A competitive hyper-immune yolk Immunoglobulin Y - Enzyme Linked Immune Sorbent Assay (CIgY-ELISA), was developed as an alternative method to detect zeatin and 2ip in plantlets of gerbera. The endogenous level of hormones in the plantlets in vitro of gerbera with one or six weeks after replication was determined with competitive IgY-ELISA set to detect between 1 and 100 pmoles of plant hormone for each 1.0 g tissue. The plantlets of six weeks presented sprouts and root, while the plantlets of one week presented only sprouts. The CIgY-ELISA was set with high independent variables values of sensitivity/specificity of 96/89 % for zeatin and 94/78 % for 2ip, with high values of reproducibility (up to 90 %) for both the cytokinins. Zeatin content varied from 2.2 to 2.8 pmoles.g-1 and from 2.7 to 3.3 pmoles.g-1 on the plantlet with one and six weeks, respectively. The 2ip content did not vary and was detected near the detection limit in all the assays. It was concluded that the observed capabilities of CIgY-ELISA were putative and the competitive assay was a highly robust and stable method, which could be used for the studies on plant physiology for endogenous cytokinins.

Fagócitos mononucleares (Mø) e polimorfo-nucleares neutrófilos (PMN) foram analisados durante o curso da infecção por Toxoplasma gondii em camundongos C57 BL/6, fêmeas, portadores de inflamações crônicas focais induzidas por material inerte. A dinâmica populacional de células mielóides dirigidas ao suporte inerte foi medida a partir do quarto dia após inoculação intraperitonial de 90-100 cistos de T. gondii, ao longo de vinte dias, e comparada com o percentual relativo de leucócitos sangüíneos. Observou-se uma diminuição do percentual de PMN e um aumento significativo de Mø no sangue dos animais infectados. A infecção assintomática por T. gondii em camundongos resultou em um maior esforço do sistema mielóide na produção de células destinadas a combater focos inflamatórios, tornando-as menos aptas a responder adequadamente a diferentes fontes de infecção crônica. A mudança na maturidade e nas proporções normais de células mielóides indicou haver um bloqueio da extrusão de quimiocinas responsáveis pela atração quimiotática de PMN com comprometimento da resolução de focos inflamatórios durante infecções crônicas e assintomáticas por Toxoplasma gondii.

Cells of the mononuclear and polymorphonuclear series were analyzed throughout the course of Toxoplasma gondii infections of C57 BL/6 female mice with chronic inflammatory foci. The animals were inoculated intraperitoneally with 90-100 parasitic cysts followed by subcutaneous implantation of cover glasses (after 4 days). The shift of leukocytes from the blood to the inflammatory focus, corresponding to a prolonged infiltration of myeloid cells, was measured in a time course of 20 days. A significant decline was observed in the mature polymorphonuclear cells in the blood and a concomitant development of a marked monocytosis. Infections with T. gondii in mice with chronic inflammatory foci resulted in dedication of the entire myeloid cells system to the generation of cells that were exported to combat the inflammation, which occurred in those places, and to the chronic infection itself. The shift in the normal proportions of cells and in cells of different degrees of maturity, suggesting that a blockade in the chemokines extruded to attract polimorphonuclear cells, might account to a large extent for the failure of immune responses commonly associated with Toxoplasma subclinical infections.

O presente experimento descreve, pela primeira vez, a elaboração de um ensaio imunoenzimático (ELISA de captura) com anticorpos IgY para a detecção e quantificação de acetato de lupeol (LAc) em Vernonia scorpioides Lam. Pers (Asteraceae). Anticorpos anti-LAc, obtidos partir do soro das aves e gemas de seus ovos, após imunização com o conjugado acetato de lupeol-cBSA, foram concentrados em coluna contendo LAc-BSA acoplado a sepharose. A eficácia da metodologia de detecção imunológica foi de 97%, com índices de sensibilidade e especificidade de 99% e 95%, respectivamente com limites de detecção do ensaio entre 0,02µg.g-1 (inferior) e 10 µg.g-1 (superior). A robustez do método foi atestada pela sua elevada reprodutibilidade (entre 94,75% e 96,81%), e pelo baixo coeficiente de variação interna (4,22 ± 1,03%) nas condições de execução descritas.

For the first time, a yolk immunoglobulins-based immunenzymatic assay (capture IgY-ELISA) was carried out to detect Lupeol acetate (LAc) from Vernonia scorpioides Lam. Pers (Asteraceae). Antibodies (IgY) against lupeol acetate (anti-LAc antibodies) were raised in White Leghorn hens immunized with LAc conjugated to the bovine serum albumin (LAc-BSA). The anti-LAc antibodies were recovered by cleanup columns containing LAc-BSA coupled to sepharose. The capture IgY-ELISA efficacy was of 97% when the predictive indices of sensitivity and specificity were 99.0% and 95%, respectively. The lowest and highest detection limits were of 0.02µg g-1 and 10µg.g-1 of plant extract, respectively. The strength of this method was attested by its high reproducibility (between 94.75% and 96.81%), and a low internal variation (4.22 ± 1.03%), under the described conditions.

Macrophages exposed to 10 mug/mL citrinin (CTR) or 0.01 mug CTR mixed with 0.04 mug aflatoxin B1 (AFB1) for a period of 2 h at 37ºC, were infected with 10(6) Toxoplasma gondii tachyzoites/muL. The parasites were treated with mycotoxins (2 h at 37ºC) before being added to the macrophage culture. The number of tachyzoites was quantified 2, 24, 48, 72 and 96 h after infection. During the first 2 hours, 59% infectivity was observed in the control. After exposure to CTR or the mixture of toxins (CTR-AFB1), macrophages were infected with 77.5% and 75% of the inoculated tachyzoites, respectively. Similarly, 72.3% of the cells were infected when cultured together with previously treated parasites. The treatment with CTR-AFB1 gave rise to 2.9 times more tachyzoites than the control at 72 h. An increased number of parasites was recovered from macrophages exposed to CTR after 96 h, and to CTR-AFB1 after 72 h of culture; The number of tachyzoites recovered from the supernatant was 1.94 and 2.06 times higher, respectively, than in the control (5 x 10(5) ± 0.054 /mL).

Foram estudadas as alterações imunológicas em um grupo de seis bovinos infestados, experimentalmente, com 60 larvas de primeiro ínstar (L1) de Dermatobia hominis por animal, enquanto que outro grupo de seis animais foi utilizado como controle. Amostras de sangue tomadas, semanalmente, durante a infestação experimental, foram analisadas para se detectar anticorpos anti-L1, anti-L2 e anti-L3 de D. hominis, utilizando-se a técnica de ELISA, na qual utilizou microplacas contendo amostras de soro dos animais e preparações antigênicas de L1, L2 e L3. Observou-se nos animais infestados, o aparecimento de anticorpos anti-L1 desde o primeiro até o 21º dia pós-infestação (DPI) e após o 42º DPI, enquanto que os anticorpos anti-L2 foram detectados no 21º DPI, e no período do 35º ao 49º DPI, seguido de declínio até atingirem valores semelhantes aos animais controles. Não se detectaram níveis expressivos de anticorpos para antígenos de L3. Os níveis de anticorpos anti-L1 dos animais infestados revelaram absorbância (D.O.) alta, ou seja, acima de 1,500 a 492nm quando comparado ao valor médio de 0,096 obtido nos animais controles, o que também ocorreu com os níveis de anticorpos anti-L2 no 21º DPI, nos quais dois animais apresentaram valores de D.O. de 0,450 e 0,900 a 492nm, em relação ao valor discriminante (cut-off) estimado em 0,110 dos animais controles. Verificou-se um aumento dos níveis de anticorpos anti-L2 em quatro animais que também apresentaram resposta contra antígenos de L1. Os resultados obtidos com a técnica de ELISA, pressupondo-se uma prevalência de 50%, quando comparados com a técnica padrão de imunodifusão dupla, revelaram uma concordância de 98%. Discutiu-se ainda a associação entre infestação e presença de anticorpos específicos.

Six bovines were infested with 60 first instar larvae of Dermatobia hominis. The animals were bleed weekly, and their antibodies levels to D. hominis L1, L2 and L3 instars measured during the time, following the infestation course. The antisera were submitted to a titration against optimal dilutions of antigen coated wells of microplates, previously sensitized with L1, L2 and L3 preparations, respectively. The ELISA assay was used to test single dilutions of antisera, which results were comparatively analyzed with a control of not infested animals. Antibodies against L1 were detected between the first and 21st day post-infestation (DPI) and, from the 42nd DPI on. Anti-L2 antibodies, could be detected on the 21st DPI and from the 35th DPI until approximately the 49th DPI, when it was observed a decreasing of antibodies titration equivalent to the control group. No antibodies were detected against the L3 instar-antigens. Antibodies levels against L1 showed absorbance higher than 1.500 O.D. at 492nm in the ELISA assay, when compared to the 0.096 O.D. observed to the negative animals. High anti-L2 antibodies were also detected on the 21st DPI, where two animals showed O.D. of 0.450 and 0.900 at 492nm, with a cut-off estimated on 0.110 O.D. It was also demonstrated a rising of anti-L2 antibodies in the same four animals, which presented antibodies response against L1 instar. The obtained results, with an estimated prevalence of 50%, were comparatively evaluated, taking the double diffusion immunoassay precipitation test as a standard, and showed a concordance of 98%. The association between infestation and presence of specific antibodies was also discussed.