Eosinophils in bronchial mucosa of asthmatics after allergen challenge: effect of anti-IgE treatment

Anti-IgE, omalizumab, inhibits the allergen response in patients with asthma. This has not been directly related to changes in inflammatory conditions. We hypothesized that anti-IgE exerts its effects by reducing airway inflammation. To that end, the effect of anti-IgE on allergen-induced inflammation in bronchial biopsies in 25 patients with asthma was investigated in a randomized, double-blind, placebo-controlled study. Allergen challenge followed by a bronchoscopy at 24 h was performed at baseline and after 12 weeks of treatment with anti-IgE or placebo. Provocative concentration that causes a 20% fall in forced expiratory volume in 1 s (PC(20)) methacholine and induced sputum was performed at baseline, 8 and 12 weeks of treatment. Changes in the early and late responses to allergen, PC(20), inflammatory cells in biopsies and sputum were assessed. Both the early and late asthmatic responses were suppressed to 15.3% and 4.7% following anti-IgE treatment as compared with placebo (P < 0.002). This was paralleled by a decrease in eosinophil counts in sputum (4-0.5%) and postallergen biopsies (15-2 cells/0.1 mm(2)) (P < 0.03). Furthermore, biopsy IgE+ cells were significantly reduced between both the groups, whereas high-affinity IgE receptor and CD4+ cells were decreased within the anti-IgE group. There were no significant differences for PC(20) methacholine. The response to inhaled allergen in asthma is diminished by anti-IgE, which in bronchial mucosa is paralleled by a reduction in eosinophils and a decline in IgE-bearing cells postallergen without changing PC(20) methacholine. This suggests that the benefits of anti-IgE in asthma may be explained by a decrease in eosinophilic inflammation and IgE-bearing cells.

Activated status of basophils in chronic urticaria leads to interleukin-3 hyper-responsiveness and enhancement of histamine release induced by anti-IgE stimulus

Background Basophils and mast cells are the main target cells in chronic idiopathic urticaria (CIU). Besides the basopenia, intrinsic defects of the anti-IgE cross-linking signalling pathway of basophils have been described in CIU. Objectives We sought to investigate the profile of expression of activation markers on basophils of patients with CIU and to explore the effect of interleukin (IL)-3 priming upon anti-IgE cross-linking stimuli through expression of activation markers and basophil histamine releasability. Methods Evaluation of the surface expression of Fc epsilon RI alpha, CD63, CD203c and CD123 on whole blood basophils of patients with CIU undergoing autologous serum skin test (ASST) was performed by flow cytometry. The effect of pretreatment with IL-3 in the anti-IgE response was analysed by the expression of basophil activation markers and histamine release using enzyme-linked immunosorbent assay. Results Blood basophils of patients with CIU were reduced in number and displayed increased surface expression of Fc epsilon RI alpha, which was positively correlated with the IgE serum levels. Upregulation of expression of both surface markers CD203c and CD63 was verified on basophils of patients with CIU, regardless of ASST response. High expression of IL-3 receptor on basophils was detected only in ASST+ patients with CIU. Pretreatment with IL-3 upregulated CD203c expression concomitantly with the excreting function of blood basophils and induced a quick hyper-responsiveness to anti-IgE cross-linking on basophils of patients with CIU compared with healthy controls. Conclusions Basophils of patients with CIU showed an activated profile, possibly due to an in vivo priming. Functionally, basophils have high responsiveness to IL-3 stimulation, thereby suggesting that defects in the signal transduction pathway after IgE cross-linking stimuli are recoverable in subjects with chronic urticaria.

IgM e IgG como marcadores da infecção transplacentária por Neospora caninum em fetos bovinos

O objetivo deste estudo foi determinar a ocorrência de anticorpos anti-Neospora caninum em 260 amostras de soro coletadas de fetos bovinos de julho de 2007 a março de 2008, em abatedouro do município de Santa Maria, Rio Grande do Sul, Brasil. Para detecção de anticorpos anti-N. caninum, a técnica de imunofluorescência indireta foi utilizada tanto para a detecção de imunoglobulinas G e M. Amostras com títulos e" 25 foram consideradas positivas. Das 260 amostras testadas, 15% (39/260) foram positivas para anticorpos anti-N. caninum. Destas, em 38 (97,4%) foi detectada a presença de IgG anti-N. caninum e em seis (15,4%) de IgM. Em cinco amostras (12,8%) detectaram-se ambos, IgG e IgM. Os resultados reafirmam a habilidade do N. caninum em determinar infecção fetal. A pesquisa de IgM foi de limitada importância na detecção da infecção via transplacentária em soro fetal bovino.

Toxocariasis/cysticercosis seroprevalence in a long-term rural settlement, Sao Paulo, Brazil

Seroprevalence of Toxocara and Taenia solium and risk factors for infection with these parasites were explored in a long-term rural settlement in Sao Paula state, Brazil. An ELISA for the detection of anti-Toxocara IgG and IgE and anti-T. solium cysticerci was standardized using Toxocara excretory-secretory antigens (TES) obtained from the cultured second-stage larvae of T. canis and by vesicular fluid antigen from Taenia crassiceps cysticerci (VC). For cysticercosis, the reactive ELISA samples were assayed by Western blot using 18 kDa and 14 kDa proteins purified from VF. Out of 182 subjects, 25 (13.7%,) presented anti-Toxocora IgG and it positive correlation between total IgE and the reactive index of specific anti-TES IgE (P=0.0265) was found amongst the subjects found seropositive for anti-Toxocara IgG. In these individuals 38.0%. showed ocular manifestations. The frequency of anti-T. solium cysticerci confirmed by Western blot wits 0.6%, Seropositivity for Toxocara was correlated with low educational levels and the owning of dogs. Embryonated eggs of Toxocara spp. were found in 43.3% of the analysed areas.

Anti-inflammatory effects of Lafoensia pacari and ellagic acid in a murine model of asthma

We have shown that the ethanolic extract of Lafoensia pacari inhibits eosinophilic inflammation induced by Toxocara canis infection, and that ellagic acid is the secondary metabolite responsible for the anti-eosinophilic activity seen in a model of beta-glucan peritonitis. In the present study, we investigated the preventive and curative effects of L. pacari extract and ellagic acid on allergic lung inflammation using a murine model of ovalbumin-induced asthma. In bronchoalveolar lavage fluid, preventive (22-day) treatment with L. pacari (200 mg/kg) and ellagic acid (10 mg/kg) inhibited neutrophil counts (by 75% and 57%) and eosinophil counts (by 78% and 68%). L. pacari reduced IL-4 and IL-13 levels (by 67% and 73%), whereas ellagic acid reduced IL-4, IL-5 and IL-13 (by 67%, 88% and 85%). To investigate curative anti-inflammatory effects, we treated mice daily with ellagic acid (0.1, 1, or 10 mg/kg), also treating selected mice with L. pacari (200 mg/kg) from day 18 to day 22. The highest ellagic acid dose reduced neutrophil and eosinophil numbers (by 59% and 82%), inhibited IL-4, IL-5, and IL-13 (by 62%,61%, and 49%). Neither L. pacari nor ellagic acid suppressed ovalbumin-induced airway hyperresponsiveness or cysteinyl leukotriene synthesis in lung homogenates. In mice treated with ellagic acid (10 mg/kg) or L. pacari (200 mg/kg) at 10 min after the second ovalbumin challenge, eosinophil numbers were 53% and 69% lower, respectively. Cytokine levels were unaffected by this treatment. L. pacari and ellagic acid are effective eosinophilic inflammation suppressors, suggesting a potential for treating allergies. (c) 2007 Elsevier B.V All rights reserved.

The IgM anti-desmoglein 1 response distinguishes Brazilian pemphigus foliaceus (Fogo selvagem) from other forms of pemphigus

Fogo selvagern (FS) and pemphigus foliaceus (PF) possess pathogenic IgG anti-desmoglein 1-(Dsg1) autoantibodies. Although PF occurs sporadically, FS is endemic in Limao Verde (LV), Brazil (3.4% prevalence). IgM anti-Dsg1 were detected in 58% FS LV patients (n=31), 19% of FS patients from Hospital-Campo Grande (n=57), 19% from Hospital-Goiania (n=42), 12% from Hospital-Sao Paulo (n=56), 10% of PF patients from United States (n=20), and 0% of PF patients from Japan (n=20). Pemphigus vulgaris (n=40, USA and Japan), bullous pemphigoid (n=40, USA), and healthy donors (n=55, USA) showed negligible percentages of positive sera. High percentages of positive IgM anti-Dsg1 were found in healthy donors from four rural Amerindian populations (42% of 243) as compared with urban donors (14% of 81; P<0.001). More than 50% of healthy donors from LV (n=99, age 5-20 years) possess IgM anti-Dsg1 across ages, whereas IgG-anti-Dsg1 was detected in 2.9% (age 5-10 years), 7.3% (age 11-15 years), and 29% of donors above age 16. IgM anti-Dsg1 epitopes are Ca2+ and carbohydrate-independent. We propose that IgM anti-Dsg1 are common in FS patients in their native environment and uncommon in other pemphigus phenotypes and in FS patients who migrate to urban hospitals. Recurrent environmental antigenic exposure may lead to IgM and IgG responses that trigger TS.

Mucosal and systemic anti-GAG immunity induced by neonatal immunization with HIV LAMP/gag DNA vaccine in mice

Vaccines capable of inducing mucosal immunity in early postnatal life until adulthood, protecting early sexual initiation, should be considered as strategies to vaccination against HIV. The HIV-1 GAG protein as a chimera with the lysosome-associated membrane protein (LAMP/gag), encoded by a DNA vaccine, is targeted to the endosomal/lysosomal compartment that contains class II MHC molecules and has been shown to be immunogenic in adult mice. Assuming that one such strategy could help to overcome the immunological immaturity in the early postnatal period, we have evaluated the systemic and mucosal immunogenicity of LAMP/gag immunization in neonatal mice. Intranasal immunization with LAMP/gag vaccine induced higher levels of sIgA and IgG anti-GAG antibodies in intestinal washes than did the gag vaccine. The combination of ID injections and the IN protocol with the chimeric vaccine promoted the increase of Ab levels in sera. Both vaccines induced splenic IFN-gamma- secreting cells against GAG peptide pools, as well as in vivo cytotoxic T lymphocyte (CTL) function, and increased the percentage of CD8+ T cells to the immunodominant class I peptide in gut and spleen. However, only the chimeric vaccine was able to enhance Th1/Th2 cytokine secretion in response to class II GAG peptide and to enhance IL-4-secreting cells against GAG peptides and p24 protein stimuli. Long-lasting humoral and cellular responses were detected until adult age, following neonatal immunization with the chimeric vaccine. The LAMP/gag vaccination was able to induce potent GAG-specific T and B cell immune responses in early life which are essential to elicit sustained and long-lasting mucosal and systemic humoral response. (C) 2010 Elsevier GmbH. All rights reserved.

The frequency of anti-beta(2)-glycoprotein I antibodies is low and these antibodies are associated with pulmonary hypertension in mixed connective tissue disease

The objective of this study is to evaluate the prevalence of antiphospholipid antibodies, mainly anti-beta(2)-glycoprotein I (anti-beta(2)-GPI), and their possible clinical and laboratory relevance in mixed connective tissue disease (MCTD). This study included 39 consecutive patients with MCTD (Kasukawa`s criteria) from January, 2005, to March, 2007, and compared them with 21 age- and sex-matched healthy controls. IgG and IgM anticardiolipin (aCL) and anti-beta(2)-GPI were measured by ELISA. Lupus anticoagulant (LA) was detected by functional coagulation tests. Medium to high titres of aCL and anti-beta(2)-GPI antibodies were found in sera from four (10.2%) MCTD patients. One of these patients was found to be positive for IgM aCL, IgM anti-beta(2)-GPI and LA antibodies simultaneously. Additionally, this patient had a previous history of foetal loss in the second trimester and new-onset pulmonary arterial hypertension (PAH). The other three patients had none of the manifestations of antiphospholipid syndrome (APS) or PAH. The mean value of IgG anti-beta(2)-GPI was higher among those MCTD patients with PAH than in the group without PAH (34.2 +/- 46.8 vs 12.3 +/- 9.1, P = 0.018). None of the controls were positive for antiphospholipid antibodies. High to moderate titres of anti-beta(2)-GPI as well as APS were rare in MCTD, and these antibodies may be correlated with the development of PAH in these patients. Lupus (2009) 18, 618-621.

Lack of Antilipoprotein Lipase Antibodies in Takayasu's Arteritis

Background. Antilipoprotein lipase (anti-LPL) antibodies were described in rheumatic diseases. In systemic lupus erythematosus they were highly associated with inflammatory markers and dyslipidemia, and may ultimately contribute to vascular damage. The relevance of this association in Takayasu's arteritis, which is characterized by major inflammatory process affecting vessels, has not been determined. Objectives. To analyze the presence of anti-LPL antibodies in patients with Takayasu's arteritis and its association with inflammatory markers and lipoprotein risk levels. Methods. Thirty sera from patients with Takayasu's arteritis, according to ACR criteria, were consecutively included. IgG anti-LPL was detected by a standard ELISA. Lipoprotein risk levels were evaluated according to NCEP/ATPIII. Inflammatory markers included ESR and CRP values. Results. Takayasu's arteritis patients had a mean age of 34 years old and all were females. Half of the patients presented high ESR and 60% elevated CRP. Lipoprotein NCEP risk levels were observed in approximately half of the patients: 53% for total cholesterol, 43% for triglycerides, 16% for HDL-c and 47% for LDL-c. In spite of the high frequency of dyslipidemia and inflammatory markers in these patients no anti-LPL were detected. Conclusions. The lack of anti-LPL antibodies in Takayasu's disease implies distinct mechanisms underlying dyslipidemia compared to systemic lupus erythematosus. Copyright (C) 2009 Jozelio Freire de Carvalho et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Auto-antibodies do not influence development of atherosclerotic plaques in rheumatoid arthritis

Background: Many questions remain unanswered about premature atherosclerosis in rheumatoid arthritis (RA). Besides inflammation, some studies have suggested the role of autoantibodies on its pathogenesis. Objective: The aim of this study was to investigate the presence of antibodies against phospholipids, beta2-glycoproteinl (beta2-gpl), lipoprotein lipase, and heat shock proteins (Hsp) in RA patients and to evaluate their possible association with subclinical carotid atherosclerosis. Methods: Seventy-one RA patients and 53 age- and sex-matched controls were selected to perform anticardiolipin antibodies (aCL) (IgG and IgM), anti-beta2-gpl (IgG, IgM, and IgA), anti-lipoprotein lipase (anti-LPL), anti-Hsp 60, and anti-Hsp 65 by ELISA tests. Intima-medial thickness (IMT) of common carotid and presence of plaques were assessed by high-resolution B-mode ultrasonography. Exclusion criteria were smoking, diabetes, and arterial hypertension. Lipoproteins, erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), and fibrinogen levels, as well as health assessment questionnaire (HAQ) and disease activity score (DAS) 28 were also evaluated. Results: Age (48.93 +/- 12.31 vs. 45.37 +/- 9.37 years; p = 0.20) and body mass index (BMI) (p = 0.69) were similar in RA and controls, as well as female gender (p = 0.56). The mean IMT was similar between RA and controls (0. 721 +/- 0.16 vs. 0.667 +/- 0.14 turn, p = 0.07) but the frequency of plaques was higher in RA (14.1% vs. 1.9%; p = 0.02). In RA patients, IMT measurements did not differ according to the presence or absence of these antibodies: IgG aCL (0.62 +/- 0.64 vs. 0.72 +/- 0.17 mm, p = 0.24), IgM aCL (0.65 +/- 0.79 vs. 0.73 +/- 0.17 mm, p = 0.33), anti-Hsp 60 (0.78 +/- 0.20 vs. 0.71 +/- 0.16 mm, p = 0.27), anti-Hsp 65 (0.73 +/- 0.16 vs. 0.72 +/- 0.17 mm, p = 0.77), IgG anti-beta2-gpl (0.73 +/- 0.16 vs. 0.71 +/- 0.17 mm, p = 0.72), and anti-CCP (0.71 +/- 0.16 vs. 0.76 +/- 0.20 mm, p = 0.36). In addition, IMT did not correlate with antibodies titers: IgG aCL (r = -0.09, p = 0.47), IgM aCL (r = - 0.15, p = 0.21), anti-Hsp 60 (r = 0.10, p = 0.42), anti-Hsp 65 (r = 0.05, p = 0.69), IgG anti-beta2-gpl (r = - 0.07, p = 0.57), IgM anti-beta2-gpl (r = - 0.05, p = 0.69), IgA anti-beta2-gpl (r = 0.03, p = 0.79), and anti-CCP (r = - 0.07, p = 0.57). RA patients with plaques had a significantly higher age compared to those without plaques (p = 0.001), as well as higher mean IMT (p < 0.001), total cholesterol (p = 0.001), and LDL (p = 0.003). Conclusions: In RA a clear association between all autoantibodies studied herein and increased IMT or presence of plaques was not observed. The great prevalence of carotid atherosclerosis in RA was related to age, total and LDL cholesterol, as identified in normal population. (c) 2008 Elsevier Masson SAS. All rights reserved.

Development of an IgG4-Based Predictor of Endemic Pemphigus Foliaceus (Fogo Selvagem)

Fogo selvagem (FS) is mediated by pathogenic, predominantly IgG4, anti-desmoglein 1 (Dsg1) autoantibodies and is endemic in Limao Verde, Brazil. IgG and IgG subclass autoantibodies were tested in a sample of 214 FS patients and 261 healthy controls by Dsg1 ELISA. For model selection, the sample was randomly divided into training (50%), validation (25%), and test (25%) sets. Using the training and validation sets, IgG4 was chosen as the best predictor of FS, with index values above 6.43 classified as FS. Using the test set, IgG4 has sensitivity of 92% (95% confidence interval (95% CI): 82-95%), specificity of 97% (95% CI: 89-100%), and area under the curve of 0.97 ( 95% CI: 0.94-1.00). The IgG4 positive predictive value (PPV) in Limao Verde (3% FS prevalence) was 49%. The sensitivity, specificity, and PPV of IgG anti-Dsg1 were 87, 91, and 23%, respectively. The IgG4-based classifier was validated by testing 11 FS patients before and after clinical disease and 60 Japanese pemphigus foliaceus patients. It classified 21 of 96 normal individuals from a Limao Verde cohort as having FS serology. On the basis of its PPV, half of the 21 individuals may currently have preclinical FS and could develop clinical disease in the future. Identifying individuals during preclinical FS will enhance our ability to identify the etiological agent(s) triggering FS.

Naturally Acquired Antibodies to Plasmodium vivax Duffy Binding Protein (DBP) in Rural Brazilian Amazon

Duffy binding protein (DBP), a leading malaria vaccine candidate, plays a critical role ill Plasmodium vivax erythrocyte invasion. Sixty-eight of 366 (18.6%) subjects had IgG anti-DBP antibodies by enzyme-linked immunosorbent assay (ELISA) in a community-based cross-sectional survey ill the Brazilian Amazon Basin. Despite Continuous exposure to low-level malaria transmission, the overall seroprevalence decreased to 9.0% when the Population was reexamined 12 months later. Antibodies from 16 of 50 (360%) Subjects who were ELISA-positive at the baseline were able to inhibit erythrocyte binding to at least one of two DBP variants tested. Most (13 of 16) of these subjects still had inhibitory antibodies when reevaluated 12 months later. Cumulative exposure to malaria was the strongest predictor of DBP seropositivity identified by Multiple logistic regression models in this population. The poor antibody recognition of DBP elicited by natural exposure to P. vivax in Amazonian populations represents a challenge to be addressed by vaccine development strategies.

Comparison of Humanized IgG and FvFc Anti-CD3 Monoclonal Antibodies Expressed in CHO Cells

Two humanized monoclonal antibody constructs bearing the same variable regions of an anti-CD3 monoclonal antibody, whole IgG and FvFc, were expressed in CHO cells. Random and site-specific integration were used resulting in similar expression levels. The transfectants were selected with appropriate selection agent, and the surviving cells were plated in semi-solid medium for capture with FITC-conjugated anti-human IG antibody and picked with the robotic ClonePix FL. Conditioned media from selected clones were purified by affinity chromatography and characterized by SDS-PAGE, Western-blot, SEC-HPLC, and isoelectric focusing. Binding to the target present in healthy human mononuclear cells was assessed by flow cytometry, as well as by competition between the two constructs and the original murine monoclonal antibody. The humanized constructs were not able to dislodge the murine antibody while the murine anti-CD3 antibody could dislodge around 20% of the FvFc or IgG humanized versions. Further in vitro and in vivo pre-clinical analyses will be carried out to verify the ability of the humanized versions to demonstrate the immunoregulatory profile required for a humanized anti-CD3 monoclonal antibody.

Detection of anti-Toxoplasma gondii antibodies in experimentally and naturally infected non-human primates by Indirect Fluorescence Assay (IFA) and indirect ELISA

The Indirect Fluorescence Assay (IFA) and the indirect ELISA were comparatively used to detect IgG and IgM antibodies for Toxoplasma gondii in experimentally and naturally infected primates. In the experimentally infected group, antibodies of diagnostic value were detected at day 9 post-infection (PI) with the IFA (IgG and IgM) and with IgG-ELISA. IgM-ELISA detected antibodies for T. gondii starting at day 3 PI until the end of the experiment (102 days PI). Of the 209 naturally infected sera tested, from many zoos of State of Sao Paulo, 64.59 and 67.94% were positive in the IgG-IFA test and IgG-ELISA respectively. IgM-ELISA test detected seropositivity in 52.63% of the sera although IgM-IFA test detected it in only in 0.96% of the samples. The differential toxoplasmosis diagnosis was accomplished with Neospora caninum by IFA, observing 61 (29.2%) seropositive animals for this parasite and 149 (70.8%) negative. Sixty animals were positive for both T. gondii and N. caninum. Pneumonia, splenomegaly, and intestinal ulcers were macroscopically observed. Unremarkable interstitial pneumonia, enteritis, colitis, splenitis, and glomerulitis were microscopically observed. The immunohistochemical stain could not detect the presence of T. gondii in the tissues of the animals infected experimentally.

Sero-epidemiology of toxocariasis in a rural settlement in Sao Paulo state, Brazil

The seroprevalence of Toxocara canis and risk factors for infection with this parasite were explored in a rural settlement in Sao Paulo state, Brazil. Total IgA and IgE levels in 79 subjects were determined by turbidimetry and chemiluminescence, respectively. Total counts of leucocytes and erythrocytes and differential counts of leucocytes were made by flow cytometry. ELISA for the detection of anti-Toxocara IgG, IgA and IgE were standardized using Toxocara excretory-secretory antigens (TES) obtained from the cultured second-stage larvae of T. canis. Seventeen (21.5%) of the subjects were found positive for anti-Toxocara IgG, with no significant differences in such seropositivity with age or gender. Thirty (38%) of the subjects showed eosinophilia and 70 (89%) had elevated levels of total IgE. Among the 17 subjects found seropositive for anti-Toxocara IgG, the percentage of leucocytes represented by eosinophils (P=0.0069) and total levels of IgE (P=0.0452) were positively correlated with the levels of anti-TES IgE. Although anti-TES IgA was detected in 10 (59%) of the subjects, there was no significant correlation between the levels of total IgA and those of Toxocara-specific IgA. Only one of the 17 subjects found positive for anti-Toxocara IgG had attended a secondary school and all but two belonged to households with monthly incomes of < U.S.$100. In the study community at least, seropositivity may be related to poor living standards and lack of basic sanitary conditions. The presence of anti-Toxocara IgE and IgA may facilitate the diagnosis of toxocariasis and may well be useful for monitoring the success of treatment.