intestinal eggs and oogram count was noticed which was reflected in normalization of liver architecture.

Doença de Chagas congênita: avaliação imunocitoquímica do parasitismo em tecidos de feto macerado

O método imunocitoquímico de peroxidase anti-peroxídase é aplicado, pela primeira vez, em tecidos de natimorto macerado de II-III graus para coloração específica de amastigotas do Trypanosoma cruzi, em caso de doença de Chagas congênita. Os resultados mostram que o encontro de ninhos de amastigotas é muito facilitado pelo método, mesmo em tecidos autolisados, onde a morfologia dos parasitas geralmente se mostra muito alterada; o método é recomendável, também nestes casos, para a avaliação mais precisa da intensidade do parasitismo.

Características das células parasitadas pelo Trypanosoma cruzi na parede da veia central das supra-renais de Chagásicos crônicos

Foram analisados os aspectos das células musculares parasitadas pelo Trypanosoma cruzi, na veia da supra-renal de chagásicos crônicos, através de exame ao microscópio óptico de lâminas coradas pela hematoxilina-eosina (HE), PAS, Feulgen e peroxidase-antiperoxidase (PAP) para antigenos do T. cruzi. Além das modificações nucleares descritas anteriormente, os leiomiócitos parasitados exibem alterações citoplasmáticas que podem ser vistas mesmo em células que albergam poucos parasitas. As formas amastigotas geralmente estão envoltas por halo claro e o citoplasma restante adquire aspecto granuloso ou reticular, basófilopelo HE, sendo sempre PAS e Feulgen negativos. Estes dados sugerem que o material basófilo no citoplasma deve ser RNA ribossômico. A periferia dos ninhos que mostram uma "membrana" com reação do PAP para antigenos do T. cruzi fortemente positiva, poderia ser devida a reação cruzada de material celular rechaçado para a periferia ou a difusão de antigenos do T. cruzi e sua adsorção à periferia celular. O material citoplasmático PAP positivo poderia resultar de artefato, de reação imunocitoquimica cruzada, de antigenos tripanossomóticos difundidos ou de antigenos tripanossoma-simile resultantes de interações entre o leiomiócito e o parasita.