Human eosinophil adhesion and degranulation stimulated with eotaxin and RANTES in vitro: Lack of interaction with nitric oxide

Background: Airway eosinophilia is considered a central event in the pathogenesis of asthma. The toxic components of eosinophils are thought to be important in inducing bronchial mucosal injury and dysfunction. Previous studies have suggested an interaction between nitric oxide (NO) and chemokines in modulating eosinophil functions, but this is still conflicting. In the present study, we have carried out functional assays (adhesion and degranulation) and flow cytometry analysis of adhesion molecules (VLA-4 and Mac-1 expression) to evaluate the interactions between NO and CC-chemokines (eotaxin and RANTES) in human eosinophils. Methods: Eosinophils were purified using a percoll gradient followed byimmunomagnetic cell separator. Cell adhesion and degranulation were evaluated by measuring eosinophil peroxidase (EPO) activity, whereas expression of Mac-1 and VLA-4 was detected using flow cytometry. Results: At 4 h incubation, both eotaxin (100 ng/ml) and RANTES (1000 ng/ml) increased by 133% and 131% eosinophil adhesion, respectively. L-NAME alone (but not D-NAME) also increased the eosinophil adhesion, but the co-incubation of L-NAME with eotaxin or RANTES did not further affect the increased adhesion seen with chemokines alone. In addition, L-NAME alone (but not D-NAME) caused a significant cell degranulation, but it did not affect the CC-chemokine-induced cell degranulation. Incubation of eosinophils with eotaxin or RANTES, in absence or presence of L-NAME, did not affect the expression of VLA-4 and Mac-1 on eosinophil surface. Eotaxin and RANTES (100 ng/ml each) also failed to elevate the cyclic GMP levels above baseline in human eosinophils. Conclusion: Eotaxin and RANTES increase the eosinophil adhesion to fibronectin-coated plates and promote cell degranulation by NO-independent mechanisms. The failure of CC-chemokines to affect VLA-4 and Mac-1 expression suggests that changes in integrin function (avidity or affinity) are rather involved in the enhanced adhesion. © 2008 Lintomen et al; licensee BioMed Central Ltd.

DNA vaccine containing the mycobacterial hsp65 gene prevented insulitis in MLD-STZ diabetes

Background: Our group previously demonstrated that a DNA plasmid encoding the mycobacterial 65-kDa heat shock protein (DNA-HSP65) displayed prophylactic and therapeutic effect in a mice model for tuberculosis. This protection was attributed to induction of a strong cellular immunity against HSP65. As specific immunity to HSP60 family has been detected in arthritis, multiple sclerosis and diabetes, the vaccination procedure with DNA-HSP65 could induce a cross-reactive immune response that could trigger or worsen these autoimmune diseases. Methods: In this investigation was evaluated the effect of a previous vaccination with DNA-HSP65 on diabetes development induced by Streptozotocin (STZ). C57BL/6 mice received three vaccine doses or the corresponding empty vector and were then injected with multiple low doses of STZ. Results: DNA-HSP65 vaccination protected mice from STZ induced insulitis and this was associated with higher production of IL-10 in spleen and also in the islets. This protective effect was also concomitant with the appearance of a regulatory cell population in the spleen and a decreased infiltration of the islets by T CD8+ lymphocytes. The vector (DNAv) also determined immunomodulation but its protective effect against insulitis was very discrete. Conclusion: The data presented in this study encourages a further investigation in the regulatory potential of the DNA-HSP65 construct. Our findings have important implications for the development of new immune therapy strategies to combat autoimmune diseases. © 2009 Santos et al; licensee BioMed Central Ltd.

Epstein-Barr virus (EBV) nuclear antigen (EBNA)-4 mutation in EBV-associated malignancies in three different populations

Different ethnic groups with a high human leukocyte antigen (HLA)-A11 prevalence have been shown to experience a high rate of Epstein-Barr virus (EBV) infection, EBV-associated malignancies, and Epstein-Barr nuclear antigen (EBNA)-4 mutations. The epitopes 393-408 and 416-424 of EBNA-4 are major antigenic epitopes that elicit an HLA-A11 cytotoxic T lymphocyte (CTL) response to EBV infection. Mutations selectively involving one or more nucleotide residues in these epitopes affect the antigenicity of EBNA-4, because the mutant EBV strains are not recognized by the HLA-A11-restricted CTLs. To investigate these mutations in common EBV-associated malignancies occurring in different populations, we studied the mutation rate of epitopes 393-408 and 416-424 of EBNA-4 in 25 cases of EBV-associated Hodgkin's disease (HD), nine cases of AIDS-related non-Hodgkin's lymphoma, and 37 cases of EBV-associated gastric carcinoma (GC) from the United States, Brazil, and Japan. We found one or more mutations in these two epitopes in 50% (6/12) of United States HD, 15% (2/13) of Brazilian HD, 50% (6/12) United States GC and 28% (7/25) Japanese GC, and 22% (2/9) of United States AIDS-lymphoma. Similar mutations were found in 30% (3/10) of United States reactive, 0% (0/6) of Brazilian reactive, and 25% (2/8) Japanese reactive tissues. The most frequent amino acid substitutions were virtually identical to those seen in previously reported isolates from EBV-associated nasopharyngeal carcinomas and Burkitt's lymphomas occurring in high prevalence HLA-A11 regions. However, only 2/28 (7%) mutations occurred in HLA-A11-positive patients. Our studies suggest that: 1) EBNA-4 mutations are a common phenomenon in EBV-associated HD, GC, and AIDS-lymphoma; 2) the mutation rate does not vary in these geographic areas and ethnic groups; 3) EBNA-4 mutations in EBV-associated United States and Brazilian HD, United States and Japanese GC, and United States AIDS lymphomas are not related to patients' HLA-A11 status.

Humoral immune responses against the malaria vaccine candidate antigen Plasmodium vivax AMA-1 and IL-4 gene polymorphisms in individuals living in an endemic area of the Brazilian Amazon

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Comparison between a soluble antigen-based ELISA and IFAT in detecting antibodies against Babesia canis in dogs

O presente trabalho estudou um ensaio imunoenzimático (ELISA) indireto para a detecção de anticorpos anti-Babesia canis no soro de cães, tendo a Reação de Imunofluorescência Indireta (RIFI), como teste de referência O antígeno utilizado no ELISA do presente estudo consistiu em uma preparação antigênica solúvel de merozoítas B. canis e as diluições ótimas do antígeno, soros e conjugado foram determinadas por titulação em bloco, utilizando soros de referência positivos e negativos. A preparação antigênica solúvel de B. canis ótima foi de 10 µg.mL-1, com soros de referência positivos e negativos em uma única diluição de 1:100, e conjugado a 1:4.000. Um total de 246 amostras séricas foram colhidas em cães, durante a campanha de vacinação anti-rábica em Jaboticabal, São Paulo, Brasil e a presença de anticorpos anti-B. canis foi avaliada pelo ELISA e RIFI. Nestas condições, a média de absorbância dos soros de referência negativos foi de 0,129 ± 0,025, resultando em um ponto de corte de 0,323 (Nível de ELISA 3) e a média da absorbância dos soros de referência positivos foi de 2,156 ± 1,187. As amostras com sorologia positiva para B. canis por ELISA e RIFI foram 67,89% (n = 167) e 59,35% (n = 146), respectivamente. Os resultados obtidos sugerem que o ELISA descrito revelou-se um teste sorológico eficaz no diagnóstico da babesiose canina.

Upregulation of soluble and membrane-bound human leukocyte antigen G expression is primarily observed in the milder histopathological stages of chronic hepatitis C virus infection

Chronic hepatitis C virus (HCV) infection is a worldwide health problem that may evolve to cirrhosis and hepatocellular carcinoma. Incompletely understood immune system mechanisms have been associated with impaired viral clearance. The nonclassical class I human leukocyte antigen G (HLA-G) molecule may downregulate immune system cell functions exhibiting well-recognized tolerogenic properties. HCV genotype was analyzed in chronic HCV-infected patients. Because HLA-G expression may be induced by certain viruses, we evaluated the presence of HLA-G in the liver microenvironment obtained from 89 biopsies of patients harboring chronic HCV infection and stratified according to clinical and histopathological features. Overall, data indicated that HCV genotype 1 was predominant, especially subgenotype 1a, with a prevalence of 87%. HLA-G expression was observed in 45(51%) liver specimens, and it was more frequent in milder stages of chronic hepatitis (67.4%) than in moderate (27.8%; p = 0.009) and severe (36.0%; p = 0.021) stages of the disease. Altogether, these results suggest that the expression of HLA-G in the context of HCV is a complex process modulated by many factors, which may contribute to an immunologic environment favoring viral persistence. However, because the milder forms predominantly expressed HLA-G, a protective role of this molecule may not be excluded. (C) 2012 American Society for Histocompatibility and Immunogenetics. Published by Elsevier B.V. All rights reserved.

Differential antibody isotype expression to the major Paracoccidioides brasiliensis antigen in juvenile and adult form paracoccidioidomycosis

We investigated the relationship between antibody response to the major Paracoccidioides brasiliensis antigen, a 43-kDa glycoprotein, and the two paracoccidioidomycosis (PCM) clinical presentations, the juvenile and the adult forms. Total immunoglobulin G (IgG), IgG isotypes, and IgA anti-gp43 antibodies were determined by enzyme-linked immunosorbent assay in patients' sera. Juvenile PCM patients had higher (P =.003) IgG anti-gp43 levels than adult form patients. IgG1 subclass levels, however, were comparable between the two clinical forms. Patients with the juvenile form had higher (P <.001) IgG4, but lower(P =.03) IgG2 levels than patients with the adult form. The IgG4 isotype, regulated by interleukin 4, was found in all juvenile form patients but in only 12% of the adult form patients. In contrast, high levels of the IgG2 isotype, regulated by interferon-gamma, were found in 41% of the adult PCM patients, mainly those with a more benign disease, but in only 12% of the juvenile patients. IgG3 was either absent or detected at low levels. These results demonstrate, for the first time, specific IgG4 antibodies in the humoral immune response of patients with an endemic deep mycosis and suggest that the switch to the IgG subclasses in PCM is regulated by the patients' T-helper subset (Th-l or Th-2) dominant cytokine profile. A possible role for IgG4 in the immunopathogenesis of the juvenile, more severe form of the disease is discussed. Finally, IgA was found mainly in adult form patients, probably as a result of the chronic mucosal antigenic stimulation characteristic of this form. (C) Elsevier, Paris.

KIR genes and their human leukocyte antigen ligands in the progression to cirrhosis in patients with chronic hepatitis C

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Frequency of human leukocyte antigen (hla) in patients with malaria and in the general population of Humaitá county, Amazonas state, Brazil

Em agosto de 1983 foram observados 85 habitantes do Município de Humaitá, Estado do Amazonas, Brasil, com a finalidade de estudar a prevalência dos antígenos de HLA -A, -B, -C e DR, dentre os quais 38 eram doentes com malária causada pelo Plasmodium falciparum Todos eles foram examinados para avaliação de esplenomegalia, exame parasitológico de sangue e pesquisa de anticorpos de malária. Foram constituídos três grupos: (I) 25 indivíduos nascidos na região Amazônica que nunca tiveram malária; (II) 38 indivíduos naturais da Amazônia que tinham sido tratados de malária no passado, ou que estavam tendo malária atual, e (III) 22 doentes com malária que contraíram na Amazônia e eram procedentes de outras regiões do Brasil. Foram colhidas amostras de sangue de cada um deles, separados os linfôcitos e os antígenos de HLA foram tipados pelo teste de microlinfocitotoxidade. Houve elevada freqüência de antígenos não identificados, nos grupos estudados, o que sugere ou a existência de homozigoze, oufenôtipo não identificado nessa população. Houve alta freqüência fenotípica de antígeno deAg(W24) (44,7%) no Grupo II, quando comparado ao Grupo 1(32%) ou Grupo III (9%). Os indivíduos do Grupo II mostraram também elevada freqüência do antígeno DR4 (80%) quando comparado ao Grupo 1(36,3%) ou Grupo III(16,6%). Essas observações sugerem a possibilidade de suscetibilidadegenética ã malária entre os nativos da Amazônia e indicam a necessidade da realização de inquéritos mais extensos sobre a freqüência de antígenos de HLA em habitantes de zona endêmica de malária.

Flow cytometric assessment of canine erythrocytes and platelets for dog erythrocyte antigen 1.1

Background: In human medicine, transfusion of ABO-mismatched platelets has been associated with shortened platelet survival and refractoriness to platelet transfusion because of expression of certain blood group antigens on platelets. It remains unknown if canine platelets express dog erythrocyte antigens (DEAs). Objective: The aim of this study was to develop a flow cytometric assay for DEA 1.1 and determine whether DEA 1.1 is present on canine platelets.Methods: Blood was collected from 172 clinically healthy dogs. Platelets and erythrocytes from each dog were tested for DEA 1.1 by flow cytometry using anti-DEA 1.1 blood-typing sera. Erythrocytes from each dog were also assessed for DEA 1.1 using a standard tube-typing test (T1) and using a second tube method (T2), if the flow cytometric and T1 results differed.Results: Using flow cytometry, DEA 1.1 was detected on erythrocytes of all 110 dogs shown by T1 or T2 testing to be DEA 1.1-positive. Initial results of the T1 test had a diagnostic accuracy of 93% (160 correct/ 172 tests). The frequency of erythrocyte DEA 1.1 positivity in previously untyped dogs (n = 118) was 56%. DEA 1.1 expression was not detected on platelets from DEA 1.1-positive dogs.Conclusions: Flow cytometry was a reliable method for detection of DEA 1.1 on canine erythrocytes. The absence of DEA 1.1 on platelets from DEA 1.1-positive dogs suggests that their platelets do not express DEA 1.1 and will not induce production of anti-DEA 1.1 antibodies that might lead to platelet refractoriness or reactions to a subsequent transfusion of DEA 1.1positive erythrocytes.

Fertility-associated antigen on Nelore bull sperm and reproductive outcomes following first-service fixed-time AT of Nelore cows and heifers

The objective was to determine whether the presence of fertility-associated antigen (FAA) on sperm collected from Nelore (Bos indicus) bulls can be used to assess potential fertility of sperm for use at first-service fixed-time AI (TAI). Six Nelore bulls were selected based on FAA status (FAA-negative: N = 3; FAA-positive: N = 3) and the ability to produce neat semen with >= 70% morphologically normal sperm and 60% estimated progressive motility before cryopmservation. In Experiment 1, suckled multiparous Nelore cows (N = 835) were evaluated for body condition score (BCS) and received an intravaginal progesterone device (CIDR) and 2.0 mg of estradiol benzoate (Day 0). on Day 9 the CIDR was removed, 12.5 mg of PGF(2 alpha) and 0.5 mg of estradiol cypionate were administered, and calves were removed for 48 h. All cows received TAI on Day II (48 h after CIDR removal). Pregnancy per TAI (P/TAI) was not different between FAA-positive and FAA-negative bulls (41.5% vs. 39.3%, respectively). There was an effect of AI technician on P/TAI (36.0% vs. 43.9%; P < 0.05) and BCS tended to affect P/TAI (P = 0.09), as cows with BCS >= 2.75 were 1.4 times more likely to become pregnant compared with cows with BCS < 2.75. In Experiment 2, nulliparous Nelore heifers (N = 617) were evaluated for BCS and received a CIDR and estradiol benzoate (2.0 mg) on Day 0. on Day 7, all heifers received PGF(2 alpha) (12.5 mg). on Day 9, CIDR inserts were removed and all heifers received estradiol cypionate (0.6 mg) and 200 IU eCG. All heifers received TAI on Day 11 (48 h after CIDR removal). Pregnancy/TAI was different (P = 0.04) between FAA-positive and FAA-negative bulls (33.7% vs. 40.7%, respectively). Presence of FAA on sperm was unsuccessful in assessing the potential fertility of sperm for use in TAI. (C) 2012 Elsevier B.V. All rights reserved.

Comparative evaluation of an indirect ELISA test for diagnosis of swine cysticercosis employing antigen from Taenia solium and Taenia crassiceps metacestodes

Taenia solium cysticercosis is still a serious public health problem in several countries where poverty and lack of hygiene favor transmission. Because pigs are the primary intermediate hosts, prevalence of porcine cysticercosis is a reliable indicator of active transmission zones. Serological diagnostic methods are important tools for epidemiological studies since they can be applied to living animals on a large scale. Four antigen preparations (cyst fluid and crude) from T. solium and T. crassiceps metacestodes were compared for swine cysticercosis diagnosis by indirect ELISA (IE). Twenty-eight serum samples from swine naturally and experimentally infected by cysticerci of T. solium and 56 serum samples from swine reared in commercial herds were tested. Best results of overall sensitivity were obtained by the use of cyst fluid and crude antigen of T. crassiceps metacestode (100 and 96.4%, respectively). Using homologous antigen preparations we have observed higher specificity percentage (98.2% for cyst fluid and 96.4% for crude metacestode T. solium antigen). We concluded that sensitivity is of far more importance than specificity for identification of endemic areas in order to prevent transmission to man. We conclude, therefore, that IE performed with cyst fluid antigen of T. crassiceps metacestode is a better tool for that purpose. (C) 2000 Elsevier B.V. B.V. All rights reserved.

Comparison between ELISA using total antigen and immunochromatography with antigen rK39 in the diagnosis of canine visceral leishmaniasis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Comparative evaluation of enzyme-linked immunosorbent assay based on crude and purified antigen in the diagnosis of canine visceral leishmaniasis in symptomatic and oligosymptomatic dogs

This study evaluated the performance of crude total antigen (CTA) and fucose-mannose ligand antigen (FML) in an enzyme-linked immunosorbent assay for diagnosis of canine visceral leishmaniasis (CVL). The assays used sera from known negative controls (n = 30), clinically symptomatic (n = 30) and oligosymptomatic (n = 30) parasitologically proven infection (by microscopy). Aspirates of popliteal lymph node from infected canines were colleted to score parasitism and compared with the ELISA results. The study indicated that FML used in ELISA provided high sensitivity for detecting oligosymptomatic dogs (90%) and CTA showed greater sensitivity than FML for symptomatic canines (90%). In oligosymptomatic dogs, specificity was 100% for CTA-ELISA, but in symptomatic dogs, FML specificity was higher (96.7%) than CTA-ELISA (93.3%). A significant correlation was observed between the degree of parasitism and the results obtained in CTA-ELISA. Since no available antigen offers 100% specificity and sensitivity for CVL diagnosis, the choice of antigen used must depend on the aim of the investigation. (C) 2008 Elsevier B.V. All rights reserved.

MyoD, myogenin and proliferating cell nuclear antigen expression in growing Nile tilapia (Oreochromis niloticus L.)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)