Multiplexed electrochemical immunosensor for detection of celiac disease serological markers

Celiac disease (CD) is a gluten-induced autoimmune enteropathy characterized by the presence of antibodies against gliadin (AGA) and anti-tissue transglutaminase (anti-tTG) antibodies. A disposable electrochemical dual immunosensor for the simultaneous detection of IgA and IgG type AGA and antitTG antibodies in real patient’s samples is presented. The proposed immunosensor is based on a dual screen-printed carbon electrode, with two working electrodes, nanostructured with a carbon–metal hybrid system that worked as the transducer surface. The immunosensing strategy consisted of the immobilization of gliadin and tTG (i.e. CD specific antigens) on the nanostructured electrode surface. The electrochemical detection of the human antibodies present in the assayed serum samples was carried out through the antigen–antibody interaction and recorded using alkaline phosphatase labelled anti-human antibodies and a mixture of 3-indoxyl phosphate with silver ions was used as the substrate. The analytical signal was based on the anodic redissolution of enzymatically generated silver by cyclic voltammetry. The results obtained were corroborated with commercial ELISA kits indicating that the developed sensor can be a good alternative to the traditional methods allowing a decentralization of the analyses towards a point-of-care strategy.

Avaliação da exposição a fungos e partículas em explorações avícolas e suinícolas: estudo

O projeto “Avaliação da Exposição a Fungos e Partículas em Explorações Avícolas e Suinícolas” contemplou um elevado número de colheitas ambientais e biológicas e respectivo processamento laboratorial, sendo apenas possível a sua concretização graças ao financiamento disponibilizado pela Autoridade para as Condições de Trabalho. Foi realizado um estudo transversal para avaliar a contaminação causada por fungos e partículas em 7 explorações avícolas e 7 explorações suinícolas. No que concerne à monitorização biológica, foram medidos os parâmetros espirométricos, utilizando o espirómetro MK8 Microlab, avaliada a existência de sintomas clínicos associados com a asma e outras doenças alérgicas, através de questionário adaptado European Community Respiratory Health Survey e, ainda, avaliada a sensibilização aos agentes fúngicos (IgE). Foram ainda adicionados dois objetivos ao estudo, designadamente: aferir a existência de três espécies/estirpes potencialmente patogénicas/toxinogénicas com recurso à biologia molecular e avaliar a exposição dos trabalhadores à micotoxina aflatoxina B1 por recurso a indicador biológico de exposição. Foram colhidas 27 amostras de ar de 25 litros nas explorações avícolas e 56 de 50 litros nas explorações suinícolas através do método de impacto. As colheitas de ar e a medição da concentração das partículas foram realizadas no interior e no exterior dos pavilhões, sendo este último considerado como local de referência. Simultaneamente, a temperatura e a humidade relativa também foram registadas. As colheitas das superfícies foram realizadas através da técnica de zaragatoa, tendo sido utilizado um quadrado de metal inoxidável de 10 cm de lado, de acordo com a International Standard ISO 18593 – 2004. As zaragatoas obtidas (20 das explorações avícolas e 48 das explorações suinícolas) foram inoculadas em malte de extract agar (2%) com cloranfenicol (0,05 g/L). Além das colheitas de ar e de superfícies, foram também obtidas colheitas da cama das explorações avícolas (7 novas e 14 usadas) e da cobertura do pavimento das explorações suinícolas (3 novas e 4 usadas) e embaladas em sacos esterilizados. Cada amostra foi diluída e inoculada em placas contendo malte extract agar. Todas as amostras foram incubadas a 27,5ºC durante 5 a 7 dias e obtidos resultados quantitativos (UFC/m3; UFC/m2; UFC/g) e qualitativos com a identificação das espécies fúngicas. Para a aplicação dos métodos de biologia molecular foram realizadas colheitas de ar de 300 litros utilizando o método de impinger com a velocidade de recolha de 300 L/min. A identificação molecular de três espécies potencialmente patogénicas e/ou toxinogénicas (Aspergillus flavus, Aspergillus fumigatus e Stachybotrys chartarum) foram obtidas por PCR em tempo real (PCR TR) utilizando o Rotor-Gene 6000 qPCR Detection System. As medições de partículas foram realizadas por recurso a equipamento de leitura direta (modelo Lighthouse, 2016 IAQ). Este recurso permitiu medir a concentração (mg/m3) de partículas em 5 dimensões distintas (PM 0.5; PM 1.0; PM 2.5; PM 5.0; PM10). Nas explorações avícolas, 28 espécies/géneros de fungos foram isolados no ar, tendo Aspergillus versicolor sido a espécie mais frequente (20.9%), seguida por Scopulariopsis brevicaulis (17.0%) e Penicillium sp. (14.1%). Entre o género Aspergillus, Aspergillus flavus apresentou o maior número de esporos (>2000 UFC/m3). Em relação às superfícies, A. versicolor foi detetada em maior número (>3 × 10−2 UFC/m2). Na cama nova, Penicillium foi o género mais frequente (59,9%), seguido por Alternaria (17,8%), Cladosporium (7,1%) e Aspergillus (5,7%). Na cama usada, Penicillium sp. foi o mais frequente (42,3%), seguido por Scopulariopsis sp. (38,3%), Trichosporon sp. (8,8%) e Aspergillus sp. (5,5%). Em relação à contaminação por partículas, as partículas com maior dimensão foram detectadas em maiores concentrações, designadamente as PM5.0 (partículas com a dimensão de 5.0 bm ou menos) e PM10 (partículas com a dimensão de 10 bm ou menos). Neste setting a prevalência da alteração ventilatória obstrutiva foi superior nos indivíduos com maior tempo de exposição (31,7%) independentemente de serem fumadores (17,1%) ou não fumadores (14,6%). Relativamente à avaliação do IgE específico, foi apenas realizado em trabalhadores das explorações avícolas (14 mulheres e 33 homens), não tendo sido encontrada associação positiva (p<0.05%) entre a contaminação fúngica e a sensibilização a antigénios fúngicos. No caso das explorações suinícolas, Aspergillus versicolor foi a espécie mais frequente (20,9%), seguida por Scopulariopsis brevicaulis (17,0%) e Penicillium sp. (14,1%). No género Aspergillus, A. versicolor apresentou o maior isolamento no ar (>2000 UFC/m3) e a maior prevalência (41,9%), seguida por A. flavus e A. fumigatus (8,1%). Em relação às superfícies analisadas, A. versicolor foi detetada em maior número (>3 ×10−2 UFC/m2). No caso da cobertura do pavimento das explorações suinícolas, o género Thicoderma foi o mais frequente na cobertura nova (28,0%) seguida por A. versicolor e Acremonium sp. (14,0%). O género Mucor foi o mais frequente na cobertura usada (25,1%), seguido por Trichoderma sp. (18,3%) e Acremonium sp. (11,2%). Relativamente às partículas, foram evidenciados também valores mais elevados na dimensão PM5 e, predominantes nas PM10. Neste contexto, apenas 4 participantes (22,2%) apresentaram uma alteração ventilatória obstrutiva. Destes, as obstruções mais graves encontraram-se nos que também apresentavam maior tempo de exposição. A prevalência de asma na amostra de trabalhadores em estudo, pertencentes aos 2 contextos em estudo, foi de 8,75%, tendo-se verificado também uma prevalência elevada de sintomatologia respiratória em profissionais não asmáticos. Em relação à utilização complementar dos métodos convencionais e moleculares, é recomendável que a avaliação da contaminação fúngica nestes settings, e, consequentemente, a exposição profissional a fungos, seja suportada pelas duas metodologias e, ainda, que ocorre exposição ocupacional à micotoxina aflatoxina B1 em ambos os contextos profissionais. Face aos resultados obtidos, é importante salientar que os settings alvo de estudo carecem de uma intervenção integrada em Saúde Ocupacional no âmbito da vigilância ambiental e da vigilância da saúde, com o objetivo de diminuir a exposição aos dois factores de risco estudados (fungos e partículas).

Amplificação em imunohistoquímica: estudo comparativo de sistemas de polímeros

Desde o início da utilização da imunohistoquímica em anatomia patológica, um dos objetivos tem sido detetar as quantidades mais ínfimas de antigénio, tornando-o visível ao microscópio ótico. Vários sistemas de amplificação têm sido aplicados de forma a concretizar este objetivo, tendo surgido um grupo genérico de métodos simples e que apresentam uma amplificação superior: são os denominados métodos do polímero indireto. Tendo em conta a variedade de métodos disponíveis, o autor propõe-se a comparar a qualidade de quatro sistemas de amplificação, que recorrem ao método do polímero indireto com horseradish peroxidase (HRP). Foram utilizadas lâminas de diferentes tecidos, fixados em formol e incluídos em parafina, nos quais se procedeu à identificação de 15 antigénios distintos. Na amplificação recorreu-se a quatro sistemas de polímero indireto (Dako EnVision+ System – K4006; LabVision UltraVision LP Detection System – TL-004-HD; Leica NovoLink – RE7140-k; Vector ImmPRESS Reagent Kit – MP-7402). A observação microscópica e classificação da imunomarcação obtida foram feitas com base num algoritmo que enquadra intensidade, marcação específica, marcação inespecífica e contraste, num score global que pode tomar valores entre 0 e 25. No tratamento dos dados, para além da estatística descritiva, foi utilizado o teste one-way ANOVA com posthoc de tukey (alfa=0.05). O melhor resultado obtido, em termos de par média/desvio-padrão, dos scores globais foi o do NovoLink (22,4/2,37) e o pior foi o do EnVision+ (17,43/3,86). Verificou-se ainda que existe diferença estatística entre os resultados obtidos pelo sistema NovoLink e os sistemas UltraVision (p=.004), ImmPRESS (p=.000) e EnVision+ (p=.000). Concluiu-se que o sistema que permitiu a obtenção de melhores resultados, neste estudo, foi o Leica NovoLink.

Risk factors for recurrent wheezing in infants: a case-control study

ABSTRACT OBJECTIVE To evaluate the association between recurrent wheezing and atopy, the Asthma Predictive Index, exposure to risk factors, and total serum IgE levels as potential factors to predict recurrent wheezing. METHODS A case-control study with infants aged 6-24 months treated at a specialized outpatient clinic from November 2011 to March 2013. Evaluations included sensitivity to inhalant and food antigens, positive Asthma Predictive Index, and other risk factors for recurrent wheezing (smoking during pregnancy, presence of indoor smoke, viral infections, and total serum IgE levels). RESULTS We evaluated 113 children: 65 infants with recurrent wheezing (63.0% male) with a mean age of 14.8 (SD = 5.2) months and 48 healthy infants (44.0% male) with a mean age of 15.2 (SD = 5.1) months. In the multiple analysis model, antigen sensitivity (OR = 12.45; 95%CI 1.28–19.11), positive Asthma Predictive Index (OR = 5.57; 95%CI 2.23–7.96), and exposure to environmental smoke (OR = 2.63; 95%CI 1.09–6.30) remained as risk factors for wheezing. Eosinophilia ≥ 4.0% e total IgE ≥ 100 UI/mL were more prevalent in the wheezing group, but failed to remain in the model. Smoking during pregnancy was identified in a small number of mothers, and secondhand smoke at home was higher in the control group. CONCLUSIONS Presence of atopy, positive Asthma Predictive Index and exposure to environmental smoke are associated to recurrent wheezing. Identifying these factors enables the adoption of preventive measures, especially for children susceptible to persistent wheezing and future asthma onset.

Suppression of humoral immunity and lymphocyte responsiveness during experimental trypanosoma cruzi infections

C3H/He and C57B1/6 mice were inoculated with 500 Trypanosoma cruzi trypomastigotes (Strain Y). During the acute phase infected mice presented parasitemia and enlargement of lymph nodes and spleens and intracellular parasites were observed in the heart. Examinations of cells derived from spleen and lymph nodes showed increased numbers of IgM and IgG-bearing cells. During the peak of splenomegaly, about day 17 post-infections, splenic lymphocytes showed a marked decrease in responsiveness to T and B-cell mitogens, parasite antigens and plaque forming cells (PFC) to sheep red blood cells (SRBC). Unfractionated or plastic adherent splenic cells from mice, obtained during the acute phase were able to suppress the response to mitogens by lymphocytes from uninfected mice. During the chronic phase. Disappearance of parasitemia and intracellular parasites in the hearts as well as a decrease in spleen size, was observed. These changes preceded the complete recovery of responsiveness to mitogens and T. cruzi antigens by C57B1/6 splenic lymphocytes. However, this recovery was only partial in the C3H/He mice, known to be more sensitive to T. cruzi infection. Partial recovery of humoral immune response also occurred in both strains of mice during the chronic phase.

The value of the Widal test in the diagnosis of Prolonged Septicemic Salmonellosis

Twenty patients with prolonged septicemic salmonellosis (Group 1) and 20 with schistosomiasis mansoni (Group 2) were selected for this study. In both groups, the Widal test was done using antigens of the sample Ty 901 (S. typhi). The test was also applied in 6 group 1 patients with antigens prepared from salmonellae isolated from these patients (autoantigens). Titres over 1:200 were considered significant. Ten group 1 patients (50%) were positive for antigen "H" and 5 (25%) were positive for antigen "O". Three patients with negative "H" and "O" reactions became positive with high titres when using autoantigens. Two other cases maintained the same positive titres and one case showed a fourfold increase in titres when the test was done 'with antigens of the Salmonella isolated. The Widal test was positive in most patients infected with group D Salmonellae. Considering titres above 1:200, all cases were negative in Group 2. The authors conclude that the Widal test has low positivity in prolonged septicemic salmonellosis. The test may be valuable in the diagnosis of this disease when using S. paratyphi "A" and "B" antigens and a mixture of Salmonella antigens taken from other groups.

Anaphylaxis with Schistosoma mansoni extracts in normal and infected mice

Methods generally utilized for studies on anaphylaxis to protein antigens such as determination of histamine release to the blood, hemoconcentration, histamine release from peritoneal mast cells and passive cutaneous anaphylaxis (PCA) were used to investigate some aspects of the anaphylaxis to parasite antigens in Schistosoma mansoni infected mice. The release of histamine to the blood and significant rates of hemoconcentration were induced by intravenous injection of schistosomula or cercarial extracts into 10-13 weeks infected mice. Cercarial, schistosomula, worm tegument and soluble egg antigens were able to trigger histamine release from peritoneal mast cells from chronically infected mice. In spite of the PCA reaction beeing detected within 2 hours of sensitization (IgG1antibodies) in 6 of 8 tested sera from chronically infected mice, no detectable reactions were obtained after 48 hours sensitization (IgE antibodies). Although IgE was not detected in the circulation, by the PCA technique, the results indicate that the infected mice contained IgE antibodies bound to their mast cells.

Cell mediated immune response in human antirabies revaccination

The occurrence of secondary cell mediated immune response (CMI) in human antirabies immunization was studied. The Puenzalida & Palácios vaccine was used because it is routinely used in Brazil. CMI was evaluated by lymphoblastic transformation indices obtained in whole blood culture in the presence of rabies and control (nervous tissue) antigens. Eleven volunteers submitted to revaccination constituted the group under study, while three other volunteers submitted primo vaccination were utilized as control group. A clear secondary CMI to rabies antigen was detected in all the revaccinated volunteers who showed earlier and more intense response than the control group. Response to the control antigen, however, present in all the components of the first group was not detectable in two out of the three primovaccinated and very low in the third one.

Approaches to tuberculosis mucosal vaccine development using nanoparticles and microparticles: a review

Next-generation vaccines for tuberculosis should be designed to prevent the infection and to achieve sterile eradication of Mycobacterium tuberculosis. Mucosal vaccination is a needle-free vaccine strategy that provides protective immunity against pathogenic bacteria and viruses in both mucosal and systemic compartments, being a promising alternative to current tuberculosis vaccines. Micro and nanoparticles have shown great potential as delivery systems for mucosal vaccines. In this review, the immunological principles underlying mucosal vaccine development will be discussed, and the application of mucosal adjuvants and delivery systems to the enhancement of protective immune responses at mucosal surfaces will be reviewed, in particular those envisioned for oral and nasal routes of administration. An overview of the essential vaccine candidates for tuberculosis in clinical trials will be provided, with special emphasis on the potential different antigens and immunization regimens.

Growth curves of Leishmania braziliensis braziliensis Promastigotes and surface antigen expression before and after adaptation to Schneider's drosophila medium as assessed by anti-Leishmania human sera

Leishmania braziliensis braziliensis(MHOM/BR/75/M2903) was grown in Schneider's Drosophila medium. In one set of experiments promastigotes were already adapted to the medium by means of serial passages whereas in the second cells were grown in a biphasic medium and transfered to the liquid. Growth was more abundant for culture medium adapted cells; degenerate cells in small numbers as well as dead ones were present from day 5 for promastigotes adapted to liquid medium and from day 3 for newly adapted cells. Synthesis of surface antigens differed according to length of cell culture as assessed by the titer of five mucocutaneous leishmaniasis sera on subsequent days. Five days of culture for cells already adapted to the culture medium and 3 days for newly adapted ones were judged to be the best for the preparation of immunofluorescence antigens.

HLA and malaria in four colombian ethnic groups

HLA antigens and their relationship with malaria infection were studied in four different ethnic groups in Colombia (South America): two groups of indians (Kunas and Katios), one of negroes and a group of mixed ancestry. A total of 965 persons were studied, 415 with malaria and 550 as controls. HLA-A,B, and C antigen frequencies in the four groups are reported. The association of each HLA antigen with malaria infection due to P. vivax and to P. falciparum was evaluated. Negroes, Kunas and Katios indians variously lack from 6 to 9 of the HLA antigens found in the mixed group. In the designated ethnic groups, antigens B5, B13, B15, Cw2 and Cw4 showed borderline association with malaria infection. However, in the mixed ethnic group, statistically significant associations were found with malaria infection and the presence of A9, Aw19, B17, B35, and Z98 (a B21-B45: crossreacting determinant) with few differences when P. vivax infection and P. falciparum infection were considered individually. This finding may represent a lack of general resistance to malaria in the group that harbors antigens of Caucasian origin. These individuals have been in direct and permanent contact with malaria only in the past 65 years. In contrast, indians, both Kunas and Katios, and Negroes have lived for centuries in malaria endemic areas, and it is possible that a natural selection system has developed through which only those individuals able to initiate an acute immune response to malaria have survived.

Lytic antibodies elicited by Trypanosoma cruzi infection recognize epitopes present on both bloodstream trypomastigote and epimastigote forms of parasite

Sera of Chaga's disease patients containing anti-T. cruzi lytic antibodies were submitted to affinity chromatography using Sepharose 4B conjugated with antigen extracted from epimasiigote or trypomasiigote forms of the parasite. Epimastigotes were obtained from culture at the exponential growth phase and the trypomastigotes from blood of infected and immunosuppressed mice. Antigen of both parasite forms was obtained by sonication of the parasites followed by centrifugation. Both antigens were then conjugated to activated Sepharose 4B. Affinity chromatography was performed by passing sera from chagasic patients through an immunoadsorbent column containing either epimasiigote or trypomasiigote antigens. Antibodies bound to the column were eluted with cold 0,2 M glycine buffer pH 2,8. The eluted antibodies were analysed regarding their isotype and lytic activity. The results showed that anti-T. cruzi lytic antibodies present in sera from chagasic patients are mainly located in the IgG isotype and recognize epitopes present in both trypomasiigote and epimastigote forms. A brief report of this work has already been published12.

Immunopathology of human schistosomiasis mansoni: I. Immunomodulatory influences on T cell function

Cell mediated immune response was studied in patients with recent and chronic Schistosoma mansoni infection. Precultured peripheral mononuclear cells showed significantly higher responses to S. mansoni adult worm antigen (SAWA) when compared to fresh cell preparations. The addition of each patient serum to the precultured cells reactions to SAWA or recall antigens demonstrated a strong inhibitory serum action, which was also noted on allogeneic cells derived from healthy subjects. The CD4 subset was the main responding cell to SAWA being this reactivity highly suppressed by the presence of the monocyte macrophage accessory cells. We stressed the simultaneous inhibitory action of humoral and cellular factors on the specific cell response to S. mansoni.

Immunopathology of human schistosomiasis mansoni: II. NK activity and stimulation by specific antigen

Sixteen S. mansoni infected and untreated patients (5 with recent infection and 11 with chronic disease) were evaluated for their in vitro natural killer (NK) activity against the NK sensitive target K562 cell line. NK levels in 9 out of 11 patients (82%) with chronic disease were significantly lower (mean = 15 ± 6%),compared with patients recently infected (mean = 41 ± 9% p < 0.001) and with the control group (mean = 38 ± 13% p < 0.001). However, both patients and controls NK activity was stimulated by soluble adult worm antigens (SAWA), indicating that NK function even in the chronic stage of the infection is able to respond to the parasite antigens. These results suggest the possibility of NK cell participation as effector mechanism against S. mansoni.

Differences in the antigenic profile of bloodstream and cell culture derived trypomastigotes of Trypanosoma cruzi

A comparative study of the antigenic profile of bloodstream and cell culture derived trypomastigotes showed many differences in their components. Using mouse anti-T. cruzi antibodies the differences were located mostly in the 120 kDa band, whereas using chagasic patient sera the differences were located in the 85 and 52 kDa bands. These findings might explain known physiological differences between trypomatigotes obtained from cell culture and from infected blood. A brief report of this work has already been published9.