Biomedical subjects

M S Macedo

Publications and source records attributed to M S Macedo.

At least 19 recordsLinked to original sources

PubMed2006

Early stages of Ascaris suum induce airway inflammation and hyperreactivity in a mouse model.

The inflammatory and functional changes that occur in murine lung after infection with 2500 infective Ascaris suum eggs were studied in this work. A sequential influx of neutrophils, mononuclear cells and eosinophils occurred into airways concomitantly with migration of larvae from liver to the lungs. Histological analysis of the lung showed a severe intra-alveolar haemorrhage at the peak of larval migration (day 8) and the most intense inflammatory cell infiltrate on day 14. Ascaris L3 were found in alveolar spaces and inside bronchioles on day 8. The number of eosinophils was elevated in the blood on days 8 and 14. The peak of eosinophil influx into the lung was at day 14, as indicated by the high levels of eosinophil peroxidase activity, followed by their migration into the airways. The antibody response against egg and larval antigens consisted mainly of IgG1 and IgM, and also of IgE and anaphylactic IgG1, that cross-reacted with adult worm antigens. Total IgE levels were substantially elevated during the infection. Measurement of lung mechanical parameters showed airway hyperreactivity in infected mice. In conclusion, the murine model of A. suum infection mimics the Th2-induced parameters observed in pigs and humans and can be used to analyse the immunoregulatory properties of this helminth.

Anaphylaxis↗

PubMed2005

Anti-inflammatory activity of PAS-1, a protein component of Ascaris suum.

BACKGROUND: Recently, we identified a 200 kDa protein (PAS-1) from Ascaris suum worms, that suppresses the humoral immune response. Here, the effect of PAS-1 on inflammatory leukocyte migration induced by bacterial lipopolysaccharide (LPS) was investigated. METHODS: Cellular migration and cytokine release, stimulated by LPS or LPS+PAS-1, were analyzed in air pouches induced in the shaved back of BALB/c mice. Cytokines were determined by ELISA and RT-PCR on air pouch exudates and in vitro stimulated peritoneal macrophages. RESULTS: The significant cellular influx induced by LPS, consisting predominantly of neutrophils, was highly suppressed in the presence of PAS-1, but not a non-related protein. PAS-1 led also to a marked reduction of TNF-alpha, IL-1 beta and IL-6 levels in both LPS-stimulated air pouches and peritoneal macrophage cultures. In contrast, PAS-1 induced a significant increase of IL-10 and TGF-beta production. CONCLUSIONS: These results demonstrate that PAS-1 has a potent anti-inflammatory activity, probably due to the stimulation of regulatory cytokines in macrophages, thus leading to the inhibition of pro-inflammatory cytokine production.

Animals↗

PubMed2005

Modulation of murine experimental asthma by Ascaris suum components.

BACKGROUND: We have recently isolated two distinct components from Ascaris suum adult worms with different effects on the immune system: the allergenic protein of A. suum (APAS-3), which induces IgE antibody production, and suppressive protein of A. suum (PAS-1), which inhibits humoral and cellular immune responses induced by unrelated antigens. In this study, we investigated the immunomodulatory effect of PAS-1 on a murine model of asthma induced by APAS-3. METHODS: BALB/c mice were immunized twice with APAS-3 or APAS-3 plus PAS-1 by the intraperitoneal and subcutaneous route (on days 0 and 7) and challenged twice with the same antigens intranasally (days 14 and 21). Two days after the last challenge, the allergic airway inflammation was evaluated by cellular migration, eosinophil peroxidase (EPO) activity, cytokine and chemokine production and pulmonary mechanical parameters. RESULTS: The allergenic properties of APAS-3 were confirmed by the stimulation of anaphylactic IgE and IgG1 antibody production and eosinophilic airway inflammation and hyper-responsiveness. On the other hand, PAS-1-treated mice showed a marked suppression of cellular migration and EPO activity that correlated well with a significant reduction in the levels of IL-4, IL-5, eotaxin and RANTES in the bronchoalveolar lavage (BAL) fluid. In contrast, considerable amounts of IL-10 were observed in the BAL fluid of PAS-1-treated mice. Airway hyper-responsiveness was obtained in APAS-3-immunized mice, but the conductance of the respiratory system was restored to normal values in the presence of PAS-1. CONCLUSION: These results indicate that A. suum allergenic protein APAS-3 induces a T helper 2-type immune response and, consequently, eosinophilic airway inflammation and hyper-responsiveness. Moreover, the modulatory protein PAS-1 has a marked suppressive effect on this response, and the inhibition of cytokine (IL-4, IL-5) and chemokine (eotaxin and RANTES) release, probably because of the presence of IL-10, may contribute to this effect.

Allergens↗

PubMed2003

Immunoglobulin E is not required for but enhances airway inflammation and hyperresponsiveness.

The aim of this study was to investigate the role of immunoglobulin E (IgE) in the late phase reaction (LPR) of murine experimental asthma. Our model consisted of an implant of DNP-conjugated, heat-coagulated hen's egg white (DNP-EWI), followed 14 days later by an intratracheal challenge with aggregated DNP-ovalbumin. Airway inflammation was analyzed 48 h after challenge and compared with a similarly immunized group of mice with highly suppressed humoral response due to anti-micro and anti-delta antibody treatment. Total number of cells in the bronchoalveolar lavage (BAL) (with predominance of eosinophils) and EPO activity in the lung homogenate were increased in the DNP-EWI-immunized group compared with immunosuppressed or nonimmunized mice. However, the cellular infiltration and EPO activity observed in the immunosuppressed group were still significantly above those obtained in the nonimmunized group, indicating that inhibition of antibody production did not completely prevent the inflammatory manifestations in BAL and lung. Airway hyperresponsiveness to methacoline was obtained in DNP-EWI-immunized mice, but the respiratory mechanical parameters returned to normal levels in the immunosuppressed group. When these mice were reconstituted with monoclonal anti-DNP antibodies, only IgE, but not IgG1, restored lung inflammation and decreased the conductance of the respiratory system, therefore, increasing hyperresponsiveness. These results indicate that antibodies are not essential for induction of LPR in the lung. However, IgE enhances pulmonary inflammation and hyperresponsiveness.

Animals↗

PubMed2002

Thymic epithelial cells mediate a Bcl-2-independent protection of single-positive thymocytes from dexamethasone-induced apoptosis.

Intrathymic maturation of thymocytes is essential for the proper formation of T-cell repertoire. This process involves two major biochemical pathways, one initiated by the recognition of MHC/peptide by the T-cell receptor and the other mediated by glucocorticoids. These hormones seem to affect thymocyte maturation by increasing the threshold of TCR-mediated positive and negative selection, and by inducing apoptosis of nonselected thymocytes. We have previously reported that an SV40-immortalized murine thymic epithelial cell line, namely 2BH4, was able to protect thymocytes from dexamethasone-induced apoptosis. Here we show that this protection is independent of cell-to-cell contact and does not seem to involve a Bcl-2-mediated resistance, since incubation of thymocytes with 2BH4 cells or its supernatant does not interfere with the levels of this antiapoptotic molecule. The protection conferred by 2BH4 cells, or by a primary culture of thymic stromal cells, is specific for the CD4(+)CD8(-) and CD4(-)CD8(+) single-positive thymocytes, whereas the broad-spectrum caspase inhibitor z-VAD-fmk blocks apoptosis induced by dexamethasone in all thymocyte subpopulations. Our results suggest that positively selected single-positive thymocytes are still susceptible to glucocorticoid-induced apoptosis but are protected from it through the action of a heat-stable protein(s) released by thymic stromal cells.

Animals↗

PubMed2002

Eosinophilic inflammation and airway hyper-responsiveness are profoundly inhibited by a helminth (Ascaris suum) extract in a murine model of asthma.

BACKGROUND: The increase of atopic disorders in developed countries has been associated with the decline of infectious diseases, including helminthic infections. We have already demonstrated that adult worm extracts from Ascaris suum (ASC) suppress the IgE antibody production against unrelated antigens. OBJECTIVE: Here we investigated the influence of ASC on the development of pulmonary eosinophilic inflammation in a murine model of asthma. METHODS: Heat-coagulated egg white alone (EWI) or mixed with ASC (EWI + ASC) was implanted subcutaneously in B10.A or C57BL/6 mice, and 14 days later they were challenged intratracheally with OVA or exposed to aerosolized OVA for 4 days. RESULTS: The suppressive effect of ASC was demonstrated on the accumulation of cells into airways, with reduction of eosinophil numbers and of eosinophil peroxidase activity in EWI + ASC-immunized mice. This effect correlated with a marked reduction of IL-5 and IL-4 levels in the BAL from C57BL/6 and B10. A mice, respectively, and of eotaxin in BAL and lung tissue from both strains. OVA-specific IgG1 and IgE levels were also impaired in serum and BAL from these mice. Airway hyper-reactivity to methacholine was obtained in B10. A mice sensitized with EWI, but the respiratory mechanical parameters returned to normal levels in EWI + ASC-immunized mice. CONCLUSION: These results indicate that ASC has a profound inhibitory effect on lung inflammation and hyper-responsiveness and that suppression of IL-5 or IL-4 and of eotaxin contributes to this effect.

Animals↗

PubMed2002

Extracts of Ascaris suum egg and adult worm share similar immunosuppressive properties.

Adult Ascaris suum body extract (Asc) prepared from male and female worms (with stored eggs) down-regulates the specific immune response of DBA/2 mice to ovalbumin (OA) and preferentially stimulates a Th2 response to its own components, which is responsible for the suppression of the OA-specific Th1 response. Here, we investigated the participation of soluble extracts prepared from male or female worms or from eggs (E-Asc) in these immunological events. Extracts from either sex (1 mg/animal) or E-Asc (0.35 or 1 mg protein/animal) suppressed the delayed-type hypersensitivity (DTH) reaction (60-85%), proliferative response (50-70%), IL-2 and IFN-gamma secretion (below detection threshold) and IgG1 antibody production (70-90%) of DBA/2 mice to OA. A dose of 0.1 mg E-Asc/animal did not change DTH or proliferation, but was as effective as 0.35 mg in suppressing IL-2 and IFN-gamma, and OA-specific IgG1 antibodies. Lymph node cells from DBA/2 mice injected with Asc (1 mg/animal) or a high dose of E-Asc (1 mg protein/animal) secreted IL-4 upon in vitro stimulation with concanavalin A. As previously demonstrated for Asc, the cytokine profile obtained with the E-Asc was dose dependent and changed towards Th1 when a low dose (0.1 mg protein/animal) was used. Taken together, these results suggest that adult worms of either sex and eggs induce the same type of T cell response and share similar immunosuppressive properties.

Animals↗

PubMed2001

Modulation of delayed-type hypersensitivity during the time course of immune response to a protein antigen.

Delayed-type hypersensitivity reactions elicited in the footpad of ovalbumin-sensitized mice after challenge with aggregated ovalbumin on day 4 or 8 of immunization are distinct. The former was characterized by a dense mononuclear infiltrate and, macroscopically, the reaction peaked at 48 hr after antigen challenge; the latter was preceded by immediate-type reactions, reached the maximum at 24 hr and faded drastically later. Histologically, oedema and a mixed granulocytic-lymphocytic infiltrate was found at this time-point. Immunoglobulin G1 (IgG1), IgG2a and IgE antibodies were detected only in plasma obtained after 8 days of immunization. Regarding the cytokines produced by draining lymph node cells after in vitro restimulation, interleukin-4 (IL-4) and IL-10 were predominant after 4 days and interferon-gamma and IL-2 after 8 days of immunization. These two types of delayed-type hypersensitivity (DTH) were used to study the influence of antibody-mediated responses on the inductive and effector phases of cell-mediated immunity. The effector phase of DTH was not affected by immediate-type reactions, as abrogation of these reactions by mediators' antagonists on day 8 or induction of passive reactions by transfer of immune serum on day 4 did not change the extent or kinetics of either type of DTH. Only transfer, before immunization, of whole or T-cell-enriched spleen cells, but not sera, from hyperimmunized donors (high antibody producers) abolished the induction of pure DTH in 4-day immunized recipient mice and changed their cytokine profile to a T helper 2 type. These results indicate that in a non-polarized immune response to a protein antigen there is initially a bias towards cell-mediated immunity, which is gradually dampened by the development of antibody-mediated immunity.

Animals↗

PubMed2001

Role of crotoxin, a phospholipase A2 isolated from Crotalus durissus terrificus snake venom, on inflammatory and immune reactions.

BACKGROUND: Crotoxin (CTX) is a potent neurotoxin from Crotalus durissus terrificus snake venom (CdtV) composed of two subunits: one without catalytic activity (crotapotin), and a basic phospolipase A2. Recent data have demonstrated that CdtV or CTX inhibit some immune and inflammatory reactions. AIM: The aim of this paper was to investigate the mechanisms involved in these impaired responses. MATERIALS AND METHODS: Male Swiss mice were bled before and at different intervals of time after subcutaneous injection of CTX or bovine serum albumin (BSA) (control animals). The effect of treatments on circulating leukocyte mobilisation and on serum levels of interleukin (IL)-6, IL-10, interferon (IFN)-gamma and corticosterone were investigated. Spleen cells from treated animals were also stimulated in vitro with concanavalin A to evaluate the profile of IL-4, IL-6, IL-10 or IFN-gamma secretion. Cytokine levels were determined by immunoenzymatic assay and corticosterone levels by radioimmunoassay. To investigate the participation of endogenous corticosteroid on the effects evoked by CTX, animals were treated with metyrapone, an inhibitor of glucocorticoid synthesis, previous to CTX treatment. RESULTS: Marked alterations on peripheral leukocyte distribution, characterised by a drop in the number of lymphocytes and monocytes and an increase in the number of neutrophils, were observed after CTX injection. No such alteration was observed in BSA-treated animals. Increased levels of IL-6, IL-10 and corticosterone were also detected in CTX-injected animals. IFN-gamma levels were not modified after treatments. In contrast, spleen cells obtained from CTX-treated animals and stimulated with concanavalin A secreted less IL-10 and IL-4 in comparison with cells obtained from control animals. Metyrapone pretreatment was effective only to reverse the neutrophilia observed after CTX administration. CONCLUSIONS: Our results suggest that CTX may contribute to the deficient inflammatory and immune responses induced by crude CdtV. CTX induces endogenous mechanisms that are responsible, at least in part, for these impaired responses.

Animals↗

PubMed2000

Induction of IL-4-dependent, anaphylactic-type and IL-4-independent, non-anaphylactic-type IgG1 antibodies is modulated by adjuvants.

Adjuvants can modulate the levels of anaphylactic- and non-anaphylactic-type IgG1 antibodies produced in response to a particular antigen. Mice immunized with ovalbumin (OVA) in Al(OH)(3) gel (alum) produced mostly the anaphylactic type, irrespective of the s.c. or i.p. route used, and this antibody was not detectable in IL-4(-/-) mice. In contrast, when OVA was injected in complete Freund's adjuvant (CFA), it induced substantial amounts of non-anaphylactic-type IgG1 in both IL-4(+/+) and IL-4(-/-) mice, and some anaphylactic IgG1 antibody in IL-4(+/+) mice only. When IFN-gamma was neutralized by specific mAb in wild-type mice immunized with OVA in CFA, the anaphylactic-type IgG1 antibody increased reaching the same levels as in alum-injected mice. This result indicates that the induction of IFN-gamma by the immunization with CFA down-regulates the production of IL-4-dependent, anaphylactic-type IgG1. Despite their different effects on IgG1 antibody production, both adjuvants dramatically increased the production of IgG2a in IL-4-deprived mice and did not induce any detectable IgE in these mice.

Adjuvants, Immunologic↗

PubMed1999

Cutting edge: mouse IgG1 antibodies comprise two functionally distinct types that are differentially regulated by IL-4 and IL-12.

IL-4-dependent and -independent IgG1 Abs differ in their ability to induce mast cell degranulation as measured by passive cutaneous anaphylaxis (PCA). Mice immunized with OVA or PIII (fraction of Ascaris suum) produced high titers of IgG1 as shown by ELISA and PCA. In contrast, another A. suum fraction, PI, elicited IgG1 Abs with no PCA activity. IgG1 with anaphylactic activity required IL-4, as IgG1 responses to OVA and PIII in IL-4-/- mice gave no PCA. PI-specific IgG1 was IL-4-independent, because no difference was found between the responses of IL-4-/- and IL-4+/+ mice. Significant PCA reactions were elicited, however, with PI-specific IgG1 from IL-12-/- or anti-IFN-gamma Ab-treated mice, although less Ab was measured by ELISA. These results indicate that one type of IgG1 has anaphylactic activity and its synthesis is IL-4-dependent, being inhibited by IL-12 or IFN-gamma; the other lacks this activity and its synthesis is stimulated by IL-12 or IFN-gamma.

Adjuvants, Immunologic↗

PubMed1998

The immunosuppressive activity of Ascaris suum is due to high molecular weight components.

The role of isolated components obtained by gel filtration chromatography of Ascaris suum body extract (Asc) on the modulation of the immune response to ovalbumin (OvA) was evaluated and correlated with the immunogenic properties of such components. We showed that high (PI), but not low (PIII), molecular weight components have the ability to inhibit OvA-induced immediate and DTH reactions, lymph node (LN) cell proliferation, cytokine (IL-2, interferon-gamma (IFN-gamma), IL-4 and IL-10) and antibody (IgG1, IgG2a, IgM and IgE) production in mice concomitantly immunized with OvA and these high mol. wt components. The pattern of cytokines synthesized in response to PI or PIII was totally different: the former induced more IL-4 and IL-10 and the latter more IL-2 and IFN-gamma. The levels of Asc-specific IgG1 antibodies were higher in mice immunized with OvA plus PI and IgG2a anti-Asc antibodies predominated in those immunized with PIII. IgE antibody production, however, was low in the former group of mice. These results indicate that the high mol. wt components present in the body extract from the helminth A. suum are responsible for its suppressive effect upon Th1- and Th2-dependent immune responses to an unrelated antigen. The suppression of the Th1-dependent parameters could be related to high-level expression of IL-4 and IL-10 induced by such components.

Animals↗

PubMed1998

Immunomodulation induced by Ascaris suum extract in mice: effect of anti-interleukin-4 and anti-interleukin-10 antibodies.

Simultaneous immunization of mice with an Ascaris suum extract (Asc) and ovalbumin (OA) markedly affects the immune response to OA. The role of interleukin (IL)-4 and IL-10 induced by Asc immunization on the modulation of antigen-specific and mitogen-induced responses was investigated following single or combined cytokine-specific monoclonal antibody (MoAb) treatment of mice before immunization with OA + Asc. Immediate hypersensitivity reactions to aggregated OA and OA-specific immunoglobulin (Ig)G2a antibody production were completely restored only when both IL-4 and IL-10 were neutralized. These findings were associated with enhanced interferon (IFN)-gamma secretion by OA-stimulated lymph node (LN) cells. In addition, the Asc-specific cytokine response in anti-IL-4 plus anti-IL-10 MoAb treated mice was shifted towards a Th1 phenotype, with an increase in IFN-gamma and IL-2 levels and a decrease in IL-4, but not in IL-10, levels. Consequently, Asc-specific IgG2a antibody production increased, whereas IgE titres diminished in these animals. These results indicate that IL-4 and IL-10 act together in the Asc-induced mechanism of antigen-specific pansuppression. In contrast, modulation of Concanavalin A (Con A)-induced cytokine responses in Asc-immunized mice appears to be essentially mediated by an IL-4-dependent mechanism, since the neutralization of just IL-4 (and not of IL-10), either in vivo or in vitro, changed the cytokine profile from a Th2 towards a Th1 type. However, OA and Asc-specific cell responses were not modified by either anti-IL-4 or by anti-IL-4 + anti-IL-10 MoAbs in vitro treatments, suggesting that the induction of a Th2 response to Asc components concomitant to OA immunization has a strong suppressive effect on the priming stage of OA-specific Th1 type response.

Adjuvants, Immunologic↗

PubMed1996

Saponin adjuvant primes for a dominant interleukin-10 production to ovalbumin and to Trypanosoma cruzi antigen.

The adjuvant activity of saponin for T-cell responses was evaluated and compared with that of complete Freund's adjuvant (CFA) in two antigen systems: a lysate of the protozoa Trypanosoma cruzi and ovalbumin (OA). Strong delayed-type hypersensitivity and T-cell proliferate responses, comparable with those stimulated by CFA, were observed for both antigens following immunization with saponin as adjuvant. Upon in vitro secondary antigen stimulation, high interleukin-10 (IL-10) and low interferon-gamma (IFN-gamma) levels were observed in lymph node (LN) cell cultures from saponin-immunized mice in contrast with the high IFN-gamma and decreased IL-10 production by LN cells from CFA-immunized mice. Production of IL-10 and IFN-gamma in these conditions was CD4-activation dependent. Concanavalin A (Con A)-stimulated interleukin-4 (IL-4) production was higher in saponin-immunized mice than in CFA-immunized mice. IL-10 produced by LN cells from saponin-immunized mice suppressed IFN-gamma production and Con A-induced proliferation. Taken together, these data are consistent with in vivo stimulation of both T-helper (Th)1 and Th2-type cells by immunization with saponin; in vitro a Th2-type cytokine response with high IL-10 production predominates, indicating preferential priming towards a Th2-type response. Immunization with CFA induced a Th1-type cytokine response. To our knowledge this is the first report in which an adjuvant is shown to prime for a dominant IL-10 production.

Adjuvants, Immunologic↗

PubMed1995

Cloning of a thymic stromal cell capable of protecting thymocytes from apoptosis.

A clone of thymic stromal cells, namely 2BH4, was established by primary culture, cellular transfection and limiting dilution. Morphological analysis by transmission electron microscopy revealed that these cells grow as multilayers, producing a well-defined basement membrane to which they attach and frequently form structures similar to hemidesmosomes. The adjoining cells are connected by intercellular junctions, as tight junctions, intermediate junctions, and desmosome-like junctions, as well as interdigitations. Their cytoplasm contains microtubules, strands of actin filaments, and scarce intermediate filaments. Fluorescence microscopy revealed that 2BH4 cells stain with anti-cytokeratin antibodies, the majority of them giving a faint reaction. In addition, they express Thy-1.1, LFA-1, ICAM-1, and the gp23 epithelial antigen, and synthesize laminin. They have a doubling time of 16 hr and are able to bind thymocytes. Thymocytes cultured in the presence of 2BH4 cells are partially protected from both spontaneous and PMA- or dexamethasone-induced apoptosis. This protection is conferred neither by soluble factors normally produced by the 2BH4 cells nor by the sole contact with fixed 2BH4 cells. Rather, thymocytes must interact with metabolically active 2BH4 cells in order to receive the protective signal(s).

Animals↗

PubMed1995

Immunization with Ascaris suum extract impairs T cell functions in mice.

The effect of an Ascaris suum extract (Asc) on several T cell functions was studied in mice immunized with Asc and ovalbumin (OA) in complete Freund's adjuvant. Delayed-type hypersensitivity reactions following challenge with aggregated OA were markedly diminished in mice injected with OA plus a high dose of Asc compared to OA-immunized animals. Proliferation and IL-2, IFN-gamma, IL-4, and IL-10 production in OA-stimulated lymph node cells from the OA + Asc-immunized group were also inhibited. Titration of anti-OA antibodies also showed suppression of IgG1, IgG2a, and IgE isotypes in the animals injected simultaneously with the extract. The degree of suppression induced by Asc on OA-specific cell-mediated responses was dose dependent. The profile of cytokines synthesized in response to Asc also changed depending on the injected dose. IL-4 and IL-10 were mainly produced in response to high doses, whereas IL-2 and IFN-gamma were greatly enhanced at a low dose of Asc. These findings indicate that the A. suum extract may impair crucial T cell functions for cell-mediated as well as humoral immune responses to other antigens through the induction of a predominantly Th2-like response.

Animals↗

PubMed1995

Biochemical, antigenic and allergenic characterization of crude extracts of Drechslera (Helminthosporium) monoceras.

In a previous study with airborne mould extracts we verified that Drechslera (Helminthosporium) monoceras presented stronger reactions than those presented by 42 other moulds isolated in São Paulo city. In the present study, we evaluated the biochemical composition and the antigenicity of crude extracts obtained from vegetative and conidial stage of D. monoceras using Czapeck broth (CB) modified and tris-HCl for extraction. The maximum values of total proteins and lipids were verified in the crude extract obtained in the 28th day of growth, and maximum values of carbohydrates were observed in the extracts of the 16th, 22nd and 26th days. The fractionated proteins by SDS-PAGE presented bands with molecular weights between 14.4 to 67 Kd; the 28th day extract showed a larger number of bands. The carbohydrates and amino acids were characterized by thin-layer chromatography. The antigenicity of the crude extracts was verified by immunodiffusion reaction in agar against rabbit hyperimmune sera. Precipitation lines were observed in all studied extracts and common antigenic molecular populations. Based on the above results, the 28th day extract was selected to verify the induction of IgE antibody responses in immunizations of Balb/c and cAF-1 mice, and titer by passive cutaneous anaphylaxis test using Wistar rats. The maximum titers obtained were 160 in cAF-1 mice and 1.280 in Balb/c mice. The results suggest that the 28th day extract contains allergenic fractions and should be chosen for future studies related to fractionation, characterization and standardization in diagnostic methods and immunotherapy.

Allergens↗

PubMed1994

Colorimetric assay for the measurement of thymocyte/thymic stromal cell adhesion.

1. We describe a simple and accurate colorimetric adhesion assay (CAA) and illustrate the assay by measuring the adhesion of mouse thymocytes to mouse 2BH4 cells. 2. The assay is based on the crystal violet staining of thymocytes adhered to a subconfluent layer of 2BH4 cells (plated at 2 x 10(4) cells/well for 24 h). The optimal incubation time was shown to be 1 h and washing in PBS of non-bound and non-specifically bound thymocytes is the critical step for the precision and accuracy of the assay. 3. Saturation curves were obtained for thymocytes adhered to plated 2BH4 cells. The blank (only 2BH4 cells) was near 0.200 +/- 0.010 (mean +/- SD) and was quite reproducible. As expected, the extent of adhesion was also dependent on the number of plated 2BH4 cells. Standard curves need to be run with each assay for quantitative measurements. The intra-assay and interassay coefficients of variation were 5% and 20%, respectively. 4. The specificity of the reaction was demonstrated by the reduction of adherence by trypsin pretreatment of thymocytes, and the dose-dependent inhibition of adherence by rabbit anti-mouse thymocyte antisera but not rabbit anti-mouse immunoglobulin antisera. 5. The proposed method is simple and requires less effort than the counting of adhered cells with the light microscope and does not require the use of radioactive material as when labelling with Na2(51)CrO4 is utilized.

Analysis of Variance↗