CHEMICAL CONSTITUENTS, ANTI-INFLAMMATORY, AND FREE-RADICAL SCAVENGING ACTIVITIES OF Guettarda viburnoides CHAM. & SCHLTDL. (RUBIACEAE)

Chemical investigation of Guettarda viburnoides (leaves) led to the isolation of ursolic acid, uncaric acid, secoxyloganin, and grandifloroside, along with a mixture of quercetin-3-O-β-D-galactopyranoside and quercetin-3-O-β-D-glucopyranoside, and of β-sitosterol and stigmasterol. The structures of the isolated compounds were elucidated on the basis of their NMR data. The crude extract, ethyl acetate fraction, aqueous-methanol fraction, and grandifloroside showed significant DPPH free-radical scavenging activities with IC50 ranging from 18.92 to 26.47 µg mL-1. The topical administration of the crude extract and fractions markedly reduced the croton oil-induced mice ear edema in 67.0%-99.0%. Inhibition of tissue MPO activity was also observed, which demonstrated an anti-inflammatory effect of the G. viburnoides species.

Especificidade de anti-soro policlonal à Leifsonia xyli subsp. xyli

Detectar a presença da bactéria Leifsonia xyli subsp. xyli em material de propagação da cana-de-açúcar (Saccharum sp.) é importante para direcionar o controle do raquitismo-da-soqueira. Neste trabalho, objetivou-se produzir anticorpo policlonal específico contra Leifsonia xyli subsp. xyli (Lxx), visando utilizá-lo em método sorológico para detecção do patógeno. Para isso, o antígeno foi preparado a partir de células intactas, após lavagem por centrifugação de cultura-pura em tampão fosfato salino 0,01 M (PBS) e diálise em glutaraldeido 2% em PBS. O plano de imunização em coelho consistiu de duas injeções intramusculares da mistura 1:1 do antígeno com adjuvante Freund (completo e incompleto, a intervalos de 21 dias) e duas injeções subcutâneas do antígeno puro, a intervalos de dez dias. O anti-soro foi testado pelo método de Dot Blot com revelação por peroxidase para se determinar: (i) título do anticorpo e (ii) reação contra Lxx, Xanthomonas axonopodis pv. vesicatoria e bactérias endofíticas de cana-de-açúcar (Azospirillum brasilense, A. lipoferum, Herbaspirillum rubrisubalbicans, H. seropedicae e Gluconacetobacter diazotrophicus). A maior diluição analisada do anti-soro 1:20.000 mostrou reação fortemente positiva e específica contra Lxx e ausência de reação contra as demais bactérias. A purificação da fração IgG (Imunoglobulina G) não resultou em melhoria na reatividade e especificidade do anti-soro. Estimou-se o nível de detecção do método a partir de suspensão bacteriana em 2x10(6) células/ml.

Freqüência de anticorpos homólogos anti-Borrelia burgdorferi em eqüinos na mesorregião metropolitana de Belém, Estado do Pará

Espiroquetas transmitidas por carrapatos são microrganismos de ampla distribuição geográfica e acometem animais silvestres, domésticos e seres humanos. Procedeu-se a análise sorológica de 300 soros de eqüinos onde 58 animais eram do município Ananideua, 61 eram de Belém, 131 de Castanhal e 50 eram do município de Santa Izabel do Pará para Borrelia burgdorferi através do teste ELISA indireto. Não foram observadas diferenças significativas (P < 0,05) entre os municípios, nem quanto à raça, sexo e função dos animais. Um total de 80 (26,7%) animais foram positivos para B. burgdorferi com os títulos de 1:800, 72 (90%) eqüinos; 1:1.600, 6 (7,5%) eqüinos; e 1:3.200, 2 (2,5%) eqüinos. Os resultados observados foram similares aos descritos nos EUA, onde foram relatadas freqüências de soropositivos variando entre 7 e 75% em eqüinos assintomáticos. A presença de anticorpos homólogos contra B. burgdorferi em eqüinos na mesorregião metropolitana de Belém é indicativo da ampla distribuição do agente e da possibilidade de ocorrerem casos humanos deste agente na região.

Nematóides resistentes a alguns anti-helmínticos em rebanhos caprinos no Cariri Paraibano

Objetivou-se avaliar a resistência de nematódeos a alguns anti-helmínticos em rebanhos caprinos no Cariri Paraibano. Foram utilizados 144 animais no período seco e 120 animais no período chuvoso, fêmeas com idade acima de oito meses de vida e lactantes, distribuídos em 4 grupos: Grupo I não tratado, Grupo II tratado com albendazole a 10%, Grupo III tratado com ivermectina a 1% e Grupo IV tratado com fosfato de levamisole a 18,8%, nas doses recomendadas pelos fabricantes. Para avaliar a resistência, aplicou-se o teste de redução na contagem de ovos por grama de fezes (RCOF) e o cultivo de larvas de helmintos. As amostras fecais foram coletadas no dia do tratamento (dia base) e 7, 14 e 21 dias após o tratamento. No grupo tratado com Albendazole, observaram-se eficácias de 61%, 11% e 24% no período seco e de 55%, 14% e 12% no período chuvoso, aos 7, 14, e 21 dias, respectivamente. No grupo tratado com Ivermectina, a eficácia foi de 14%, 70% e 66% para o período seco, e de 76%, 34% e 71% para o período chuvoso, aos 7, 14 e 21 dias, respectivamente. O grupo tratado com fosfato de Levamisole apresentou percentuais de eficácia de 89%, 79% e 73% no período seco e de 76%, 69% e 67% no período chuvoso, aos 7, 14 e 21 dias, respectivamente. Os resultados obtidos indicam que os nematódeos gastrintestinais de alguns rebanhos caprinos no Cariri Paraibano não são sensíveis aos princípios ativos Albendazole, Levamisole e Ivermectina. Durante o período de estudo foi identificada a presença de parasitas dos gêneros Haemonchus, Trichostrongylus, Oesophagostomum e Strongyloides.

Comparação das técnicas de ELISA indireto e Imunofluorescência indireta na detecção de anticorpos anti-Neospora caninum em búfalas (Bubalus bubalis)

Para comparar dois testes sorológicos na detecção de anticorpos anti-Neospora caninum em soros sanguíneos de búfalas, foram coletados amostras de 288 búfalas entre dois a dez anos de idade. Para identificar a presença de imunoglobulina G anti-N. caninum utilizou-se à reação de imunofluorescência indireta (RIFI), tendo o título 200 como ponto de corte, e o Ensaio Imunoenzimático indireto (ELISA-indireto), considerando-se positiva as amostras que obtiveram razão S/P>0,5. Observaram-se 153 (53,12%) animais soropositivos para N. caninum, através da RIFI, enquanto que 50 (17,36%) animais foram reagentes no ELISA. A ocorrência de anticorpos anti-N. caninum demonstram que o parasito esta circulando entre búfalas criadas no estado do Pará, sendo que ambos os teste de RIFI e ELISA podem ser utilizados para diagnosticar imunoglobulinas contra este agente. No entanto observou-se uma fraca correlação (Kappa=0,36) entre ambos os testes, considerando a RIFI como padrão ouro.

Ação anti-helmíntica do extrato hidroalcóolico da raiz da Tarenaya spinosa (Jacq.) Raf. no controle de Haemonchus contortus em ovinos

Para investigar o potencial anti-helmíntico do extrato hidroalcóolico da raiz de Tarenaya spinosa, como alternativa no controle do nematóide gastrintestinal Haemonchus contortus de ovinos, foram realizados testes in vitro com Artemia salina, em seguida, sobre coproculturas contendo ovos do helminto, avaliando a ação ovicida e larvicida do extrato. Após os testes preliminares, foi realizado o teste in vivo em 20 ovinos machos os quais foram divididos em quatro tratamentos Grupo 1: ivermectina 1%; Grupo 2: sem tratamento; Grupo 3: extrato hidroalcóolico de T. spinosa 150μg/mL; Grupo 4: extrato hidroalcóolico de T. spinosa 300μg/mL, amostras de fezes e de sangue foram coletadas para exames parasitológicos e hematológicos. No teste in vitro, a toxicidade frente a A. salina foi de 150μg/mL, obteve-se eficiência de 81,53% sobre ovos e larvas do H. contortus. No teste in vivo, a avaliação da eficácia dos tratamentos foi realizada pelo teste de redução na contagem de ovos fecais (RCOF), os grupos tratados com Ivermectina e T. spinosa a 150μg/mL e 300μg/mL obtiveram os melhores resultados, reduzindo o OPG após 28 dias em 40,6, 41 e 40,2% respectivamente, revelando assim seu potencial fitoterápico para fins de controle de nematódeos gastrintestinais em ovinos.

Estudo da atividade anti-helmíntica do extrato etanólico de Jatropha mollissima (Pohl) Baill. (Euphorbiaceae) sob Haemonchus contortus em ovinos no semiárido paraibano

Objetivou-se com o presente trabalho, avaliar o efeito anti-helmíntico de Jatropha mollissima por meio de experimentos in vitro e in vivo. Inicialmente foi investigada a concentração de extrato com efeito bioativo, pelo teste de evolução da toxicidade do extrato etanólico de J. mollissima sobre o microcrustáceo Artemia salina, obtendo uma CL50 de 660,80µg/ml, que foi testada em coproculturas contendo larvas infectantes de Haemonchus contortus e em animais para a verificação da redução do OPG. Para o teste in vivo o extrato foi dissolvido em água para se obter as concentrações 660,80µg/ml e 1321,6µg/ml, foram coletadas fezes semanalmente e sangue quinzenalmente. Como resultados dos testes in vitro, o extrato etanólico do caule de Jatropha mollissima mostrou-se tóxico sobre A. salina, com CL50 abaixo de 1000 µg/ml e inibiu a eclosão de ovos e o desenvolvimento de larvas de H. contortus, apresentando uma eficiência de 70,77%. O teste in vivo revelou que o extrato é também eficaz em ovinos, com redução significativa na contagem de OPG após 28 dias de experimento, 47 e 44% de redução nos grupos tratados com o extrato, 7,5% no grupo de animais não tratados e 40,6% com a ivermectina. Mesmo parasitados, os animais permaneceram clinicamente saudáveis e sem anemia. O extrato etanólico do caule de Jatropha mollissima pode representar uma alternativa ao controle da verminose ovina, pois retarda a resistência parasitária.

Occurrences of anti-Toxoplasma gondii and anti-Neospora caninum antibodies in sheep from four districts of Tocantins state, Brazilian Legal Amazon Region

Toxoplasmosis and neosporosis have been recognized as economically important diseases with considerable impact on the livestock industry. Little is known concerning the occurrence of Toxoplasma gondii and Neospora caninum in sheep from Tocantins state, Brazil. Here, we investigated antibodies against these parasites and associated factors in 182 sheep from Araguaína, Santa Terezinha do Tocantins, Arguianópolis and Palmeiras do Tocantins districts, Tocantins. Sheep sera were assayed for T. gondii and N. caninum IgG antibodies by indirect fluorescence antibody test (IFAT), using cut-off point at a dilution of 1:40 and 1:25 respectively. The prevalence of seropositive animal for T. gondii was 13.74% and 13.74% for N. caninum. None of the characteristics studied including reproductive problems, presence of cats, presence of dogs and veterinary care (p>0.05) was associated with occurrence of T. gondii or N. caninum infection. Only breed was identified as associated factor for the occurrence of toxoplasmosis in sheep (p<0.05). The present study is the first report on serum occurrence of T. gondii and N. caninum in sheep from the state of Tocantins, Brazil.

Evaluation of an anti-carcinoembryonic monoclonal antibody suitable for immunoscintigraphy

An anti-carcinoembryonic antigen (CEA) monoclonal antibody (mAb 6D1.1) was evaluated in vitro and in vivo to determine its suitability as a tracer for immunoscintigraphy of colorectal carcinomas. Determination of mAb affinity for CEA showed a constant of association of 0.63 ± 0.11 x 109 M-1. Binding of technetium-99m (99mTc)-6D1.1, labeled by a direct method, to human cultured lineages was highly specific. Binding to only CEA-positive LS-174T cells resulted in a saturable curve inhibited by pre-incubation with unlabeled mAb. No binding at all was observed for the human lineages MeWo (melanoma) or ZR75-30 (breast carcinoma), neither of them expressing CEA cells. Intravenous injection of 99mTc-6D1.1 into nude mice xenografted with human LS-174T tumors resulted in planar images of excellent quality. Localization of an irrelevant mAb labeled with either 99mTc or iodine-125 was never observed in tumor masses. Biodistribution studies on excised tumoral tissue showed retention of 28.48% of the injected dose per gram of LS-174T tumor. The tumor-to-blood ratio was 3.46. The same analysis performed on the other three human xenografted tumors studied demonstrated that only the CEA-producing HT-29 (colorectal adenocarcinoma) retained 99mTc-6D1.1 while the other two (ZR75-30 and MeWo) did not. These data demonstrate that this mAb is an adequate tool for targeting CEA-expressing tumors in experimental models.

Relationship between the prevalence of anti-glutamic acid decarboxylase autoantibodies and duration of type 1 diabetes mellitus in Brazilian patients

The objective of the present study was to determine whether the duration of disease has any influence on the prevalence of glutamic acid decarboxylase autoantibodies (GADA) in Brazilian patients with type 1 diabetes (T1D) and variable disease duration. We evaluated 83 patients with T1D. All participants were interviewed and blood was obtained for GADA measurement by a commercial radioimmunoassay (RSR Limited, Cardiff, UK). Four groups of patients were established according to disease duration: A) 1-5 years of disease (N = 24), B) 6-10 years of disease (N = 19), C) 11-15 years of disease (N = 25), and D) >15 years of disease (N = 15). GADA prevalence and its titers were determined in each group. GADA was positive in 38 patients (45.8%) and its frequency did not differ between the groups. The prevalence was 11/24 (45.8%), 8/19 (42.1%), 13/25 (52%), and 6/15 (40%) in groups A, B, C, and D, respectively (P = 0.874). Mean GADA titer was 12.54 ± 11.33 U/ml for the sample as a whole and 11.95 ± 11.8, 12.85 ± 12.07, 10.57 ± 8.35, and 17.45 ± 16.1 U/ml for groups A, B, C, and D, respectively (P = 0.686). Sex, age at diagnosis or ethnic background had no significant effect on GADA (+) frequency. In conclusion, in this transversal study, duration of disease did not affect significantly the prevalence of GADA or its titers in patients with T1D after one year of diagnosis. This was the first study to report this finding in the Brazilian population.

Trypanosoma cruzi infection and/or administration of the nonsteroidal anti-inflammatory nimesulide increase the number of colonic crypts overexpressing metallothioneins in rat colon carcinogenesis

Trypanosoma cruzi infection and nonsteroidal anti-inflammatory drugs inhibit colorectal carcinogenesis by mechanisms not completely known and metallothionein proteins (MTs) may be involved in this process. Sixty-six male Wistar rats weighing 90 to 120 g were randomly divided into seven groups (GI to GVII). GI, GII and GIII animals were subcutaneously infected with 200,000 trypomastigote forms of the Y strain of T. cruzi. After 8 weeks, GI, GII, GIV, and GVI were injected with one weekly subcutaneous dose of 12 mg/kg dimethylhydrazine for 4 weeks. In sequence, GI, GIV and GV were treated with nimesulide (10 mg/kg per dose, five times per week for 8 weeks). Groups I, III, IV, and VI had 12 animals, and each of the other groups had 6 animals. All the animals were euthanized 8 weeks after the last dimethylhydrazine injection. The colons were fixed and processed for MT immunohistochemistry. The index of MT-overexpressing colonic crypts (MTEC) was estimated as the percentage of MT-stained crypts in relation to the total number of crypts scored. Five hundred crypts per animal were scored. Data were analyzed by the Kruskal-Wallis test followed by the Dunn test. There was an increase in MTEC index in the groups either infected with T. cruzi or treated with nimesulide or both infected and treated when compared to control (401, 809, and 1011%, respectively). We suggest that the increased formation of MTEC may be related to the protection against carcinogenesis provided both by T. cruzi infection and nimesulide.

Anti-tumor necrosis factor-a for the treatment of steroid-refractory acute graft-versus-host disease

Allogeneic stem cell transplantation has been increasingly performed for a variety of hematologic diseases. Clinically significant acute graft-versus-host disease (GVHD) occurs in 9 to 50% of patients who receive allogeneic grafts, resulting in high morbidity and mortality. There is no standard therapy for patients with acute GVHD who do not respond to steroids. Studies have shown a possible benefit of anti-TNF-a (infliximab)for the treatment of acute GVHD. We report here on the outcomes of 10 recipients of related or unrelated stem cell transplants who received 10 mg/kg infliximab, iv, once weekly for a median of 3.5 doses (range: 1-6) for the treatment of severe acute GVHD and who were not responsive to standard therapy. All patients had acute GVHD grades II to IV (II = 2, III = 3, IV = 5). Overall, 9 patients responded and 1 patient had progressive disease. Among the responders, 3 had complete responses and 6 partial responses. All patients with cutaneous or gastrointestinal involvement responded, while only 2 of 6 patients with liver disease showed any response. None of the 10 patients had any kind of immediate toxicity. Four patients died, all of them with sepsis. Six patients are still alive after a median follow-up time of 544 days (92-600) after transplantation. Considering the severity of the cases and the bad prognosis associated with advanced acute GVHD, we find our results encouraging. Anti-TNF-a seems to be a useful agent for the treatment of acute GVHD.

The husk fiber of Cocos nucifera L. (Palmae) is a source of anti-neoplastic activity

In the present study, we investigated the in vitro anti-tumoral activities of fractions from aqueous extracts of the husk fiber of the typical A and common varieties of Cocos nucifera (Palmae). Cytotoxicity against leukemia cells was determined by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. Cells (2 x 104/well) were incubated with 0, 5, 50 or 500 µg/mL high- or low-molecular weight fractions for 48 h, treated with MTT and absorbance was measured with an ELISA reader. The results showed that both varieties have almost similar antitumoral activity against the leukemia cell line K562 (60.1 ± 8.5 and 47.5 ± 11.9% for the typical A and common varieties, respectively). Separation of the crude extracts with Amicon membranes yielded fractions with molecular weights ranging in size from 1-3 kDa (fraction A) to 3-10 kDa (fraction B) and to more than 10 kDa (fraction C). Cells were treated with 500 µg/mL of these fractions and cytotoxicity was evaluated by MTT. Fractions ranging in molecular weight from 1-10 kDa had higher cytotoxicity. Interestingly, C. nucifera extracts were also active against Lucena 1, a multidrug-resistant leukemia cell line. Their cytotoxicity against this cell line was about 50% (51.9 ± 3.2 and 56.3 ± 2.9 for varieties typical A and common, respectively). Since the common C. nucifera variety is extensively cultured in Brazil and the husk fiber is its industrial by-product, the results obtained in the present study suggest that it might be a very inexpensive source of new antineoplastic and anti-multidrug resistant drugs that warrants further investigation.

Influence of He-Ne laser therapy on the dynamics of wound healing in mice treated with anti-inflammatory drugs

We determined the effects of helium-neon (He-Ne) laser irradiation on wound healing dynamics in mice treated with steroidal and non-steroidal anti-inflammatory agents. Male albino mice, 28-32 g, were randomized into 6 groups of 6 animals each: control (C), He-Ne laser (L), dexamethasone (D), D + L, celecoxib (X), and X + L. D and X were injected im at doses of 5 and 22 mg/kg, respectively, 24 h before the experiment. A 1-cm long surgical wound was made with a scalpel on the abdomens of the mice. Animals from groups L, D + L and X + L were exposed to 4 J (cm²)-1 day-1 of He-Ne laser for 12 s and were sacrificed on days 1, 2, or 3 after the procedure, when skin samples were taken for histological examination. A significant increase of collagen synthesis was observed in group L compared with C (168 ± 20 vs 63 ± 8 mm²). The basal cellularity values on day 1 were: C = 763 ± 47, L = 1116 ± 85, D = 376 ± 24, D + L = 698 ± 31, X = 453 ± 29, X + L = 639 ± 32 U/mm². These data show that application of L increases while D and X decrease the inflammatory cellularity compared with C. They also show that L restores the diminished cellularity induced by the anti-inflammatory drugs. We suggest that He-Ne laser promotes collagen formation and restores the baseline cellularity after pharmacological inhibition, indicating new perspectives for laser therapy aiming to increase the healing process when anti-inflammatory drugs are used.

A majority of Brazilian patients with rheumatoid arthritis HLA-DRB1 alleles carry both the HLA-DRB1 shared epitope and anti-citrunillated peptide antibodies

The objective of the present study was to evaluate the contribution of the shared epitope (SE), the rheumatoid arthritis (RA) protection model, and the occurrence of anti-cyclic citrullinated peptide (anti-CCP) antibodies in RA patients from a genetically diverse population. One hundred and forty Brazilian RA patients and 161 matched controls were typed for HLA-DRB1 alleles using amplified DNA hybridized with sequence-specific oligonucleotide probes or primers. Patients were stratified according to the presence or absence of SE (DRB1*0401, *0404, *0405, *0101, *1001, and *1402), of the DERAA alleles (DRB1*0103, *0402, *1102, *1103, *1301, *1302, and *1304), and X (all other alleles). Anti-CCP antibodies were measured by ELISA. The combined frequency of SE-positive alleles was significantly greater (76.4 vs 23.6%, P < 0.0001) than the controls. The SE/SE and SE/X genotypes were over-represented (P < 0.0001, OR = 6.02) and DERAA/X was under-represented in RA patients (P < 0.001, OR = 0.49), whereas the frequencies of the SE/DERAA, X/X and X/DERAA genotypes were not significantly different from controls. The frequency of anti-CCP antibodies was higher in SE-positive patients than in SE-negative patients (64.6 vs 44.7%, P = 0.03; OR = 2.25). Although the Brazilian population is highly miscegenated, the results of this study support the findings observed in most genetically homogeneous populations with RA; however, they are not mutually exclusive but rather complementary. The participation of DRB1-DERAA alleles in protection against RA was also observed (OR = 0.4; 95%CI = 0.23-0.68).