High prevalence of anti-apolipoprotein/A-1 autoantibodies in maintenance hemodialysis and association with dialysis vintage.

Autoantibodies to apolipoprotein/A-1 (anti-ApoA-1 IgG) have pro-atherogenic properties in patients at high cardiovascular risk, but its prevalence in patients with end-stage kidney disease is unknown. The aims of this single-center, cross-sectional study were to assess the prevalence of anti-ApoA-1 antibodies in patients on maintenance hemodialysis (MHD), and to examine its correlation with inflammatory biomarkers related to atherosclerotic plaque vulnerability and dialysis vintage. To this purpose, anti-ApoA-1 IgG levels and the concentrations of interleukin-6 (IL-6), interleukin-8 (IL-8), monocyte chemoattractant protein-1 (MCP-1), metalloproteinase-9 (MMP-9), tumor necrosis factor-α, and C-reactive protein (CRP) were assessed in the sera of 66 MHD patients (mean age: 68 ± 14 years, 36% women, 32% diabetics). Anti-ApoA-1 IgG positivity (defined as a blood value ≥ 97.5(th) percentile of the normal distribution as assessed in healthy blood donors) was 20%. Circulating levels of anti-ApoA-1 IgG correlated positively with dialysis vintage, but not with cardiovascular risk factors or previous cardiovascular events; no significant correlations were found between the anti-ApoA1 IgG levels and circulating levels of IL-6, IL-8, MCP-1, MMP-9, CRP, or low-density lipoprotein-cholesterol. In multivariable linear regression, adjusted for age and sex, only dialysis vintage remained positively and independently associated with anti-ApoA-1 titers (β = 0.05, 95% CI: 0.006; 0.28, P = 0.049). In conclusion, the prevalence of anti-ApoA-1 IgG is raised in the MHD-population, and positively associated with dialysis vintage, a major determinant of cardiovascular outcome. Whether antiApoA-1 antibodies play a role in the pathophysiology of accelerated atherosclerosis in the MHD-population merits further study.

Radioimmunotherapy of human colon carcinoma by 131I-labelled monoclonal anti-CEA antibodies in a nude mouse model.

A mixture of 3 MAbs directed against 3 different CEA epitopes was radiolabelled with 131I and used for the treatment of a human colon carcinoma transplanted s.c. into nude mice. Intact MAbs and F(ab')2 fragments were mixed because it had been shown by autoradiography that these 2 antibody forms can penetrate into different areas of the tumor nodule. Ten days after transplantation of colon tumor T380 a single dose of 600 microCi of 131I MAbs was injected i.v. The tumor grafts were well established (as evidenced by exponential growth in untreated mice) and their size continued to increase up to 6 days after radiolabelled antibody injection. Tumor shrinking was then observed lasting for 4-12 weeks. In a control group injected with 600 microCi of 131I coupled to irrelevant monoclonal IgG, tumor growth was delayed, but no regression was observed. Tumors of mice injected with the corresponding amount of unlabelled antibodies grew like those of untreated mice. Based on measurements of the effective whole-body half-life of injected 131I, the mean radiation dose received by the animals was calculated to be 382 rads for the antibody group and 478 rads for the normal IgG controls. The genetically immunodeficient animals exhibited no increase in mortality, and only limited bone-marrow toxicity was observed. Direct measurement of radioactivity in mice dissected 1, 3 and 7 days after 131I-MAb injection showed that 25, 7.2 and 2.2% of injected dose were recovered per gram of tumor, the mean radiation dose delivered to the tumor being thus more than 5,000 rads. These experiments show that therapeutic doses of radioactivity can be selectively directed to human colon carcinoma by i.v. injection of 131I-labelled anti-CEA MAbs.

Ablation of human colon carcinoma in nude mice by 131I-labeled monoclonal anti-carcinoembryonic antigen antibody F(ab')2 fragments.

Pooled F(ab')2 fragments of three MAbs against distinct epitopes of carcinoembryonic antigen (CEA) were used for radioimmunotherapy of nude mice bearing a subcutaneous human colon carcinoma xenograft. 9-10 d after transplantation when tumor nodules were in exponential growth, 36 mice were treated by intravenous injection of different amounts of 131I-labeled MAb F(ab')2. All 14 mice injected with a single dose of 2,200 (n = 10) or 2,800 microCi (n = 4) showed complete tumor remission. 8 of the 10 mice treated with 2,200 microCi survived in good health for 1 yr when they were killed and shown to be tumor free. Four of nine other mice treated with four fractionated doses of 400 microCi showed no tumor relapse for more than 9 mo. In contrast, all 15 mice injected with 1,600-3,000 microCi 131I-control IgG F(ab')2 showed tumor growth retardation of only 1-4 wk, and 15 of 16 mice injected with unlabeled anti-CEA MAb F(ab')2 showed unmodified tumor progression as compared with untreated mice. From tissue radioactivity distributions it was calculated that by an injection of 2,200 microCi 131I-MAb F(ab')2 a mean dose of 8,335 rad was selectively delivered to the tumor, while the tissue-absorbed radiation doses for the normal organs were: peripheral blood, 2,093; stomach, 1,668; kidney, 1,289; lung, 1,185; liver, 617; spleen, 501; small intestine, 427; large intestine, 367; bone, 337; and muscle, 198. These treatments were well tolerated since out of 19 mice with complete tumor remission only 4 required bone marrow transplantation and 17 were in good health for 6-12 mo of observation. The results demonstrate the selective destruction of established human colon carcinoma transplants by intravenous injection of either single or fractionated doses of 131I-MAb F(ab')2.

Comparison of five commercial serological tests for the detection of anti-Chlamydia trachomatis antibodies.

Screening for Chlamydia trachomatis-specific antibodies is valuable in investigating recurrent miscarriage, tubal infertility and extrauterine pregnancy. We compared here the performance of immunofluorescence (IF) to four other commercial tests in detecting IgG antibodies directed against C. trachomatis: two enzyme-linked immunosorbent assays (ELISAs) using the major outer membrane protein (MOMP) as the antigen, commercialised respectively by Medac and R-Biopharm (RB), one ELISA using the chlamydial heat shock protein 60 (cHSP60) as the antigen (Medac), as well as a new automated epifluorescence immunoassay (InoDiag). A total of 405 patients with (n = 251) and without (n = 154) miscarriages were tested by all five tests. The prevalence of C. trachomatis-specific IgG antibodies as determined by the IF, cHSP60-Medac, MOMP-Medac, MOMP-RB and InoDiag was 14.3, 23.2, 14.3, 11.9 and 26.2%, respectively. InoDiag exhibited the highest sensitivity, whereas MOMP-RB showed the best specificity. Cross-reactivity was observed with C. pneumoniae using IF, MOMP-RB and InoDiag, and Parachlamydia acanthamoebae using the cHSP60 ELISA test. No cross-reactivity was observed between C. trachomatis and the other Chlamydiales (Neochlamydia hartmannellae, Waddlia chondrophila and Simkania negevensis). Given its high sensitivity, the new automated epifluorescence immunoassay from InoDiag represents an interesting alternative. The MOMP-based ELISA of R-Biopharm should be preferred for large serological studies, given the high throughput of ELISA and its excellent specificity.

Anti-HLA antibody repertoire after IVIg infusion in highly sensitized patients waiting for kidney transplantation.

Polyclonal intravenous immunoglobulin (IVIg) treatment reduces crossmatch positivity and increases rates of transplantation in highly sensitised patients (HS). We quantified the panel reactive antibody (PRA) by microlymphocytotoxicity (MLCC), and we analysed anti-HLA class I and class II IgG specific antibody repertoire by Luminex before and after IVIg infusion alone in HS patients awaiting kidney transplantation. Five patients received three monthly infusions of 1 g/kg of IVIg. Serum samples collected pre and post IVIg treatment were submitted for PRA analysis by MLCC. Anti-class I and anti-class II antibody specificities were then tested by Luminex. We focused on the anti-HLA class I and class II antibodies directed against HLA expressed by a previous graft. We also analysed the anti-HLA antibody repertoire in three patients who had not received IVIg infusion. The PRA level determined by MLCC decreased significantly in one of the five patients, dropping from 40% to 17%. The Luminex assay showed fluctuations of the anti-HLA antibody levels over time, but no significant longterm modifications of the anti-HLA antibody repertoire were observed, even in the patient with a strong and prolonged reduction of the PRA determined by MLCC. Our results show that IVIg at 1 g/kg is not sufficient to reduce PRA and does not modify the repertoire of specific anti-HLA antibody determined by Luminex.

A diet enriched with cocoa prevents IgE synthesis in a rat allergy model

Previous studies in young rats reported the impact of cocoa intake on healthy immune status and allow suggesting it may have a role in the prevention of some immune-mediated diseases. The aim of this study was to ascertain the effect of a cocoa diet in a model of allergy in young rats. Three-week-old Brown Norway rats were immunized by i.p. injection of ovalbumin (OVA) with alum as adjuvant and Bordetella pertussis toxin. During the next 4 weeks rats received either a cocoa diet (containing 0.2% polyphenols, w/w) or a standard diet. Animals fed a standard diet showed high concentrations of anti-OVA IgG1, IgG2a, IgG2b and high anti-OVA IgE titres, which is the antibody involved in allergic response. In contrast, animals fed a cocoa diet showed significantly lower concentrations of anti-OVA IgG1 and IgG2a antibodies. Interestingly, the cocoa diet prevented anti-OVA IgE synthesis and decreased total serum IgE concentration. Analysis of cytokine production in lymph node cells at the end of the study revealed that, in this compartment, the cocoa diet decreased the tumor necrosis factor (TNF) - alpha and the interleukin (IL) -10 secretion but not IL-4 production. In conclusion, a cocoa-enriched diet in young rats produces an immunomodulatory effect that prevents anti-allergen IgE synthesis, suggesting a potential role for cocoa flavonoids in the prevention or treatment of allergic diseases.

Evaluation of a new serological test for the detection of anti-Coxiella and anti-Rickettsia antibodies.

Coxiella burnetii and members of the genus Rickettsia are obligate intracellular bacteria. Since cultivation of these organisms requires dedicated techniques, their diagnosis usually relies on serological or molecular biology methods. Immunofluorescence is considered the gold standard to detect antibody-reactivity towards these organisms. Here, we assessed the performance of a new automated epifluorescence immunoassay (InoDiag) to detect IgM and IgG against C. burnetii, Rickettsia typhi and Rickettsia conorii. Samples were tested with the InoDiag assay. A total of 213 sera were tested, of which 63 samples from Q fever, 20 from spotted fever rickettsiosis, 6 from murine typhus and 124 controls. InoDiag results were compared to micro-immunofluorescence. For acute Q fever, the sensitivity of phase 2 IgG was only of 30% with a cutoff of 1 arbitrary unit (AU). In patients with acute Q fever with positive IF IgM, sensitivity reached 83% with the same cutoff. Sensitivity for chronic Q fever was 100% whereas sensitivity for past Q fever was 65%. Sensitivity for spotted Mediterranean fever and murine typhus were 91% and 100%, respectively. Both assays exhibited a good specificity in control groups, ranging from 79% in sera from patients with unrelated diseases or EBV positivity to 100% in sera from healthy patients. In conclusion, the InoDiag assay exhibits an excellent performance for the diagnosis of chronic Q fever but a very low IgG sensitivity for acute Q fever likely due to low reactivity of phase 2 antigens present on the glass slide. This defect is partially compensated by the detection of IgM. Because it exhibits a good negative predictive value, the InoDiag assay is valuable to rule out a chronic Q fever. For the diagnosis of rickettsial diseases, the sensitivity of the InoDiag method is similar to conventional immunofluorescence.

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.

Prevalência de anticorpos anti-Neospora caninum (Apicomplexa: Sarcocystidae) em bovinos leiteiros de propriedades rurais em três microrregiões no estado do Maranhão

Objetivou-se, no presente estudo, pesquisar a prevalência de anticorpos anti-Neospora caninum em 812 amostras de soros sangüíneos de bovinos leiteiros procedentes de propriedades rurais de sete municípios das microrregiões de Itapecuru-Mirim, Médio Mearim e Presidente Dutra, estado do Maranhão, Brasil. Para o cálculo do tamanho da amostra, considerou-se um soroprevalência de 34,7% para N. caninum, com erro máximo de 9,5% e intervalo de confiança de 95%. Para a detecção da presença de anticorpos da classe IgG, utilizou-se a técnica de Imunofluorescência Indireta (IFI), com ponto de corte 1:200, usando como antígeno, taquizoítos da cepa NC-1, mantida em cultura celular no Laboratório de Diagnóstico das Parasitoses dos Animais da Escola de Medicina Veterinária da UFBA. Do total de amostras analisadas, encontrou-se uma prevalência de 50,74%. Os títulos variaram de 1:200 a 1:6400, assim distribuídos: 108 (26,21%) amostras de soro apresentaram título de 1:200; 132 (32,04%) 1:400; 94 (22,81%) 1:800; 46 (11,16%) 1:1600; 23 (5,58%) 1:3200 e nove (2,18%) com títulos de 1:6400. Dentre as microrregiões, a Itapecuru-Mirim apresentou o menor percentual de animais soropositivos (20,69%) e Presidente Dutra o maior (47,66%). Com relação à variável sexo, observou-se maior prevalência de sororreagentes nas fêmeas (46,80%) do que nos machos (52,46%). Não se verificou diferença significativa (P>0,05) para as variáveis microrregiões, sexo e idade. Conclui-se que os bovinos leiteiros das regiões estudadas estão expostos à infecção por N. caninum.

Detecção de anticorpos anti-Neospora caninum em amostras individuais e coletivas de leite de bovinos pela reação de imunofluorescência indireta

Neospora caninum é um agente envolvido em perdas reprodutivas em bovinos. O diagnóstico dessa infecção é de grande importância, principalmente para programas de erradicação e controle. Sendo assim, os objetivos deste estudo foram: (1) adaptar uma reação de imunofluorescência indireta (RIFI) para detecção de anticorpos anti-N. caninum no leite, a partir de uma RIFI padronizada para a detecção desses anticorpos no soro sanguíneo, (2) analisar a concordância entre a detecção desses anticorpos pela RIFI no soro sanguíneo e no leite de fêmeas bovinas, (3) avaliar a viabilidade da RIFI para a detecção de anticorpos anti-N. caninum em amostras coletivas de leite. Foram testadas amostras de soro sanguíneo e de leite, coletadas de 112 vacas em lactação, e seis amostras coletivas de leite, correspondentes a cada uma das propriedades avaliadas. Encontrou-se 78% de concordância entre a detecção de anticorpos no soro sanguíneo (com título de anticorpos >50) e no leite, com sensibilidade de 90% e especificidade de 100% para a RIFI nas amostras de leite. Entretanto, para as vacas com títulos de anticorpos >100 no soro sanguíneo, tanto a concordância como os valores de sensibilidade e especificidade da RIFI no leite foram de 100%. Todas as amostras coletivas de leite foram positivas na RIFI. Isso demonstra que, conforme a propriedade pode-se eleger com segurança qual a melhor abordagem diagnóstica a ser adotada em relação à coleta de soro sanguíneo ou de leite para a pesquisa de N. caninum pela RIFI. Além disso, a determinação da presença de anticorpos em amostras coletivas de leite pode servir para diagnóstico e triagem de rebanhos com animais infectados.

Diagnóstico molecular e frequência de anticorpos anti-Leishmania infantum chagasi em cães do município de Belém, Pará

A leishmaniose visceral é uma enfermidade cujo agente etiológico no Brasil é o protozoário Leishmania infantum chagasi. Os cães são considerados reservatórios urbanos da doença, sendo indicadores da ocorrência de casos humanos. O presente trabalho teve como objetivo diagnosticar a infecção por L. infantum chagasi em cães domiciliados e errantes do município de Belém, estado do Pará, através da reação em cadeia da polimerase (PCR) e da reação de imunofluorescência indireta (RIFI), empregando dois antígenos distintos. Amostras de sangue venoso de cães adultos, sem distinção de sexo ou raça, de diferentes bairros e épocas do ano da cidade de Belém-PA, foram colhidas em tubos sem e com anticoagulante para obtenção do soro e do DNA, respectivamente. Esses animais foram divididos em dois grupos: cães errantes capturados pelo Centro de Controle de Zoonoses (Grupo A) e cães domiciliados (Grupo B). Os soros foram analisados através do teste de RIFI para pesquisa de IgG utilizando-se dois antígenos distintos: 1) antígeno do kit Bio-Manguinhos/FIOCRUZ (Ag-PRO) contendo formas promastigotas de Leishmania sp. (complexo major-like); 2) Antígeno do Instituto Evandro Chagas (Ag-AMA) constituído por formas amastigotas de L. infantum chagasi. A avaliação dos dois antígenos foi realizada com as amostras reagentes a partir da titulação 1:80. Já a PCR foi realizada a partir do DNA extraído do sangue total dos animais e amplificado utilizando-se os iniciadores RV1e RV2. Das 335 amostras analisadas, 10,4% (35/335) foram reagentes na RIFI (Ag-PRO) e 0,9% (3/335) reagiram com o Ag-AMA. A distribuição das amostras positivas se deu da seguinte forma: Grupo A 14,8% (25/169) com Ag-PRO e 1,2% (2/169) com Ag-AMA; Grupo B 6% (10/166) com Ag-PRO e 0,6% (1/166) com Ag-AMA; sendo que todas as amostras positivas pelo teste de RIFI com o Ag-AMA também reagiram com o Ag-PRO e em nenhuma das amostras foi detectado o DNA de L. infantum chagasi. Os achados do presente estudo indicam que Belém ainda pode ser considerada área não endêmica para leishmaniose visceral canina e que a natureza do antígeno influencia no resultado da RIFI para a pesquisa de anticorpos anti-L. infantum chagasi em cães, sendo que a RIFI que utiliza formas promastigotas de Leishmania major-like como antígeno deve ser utilizada com cautela como método diagnóstico confirmatório em estudos epidemiológicos em áreas não endêmicas para LVC.

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.

Immune response induced in mice oral immunization with cowpea severe mosaic virus

There is increasing interest in the immune response induced by plant viruses since these could be used as antigen-expressing systems in vaccination procedures. Cowpea severe mosaic virus (CPSMV), as a purified preparation (300 g of leaves, 2 weeks post-inoculation), or crude extract from cowpea (Vigna unguiculata) leaves infected with CPSMV both administered by gavage to Swiss mice induced a humoral immune response. Groups of 10 Swiss mice (2-month-old females) were immunized orally with 10 daily doses of either 50 µg viral capsid protein (boosters of 50 µg at days 21 and 35 after immunization) or 0.6 mg protein of the crude extract (boosters of 0.6 mg at days 21 and 35 after immunization). Anti-CPSMV antibodies were quantified by ELISA in pooled sera diluted at least 1:400 at days 7, 14, 21, 28, 35 and 42 after the 10th dose. IgG and IgA against CPSMV were produced systemically, but IgE was not detected. No synthesis of specific antibodies against the proteins of leaf extracts from V. unguiculata, infected or not with CPSMV, was detected. The use of CPSMV, a plant-infecting virus that apparently does not induce a pathogenic response in animals, induced a humoral and persistent (at least 6 months) immune response through the administration of low antigen doses by gavage. These results raise the possibility of using CPSMV either as a vector for the production of vaccines against animal pathogens or in quick and easy methods to produce specific antisera for viral diagnosis.

Anti-leishmania antibodies in cerebrospinal fluid from dogs with visceral leishmaniasis

Visceral leishmaniasis in Brazil is caused by Leishmania (Leishmania) chagasi and the dog is its most important reservoir. The clinical features in dogs include loss of weight, lymphadenopathy, renal failure, skin lesions, fever, hypergammaglobulinemia, hepatosplenomegaly, anemia, and, rarely, neurological symptoms. Most infected animals develop active disease, characterized by high anti-leishmania antibody titers and depressed lymphoproliferative ability. Antibody production is not primarily important for protection but might be involved in the pathogenesis of tissue lesions. An ELISA test was used to determine if there is an association between neurological symptoms and the presence of anti-L. chagasi antibodies in cerebrospinal fluid (CSF). Thirty serum and CSF samples from symptomatic mixed breed dogs (three with neurological symptoms) from a region of high incidence of visceral leishmaniasis in Brazil were examined for antibody using total parasite antigen and anti-dog IgG peroxidase conjugate. A high level of L. chagasi antibodies was observed in sera (mean absorbance ± SD, 1.939 ± 0.405; negative control, N = 20, 0.154 ± 0.074) and CSF (1.571 ± 0.532; negative control, N = 10, 0.0195 ± 0.040) from all animals studied. This observation suggests that L. chagasi can cause breakdown of filtration barriers and the transfer of antibodies and antigens from the blood to the CSF compartment. No correlation was observed between antibody titer in CSF and neurological symptoms.

A model of chronic IgE-mediated food allergy in ovalbumin-sensitized mice

Food allergy is most frequently the result of IgE-mediated hypersensitivity reactions. Here, we describe a chronic model in which some of the intestinal and systemic consequences of continuous egg white solution ingestion by ovalbumin-sensitized eight-week-old BALB/c mice, 6 animals per group, of both sexes, were investigated. There was a 20% loss of body weight that began one week after antigen exposure and persisted throughout the experiment (3 weeks). The sensitization procedure induced the production of anti-ovalbumin IgG1 and IgE, which were enhanced by oral antigen exposure (129% for IgG1 and 164% for IgE, compared to sensitization values). Intestinal changes were determined by jejunum edema at 6 h (45% Evans blue extravasation) and by a significant eosinophil infiltration with a peak at 48 h. By day 21 of continuous antigen exposure, histological findings were mild, with mast cell hyperplasia (100%) and increased mucus production (483%). Altogether, our data clearly demonstrate that, although immune stimulation was persistently occurring in response to continuous oral antigen exposure, regulatory mechanisms were occurring in the intestinal mucosa, preventing overt pathology. The experimental model described here reproduces the clinical and pathological changes of mild chronic food allergy and may be useful for mechanistic studies of this common clinical condition.