Effect of vitamin D supplementation on total and allergen-specific IgE in children with asthma and low vitamin D levels.

BACKGROUND: Observational studies have yielded inconsistent findings for the relation between vitamin D level and total IgE or allergic sensitization. OBJECTIVE: To determine whether vitamin D supplementation reduces levels of total IgE and IgE to each of 2 common indoor allergens in children with asthma and low vitamin D levels. METHODS: Total IgE, IgE to Dermatophagoides pteronyssinus, and IgE to Blattella germanica were measured at the randomization and exit visits for 174 participants in the Vitamin D Kids Asthma Study, a multicenter, double-blind, randomized placebo-controlled trial of vitamin D3 supplementation (4000 IU/d) to prevent severe exacerbations in children with persistent asthma and vitamin D levels less than 30 ng/mL. Multivariable linear regression was used for the analysis of the effect of vitamin D supplementation on change in each IgE measure. RESULTS: Participants were followed for an average of 316 days. At the exit visit, more subjects in the vitamin D arm achieved a vitamin D level equal to or more than 30 ng/mL compared with those in the placebo arm (87% vs 30%; P < .001). In a multivariable analysis, vitamin D3 supplementation had no significant effect on change in total IgE, IgE to Dermatophagoides pteronyssinus, or IgE to Blattella germanica between the exit and randomization visits (eg, for log10 total IgE, ß = 0.007; 95% CI, -0.061 to 0.074; P = .85). CONCLUSIONS: Vitamin D supplementation, compared with placebo, has no significant effect on serum levels of total IgE, IgE to dust mite, or IgE to cockroach in children with asthma and low vitamin D levels.

Cruzipain and Its Physiological Inhibitor, Chagasin, as a DNA-Based Therapeutic Vaccine Against Trypanosoma cruzi.

Chagas disease caused by the protozoan parasite Trypanosoma cruzi is endemic in 21 Latin American countries and the southern United States and now is spreading into several other countries due to migration. Despite the efforts to control the vector throughout the Americas, currently, there are almost seven million infected people worldwide, causing ~10,000 deaths per year, and 70 million people at risk to acquire the infection. Chagas disease treatment is restricted only to two parasiticidal drugs, benznidazole and nifurtimox, which are effective during the acute and early infections but have not been found to be as effective in chronic infection. No prophylactic or therapeutic vaccine for human use has been communicated at this moment. Here, we evaluate in a mouse model a therapeutic DNA vaccine combining Cruzipain (Cz), a T. cruzi cysteine protease that proved to be protective in several settings, and Chagasin (Chg), which is the natural Cz inhibitor. The DNAs of both antigens, as well as a plasmid encoding GM-CSF as adjuvant, were orally administrated and delivered by an attenuated Salmonella strain to treat mice during the acute phase of T. cruzi infection. The bicomponent vaccine based on Salmonella carrying Cz and Chg (SChg+SCz) was able to improve the protection obtained by each antigen as monocomponent therapeutic vaccine and significantly increased the titers of antigen- and parasite-specific antibodies. More importantly, the bicomponent vaccine triggered a robust cellular response with interferon gamma (IFN-γ) secretion that rapidly reduced the parasitemia during the acute phase and decreased the tissue damage in the chronic stage of the infection, suggesting it could be an effective tool to ameliorate the pathology associated to Chagas disease.

Heterologous Chimeric Construct Comprising a Modified Bacterial Superantigen and a Cruzipain Domain Confers Protection Against Trypanosoma cruzi Infection.

Chagas disease is an endemic chronic parasitosis in Latin America affecting more than 7 million people. Around 100 million people are currently at risk of acquiring the infection; however, no effective vaccine has been developed yet. Trypanosoma cruzi is the etiological agent of this parasitosis and as an intracellular protozoan it can reside within different tissues, mainly muscle cells, evading host immunity and allowing progression towards the chronic stage of the disease. Considering this intracellular parasitism triggers strong cellular immunity that, besides being necessary to limit infection, is not sufficient to eradicate the parasite from tissues, a differential immune response is required and new strategies for vaccines against Chagas disease need to be explored. In this work, we designed, cloned and expressed a chimeric molecule, named NCz-SEGN24A, comprising a parasite antigen, the N-terminal domain of the major cysteine protease of T. cruzi, cruzipain (Nt-Cz), and a non-toxic form of the staphylococcal superantigen (SAg) G, SEG, with the residue Asn24 mutated to Ala (N24A). The mutant SAg SEGN24A, retains its ability to trigger classical activation of macrophages without inducing T cell apoptosis. To evaluate, as a proof of concept, the immunogenicity and efficacy of the chimeric immunogen vs. its individual antigens, C3H mice were immunized intramuscularly with NCz-SEGN24A co-adjuvanted with CpG-ODN, or the recombinant proteins Nt-Cz plus SEGN24A with the same adjuvant. Vaccinated mice significantly produced Nt-Cz-specific IgG titers after immunization and developed higher IgG2a than IgG1 titers. Specific cell-mediated immunity was assessed by in-vivo DTH and significant responses were obtained. To assess protection, mice were challenged with trypomastigotes of T. cruzi. Both schemes reduced the parasite load throughout the acute phase, but only mice immunized with NCz-SEGN24A showed significant differences against control; moreover, these mice maintained 100% survival. These results encourage testing mutated superantigens fused to specific antigens as immune modulators against pathogens.

Protective Cellular Immune Response Induction for Cutaneous Leishmaniasis by a New Immunochemotherapy Schedule.

The palladacycle complex DPPE 1.2 was previously shown to inhibit Leishmania (Leishmania) amazonensis infection in vitro and in vivo. The present study aimed to evaluate the effect of DPPE 1.2 associated with a recombinant cysteine proteinase, rLdccys1, and the adjuvant Propionibacterium acnes on L. (L.) amazonensis infection in two mouse strains, BALB/c, and C57BL/6. Treatment with this association potentiated the leishmanicidal effect of DPPE 1.2 resulting in a reduction of parasite load in both strains of mice which was higher compared to that found in groups treated with either DPPE 1.2 alone or associated with P. acnes or rLdccys1. The reduction of parasite load in both mice strains was followed by immunomodulation mediated by an increase of memory CD4+ and CD8+ T lymphocytes, IFN-γ levels and reduction of active TGF-ß in treated animals. No infection relapse was observed 1 month after the end of treatment in mice which received DPPE 1.2 associated with rLdccys1 or rLdccys1 plus P. acnes in comparison to that exhibited by animals treated with DPPE 1.2 alone. Evaluation of serum levels of AST, ALT, urea, and creatinine showed no alterations among treated groups, indicating that this treatment schedule did not induce hepato or nephrotoxicity. These data indicate the potential use of this association as a therapeutic alternative for cutaneous leishmaniasis caused by L. (L) amazonensis.

Trypanosoma cruzi Virulence Factors for the Diagnosis of Chagas' Disease.

trans-Sialidase and cruzipain are important virulence factors from Trypanosoma cruzi, the etiological agent of Chagas disease, that have highly antigenic domains in their structure and were reported as potential tools for diagnosis of the illness. The aim of the present study is to assess the possibility of using cruzipain and the catalytic domain of trans-sialidase in a Surface Plasmon Resonance-based immunosensor for the diagnosis of chronic Chagas disease. Immunoassays carried out with canine sera verified that cruzipain allows the detection of anti-Trypanosoma cruzi antibodies whereas recombinant trans-sialidase did not yield specific detections, due to the high dilutions of serum used in the immunoassays that hinder the possibility to sense the specific low titer antibodies. The developed cruzipain-based biosensor, whose price per assay is comparable to a commercial enzyme-linked immunosorbent assay (ELISA), was successfully applied for the rapid quantification of specific antibodies against Trypanosoma cruzi in fresh human sera showing an excellent agreement with ELISA.

In vitro selection of Phytomonas serpens cells resistant to the calpain inhibitor MDL28170: alterations in fitness and expression of the major peptidases and efflux pumps.

The species Phytomonas serpens is known to express some molecules displaying similarity to those described in trypanosomatids pathogenic to humans, such as peptidases from Trypanosoma cruzi (cruzipain) and Leishmania spp. (gp63). In this work, a population of P. serpens resistant to the calpain inhibitor MDL28170 at 70 µ m (MDLR population) was selected by culturing promastigotes in increasing concentrations of the drug. The only relevant ultrastructural difference between wild-type (WT) and MDLR promastigotes was the presence of microvesicles within the flagellar pocket of the latter. MDLR population also showed an increased reactivity to anti-cruzipain antibody as well as a higher papain-like proteolytic activity, while the expression of calpain-like molecules cross-reactive to anti-Dm-calpain (from Drosophila melanogaster) antibody and calcium-dependent cysteine peptidase activity were decreased. Gp63-like molecules also presented a diminished expression in MDLR population, which is probably correlated to the reduction in the parasite adhesion to the salivary glands of the insect vector Oncopeltus fasciatus. A lower accumulation of Rhodamine 123 was detected in MDLR cells when compared with the WT population, a phenotype that was reversed when MDLR cells were treated with cyclosporin A and verapamil. Collectively, our results may help in the understanding of the roles of calpain inhibitors in trypanosomatids.

Trans-sialidase overcomes many antigens to be used as a vaccine candidate against Trypanosoma cruzi.

AIM: The development of vaccines against Trypanosoma cruzi remains in an exploratory stage. Despite several antigen candidates have been evaluated, a comparison among the performance of the immunogens cannot be carried out because the available reports differ in formulations and infection model. In this work, we compared the protective capacity of seven T. cruzi antigens in the same model of five new antigens and two well-established candidates. Materials & methods: We evaluated highly immunogenic proteins that contain tandem repeats (FRA [flagelar repetitive protein], Tc3, Tc6); enzymes involved in metabolic pathways critical for parasite survival (cytosolic tryparedoxin peroxidase and tryparedoxin peroxidase); and enzymes involved in parasite invasion (trans-sialidase [TS] and cruzipain). All these antigens were formulated with Freund's adjuvant and protection against the parasite infection was assessed in BALB/c mice. RESULTS: Tc3, cytosolic tryparedoxin peroxidase, cruzipain and TS showed the best outcome after infection in survival level and parasitemia. According to these data, these groups were also assessed using the ISCOMATRIX™ adjuvant which is being used in clinical trials. CONCLUSION: Taken together, our results showed that the TS overcomes the performance of other antigens when the same model is employed, confirming that TS is a promising antigen that could be used as a vaccine against T. cruzi.

Sensitization to 10 mites in a tropic area. Der p and Der f are important risk factor for sensitization to other mites from Pyroglyphidae, Acaridae, Chortoglyphidae, and Glyciphagidae families.

BACKGROUND: Much is known about the frequency of sensitization to Blomia tropicalis, Dermatophagoides pteronyssinus and Dermatophagoides farinae, although less is known about sensitization to other species and their possible interactions. OBJECTIVE: In patients with allergic manifestations, to evaluate the frequency of sensitization to 10 species of mites in a tropical area and their possible interactions. METHODS: Cross-sectional study. Sensitization was evaluated by skin tests. A generalized linear Poisson regression model with robust variance was used. Based on the sensitization probability reasons and social networking analysis, explorations of relationship for 10 mites were performed. RESULTS: 147 patients were included. The highest sensitization was found to mites' family Pyroglyphidae (> 70 %) and less frequently was the Glycyphagidae family (< 50 %). Sensitization to any mites significantly increased the likelihood of sensitization to others. Sensitization to Der f or Der p increased, more than 20 times the likelihood of sensitization to other mites of the Pyroglyphidae family and more than 10 times to mites from other families. Sensitization to mites from Glycyphagidae, Chortoglyphidae or Acaridae family also increased the risk of sensitization to other mites but less than 5 times. CONCLUSION: Sensitization to mites is frequent in tropical area. Pyroglyphidae sensitization is the main risk factor for polysensitization with other mites from Glycyphagidae, Chortoglyphidae or Acaridae. These results must be considered at diagnosis and treatment of allergy diseases.

GRAIL and Otubain-1 are Related to T Cell Hyporesponsiveness during Trypanosoma cruzi Infection.

BACKGROUND: Trypanosoma cruzi infection is associated with severe T cell unresponsiveness to antigens and mitogens and is characterized by decreased IL-2 synthesis. In addition, the acquisition of the anergic phenotype is correlated with upregulation of "gene related to anergy in lymphocytes" (GRAIL) protein in CD4 T cells. We therefore sought to examine the role of GRAIL in CD4 T cell proliferation during T. cruzi infection. METHODOLOGY/PRINCIPAL FINDINGS: Balb/c mice were infected intraperitoneally with 500 blood-derived trypomastigotes of Tulahuen strain, and spleen cells from control non-infected or infected animals were obtained. CD4 T cell proliferation was assessed by CFSE staining, and the expression of GRAIL in splenic T cells was measured by real-time PCR, flow cytometry and Western blot. We found increased GRAIL expression at the early stages of infection, coinciding with the peak of parasitemia, with these findings correlating with impaired proliferation and poor IL-2 and IFN-γ secretion in response to plate-bound antibodies. In addition, we showed that the expression of GRAIL E3-ubiquitin ligase in CD4 T cells during the acute phase of infection was complemented by a high expression of inhibitory receptors such as PD-1 and CTLA-4. We demonstrated that GRAIL expression during infection was modulated by the mammalian target of the rapamycin (mTOR) pathway, since addition of IL-2 or CTLA-4 blockade in splenocytes from mice 21 days post infection led to a reduction in GRAIL expression. Furthermore, addition of IL-2 was able to activate the mTOR pathway, inducing Otubain-1 expression, which mediated GRAIL degradation and improved T cell proliferation. CONCLUSIONS: We hypothesize that GRAIL expression induced by the parasite may be maintained by the increased expression of inhibitory molecules, which blocked mTOR activation and IL-2 secretion. Consequently, the GRAIL regulator Otubain-1 was not expressed and GRAIL maintained the brake on T cell proliferation. Our findings reveal a novel association between increased GRAIL expression and impaired CD4 T cell proliferation during Trypanosoma cruzi infection.

Effect of the house dust mite allergen Der p 1 on tryptase release from human mast cells.

This study aimed to investigate the effects of the house dust mite allergen Der p 1 on the secretion of tryptase from the human mast cell line HMC-1. Flow cytometry was used to determine the expression levels of protease-activated receptor-2 (PAR2) on the surface of HMC-1 cells. HMC-1 cells were treated with Der p 1, SLIGRL-NH2 (PAR2 agonist), LRGILS-NH2 (control peptide for PAR2), or Der p 1 + FSLLRY (PAR2 antagonist), and the tryptase levels were measured using enzyme-linked immunosorbent assay. The biological functions of PAR2 were determined using the calcium green indicator, and intracellular calcium fluorescence intensity in the different groups (Der p 1, SLIGRL-NH2, LRGILS- NH2, Der p 1 + FSLLRY, tryptase, tryptase + FSLLRY, or cell culture medium) was detected by laser scanning confocal microscopy. The mast cells expressed PAR2 receptor on their surfaces. Der p 1 alone induced a significant release of intracellular calcium and tryptase in HMC-1 cells compared with the SLIGRL- NH2 treatment group and the control group. The combination of Der p 1 and FSLLRY partly inhibited intracellular calcium and tryptase release in HMC-1 cells compared with the Der p 1 treatment group. Moreover, tryptase induced a significant release of intracellular calcium in the HMC-1 cells. Der p 1 induced HMC-1 cell degranulation and the release of tryptase by activating the PAR2 receptor on the cell surfaces. Tryptase activated the PAR2 receptor and induced intracellular calcium release from the HMC-1 cells in a positive feedback loop.

Molecular-based diagnosis of respiratory allergic diseases in children from Curitiba, a city in Southern Brazil.

BACKGROUND: Prevalence of respiratory allergic diseases has increased worldwide. Identification of the aeroallergens involved in allergenic sensitisation is important for diagnosis, treatment and prevention. OBJECTIVE: To verify the molecular pattern of sensitisation to aeroallergens in patients with allergic respiratory diseases using microarray technique for specific IgE antibody detection. METHODS: Cross-sectional study of 101 children with allergic rhinitis was followed in an outpatient clinic. All patients had positive skin prick tests (SPT) to at least one of the following antigens: Dermatophagoides pteronyssinus, Blomia tropicalis, Blattella germanica, Lolium multiflorum, and dog and cat epithelium. Serum specific IgE antibodies (sIgE) to mites, animal epithelia, fungi, cockroach and pollens components were determined by ImmunoCAP ISAC. RESULTS: sIgE to group 1 and 2 mite allergens showed higher positive rates: Der p 1 (74.2%), Der p 2 (73.3%), Der f 1 (74.2%), Der f 2 (72.3%). sIgE to animal epithelia were less frequent, Can f 1, Can f 2, Can f 3 in 4.9%, 2.9%, 1.9% respectively and Fel d 1, Fel d 2, Fel d 4 in 16.8%, 0.9% and 1.9%. respectively. Sensitisation to fungi and cockroach were rare, except for Bla g 7, to which 16.8% were positive. There was no significant recognition for tree pollens group. For grass, sIgE were detected to Cyn d 1 in 16.8%, Phl p 1 and Phl p 4 in 14.8% and 12.9%, respectively. CONCLUSION: Knowing that the pattern of allergic sensitisation varies according to environment and population, our results reinforce the need for local studies, using molecular-based diagnosis.

Immunocapture of circulating Schistosoma mansoni cathepsin B antigen (Sm31) by anti-Sm31 polyclonal antibodies.

Adult Schistosoma mansoni parasites have the capacity to degrade ingested host hemoglobin and other host plasma proteins by using a series of gut proteolytic enzymes, including cathepsin B; this enzyme is released to the host intravascular environment during regurgitations of adult worms. Cathepsin B becomes thus a circulating parasite component that has been shown to be specifically recognized as the Sm31 antigen by antibodies present in most S. mansoni infected patients. Taking advantage of this immunological property, we attempted here to immunocapture Sm31 from sera of infected patients using specific polyclonal rabbit antibodies raised against a highly enriched preparation of Sm31 and detect its intrinsic proteolytic activity using a previously described solid-phase procedure called Cysteine Protease Immuno Assay (CPIA). To produce highly specific anti-Sm31/cathepsin B antibodies, cathepsin B (Sm31 or SmCB) was enriched more than 3000-folds from an adult worm preparation using a series of conventional biochemical steps including ion exchange and affinity chromatography. Anti-cathepsin B antibodies were generated by immunizing rabbits with the enriched cathepsin B fraction; these antibodies recognized a band of Mr.~31 kDa in Western-blot (WB) analysis of this fraction and were able to capture, in a modified CPIA procedure, Sm31/SmCB present in sera from infected Venezuelan patients living in low endemic areas for schistosomiasis. CPIA showed 100% sensitivity and 100% specificity; representing a new diagnostic tool to detect circulating Sm31 antigen in actual infections.

Genome-wide expression profiles identify potential targets for gene-environment interactions in asthma severity.

BACKGROUND: Gene-environment interaction studies using genome-wide association study data are often underpowered after adjustment for multiple comparisons. Differential gene expression in response to the exposure of interest can capture the most biologically relevant genes at the genome-wide level. OBJECTIVE: We used differential genome-wide expression profiles from the Epidemiology of Home Allergens and Asthma birth cohort in response to Der f 1 allergen (sensitized vs nonsensitized) to inform a gene-environment study of dust mite exposure and asthma severity. METHODS: Polymorphisms in differentially expressed genes were identified in genome-wide association study data from the Childhood Asthma Management Program, a clinical trial in childhood asthmatic patients. Home dust mite allergen levels (<10 or ≥10 µg/g dust) were assessed at baseline, and (≥1) severe asthma exacerbation (emergency department visit or hospitalization for asthma in the first trial year) served as the disease severity outcome. The Genetics of Asthma in Costa Rica Study and a Puerto Rico/Connecticut asthma cohort were used for replication. RESULTS: IL9, IL5, and proteoglycan 2 expression (PRG2) was upregulated in Der f 1-stimulated PBMCs from dust mite-sensitized patients (adjusted P < .04). IL9 polymorphisms (rs11741137, rs2069885, and rs1859430) showed evidence for interaction with dust mite in the Childhood Asthma Management Program (P = .02 to .03), with replication in the Genetics of Asthma in Costa Rica Study (P = .04). Subjects with the dominant genotype for these IL9 polymorphisms were more likely to report a severe asthma exacerbation if exposed to increased dust mite levels. CONCLUSIONS: Genome-wide differential gene expression in response to dust mite allergen identified IL9, a biologically plausible gene target that might interact with environmental dust mite to increase severe asthma exacerbations in children.

Requirement of MyD88 and Fas pathways for the efficacy of allergen-free immunotherapy.

BACKGROUND: We have shown that mycobacterial antigens and CpG oligodeoxynucleotides downmodulate airway allergic inflammation by mechanisms dependent on T-cell activation. Here, we investigated the participation of the innate response, particularly the role of MyD88 adaptor, and Fas molecules in the effectiveness of DNA-HSP65 or CpG/culture filtrated proteins (CFP) immunotherapy. METHODS: Mice sensitized and challenged with Der p 1 allergen were treated with DNA-HSP65, CpG/CFP, or with adoptively transferred cells from immunized mice. The treatment efficacy was assessed by evaluating eosinophil recruitment, antibody, and cytokine production. RESULTS: In addition to downregulating the Th2 response, DNA-HSP65 and CpG/CFP promoted IL-10 and IFN-γ production. Adoptive transfer of cells from mice immunized with DNA-HSP65 or CpG/CFP to allergic recipients downmodulated the allergic response. Notably, transfer of cells from DNA-HSP65- or CpG/CFP-immunized MyD88(-/-) mice failed to reduce allergy. Additionally, for effective reduction of allergy by cells from CpG/CFP-immunized mice, Fas molecules were required. Although DNA-HSP65 or CpG/CFP immunization stimulated antigen-specific production of IFN-γ and IL-10, the effect of DNA-HSP65 was associated with IL-10 while CpG/CFP was associated with IFN-γ. Moreover, after stimulation with mycobacterial antigens plus Der p 1 allergen, cells from mite-allergic patients with asthma exhibited similar patterns of cytokine production as those found in the lung of treated mice. CONCLUSIONS: This study provides new insights on the mechanisms of allergen-free immunotherapy by showing that both DNA-HSP65 and CpG/CFP downregulated house dust mite-induced allergic airway inflammation via distinct pathways that involve not only induction of mycobacterial-specific adaptive responses but also signaling via MyD88 and Fas molecules.

mAb CZP-315.D9: an antirecombinant cruzipain monoclonal antibody that specifically labels the reservosomes of Trypanosoma cruzi epimastigotes.

Reservosomes are large round vesicles located at the posterior end of epimastigote forms of the protozoan Trypanosoma cruzi, the etiological agent of Chagas disease. They are the specific end organelles of the endocytosis pathway of T. cruzi, and they play key roles in nutrient uptake and cell differentiation. These lysosome-like organelles accumulate ingested macromolecules and contain large amounts of a major cysteine proteinase (cruzipain or GP57/51 protein). Aim of this study was to produce a monoclonal antibody (mAb) against a recombinant T. cruzi cruzipain (TcCruzipain) that specifically labels the reservosomes. BALB/c mice were immunized with purified recombinant TcCruzipain to obtain the mAb. After fusion of isolated splenocytes with myeloma cells and screening, a mAb was obtained by limiting dilution and characterized by capture ELISA. We report here the production of a kappa-positive monoclonal IgG antibody (mAb CZP-315.D9) that recognizes recombinant TcCruzipain. This mAb binds preferentially to a protein with a molecular weight of about 50 kDa on western blots and specifically labels reservosomes by immunofluorescence and transmission electron microscopy. The monoclonal CZP-315.D9 constitutes a potentially powerful marker for use in studies on the function of reservosomes of T. cruzi.

B-cell epitopes of antigenic proteins in Leishmania infantum: an in silico analysis.

Serodiagnosis of visceral leishmaniasis is often hindered by cross-reactions to other parasitic diseases. Identifying specific B-cell epitopes in proteins is therefore important for immunodiagnostics, as well as for disease control by vaccines. This study aimed to identify linear and conformational B-cell epitopes and to evaluate the secondary structure of antigen proteins in Leishmania infantum using in silico analysis. Linear epitopes were predicted using the Immune Epitope Database and Analysis Resource (IEDB), BepiPred and BcePred programs. The conformational B-cell epitopes were identified using the CBTOPE server. The combination of the predictions using IEDB, BepiPred and BcePred generated 148 linear epitopes from the calpain-like cysteine peptidase (CP), thiol-dependent reductase 1 (TDR1) and HSP70 proteins. In total, 164 conformational epitopes were predicted, mostly located in the linear epitope region. The predicted epitopes are located in α helix and random coil regions in the thiol-dependent reductase 1 and HSP70 proteins. New linear and conformational B-cell epitopes of L. infantum proteins were identified in silico, and the prediction using various programs ensures greater accuracy of the results, as suggested by confirmation of previously identified HSP70 epitopes.

Respiratory allergen from house dust mite is present in human milk and primes for allergic sensitization in a mouse model of asthma.

There is an urgent need to identify environmental risk and protective factors in early life for the prevention of allergy. Our study demonstrates the presence of respiratory allergen from house dust mite, Der p 1, in human breast milk. Der p 1 in milk is immunoreactive, present in similar amounts as dietary egg antigen, and can be found in breast milk from diverse regions of the world. In a mouse model of asthma, oral exposure to Der p through breast milk strongly promotes sensitization rather than protect the progeny as we reported with egg antigen. These data highlight that antigen administration to the neonate through the oral route may contribute to child allergic sensitization and have important implications for the design of studies assessing early oral antigen exposure for allergic disease prevention. The up-to-now unknown worldwide presence of respiratory allergen in maternal milk allows new interpretation and design of environmental control epidemiological studies for allergic disease prevention.

Modulation of mucosal/systemic antibody response after sublingual immunotherapy in mite-allergic children.

BACKGROUND: There have been no data on sublingual immunotherapy (SLIT) in Brazilian patients sensitized to house dust mites. This study aimed to evaluate the mucosal/systemic antibody response changes and clinical efficacy after SLIT using Dermatophagoides pteronyssinus (Dpt) allergens with or without bacterial extracts in mite-allergic Brazilian children. METHODS: Patients with allergic rhinitis and asthma were selected for a double-blind, placebo-controlled trial randomized to three groups: DPT (Dpt extract, n = 34), DPT+MRB (Dpt plus mixed respiratory bacterial extracts, n = 36), and Placebo (n = 32). Total symptom and medication scores for rhinitis/asthma, skin prick test (SPT) to Dpt, and measurements of Dpt-, Der p 1-, Der p 2-specific serum IgE, IgG4, IgG1, and specific salivary IgA were evaluated at baseline and after 12 and 18 months of treatment. RESULTS: A significant long-term decline in total symptom/medication scores was observed only in active groups (DTP and DPT+MRB). There was no significant change in SPT results in all groups. SLIT using Dpt allergen alone induced increased levels of serum IgG4 to Dpt, Der p 1, and Der p 2, serum IgG1 and salivary IgA to Dpt and Der p 1. SLIT with Dpt plus bacterial extracts was able to decrease IgE levels, particularly to Der p 2, to increase salivary IgA levels to Der p 1, but had no changes on specific IgG4 and IgG1 levels. CONCLUSIONS: All children undergoing SLIT showed clinical improvement, but a long-term reduction in symptom/medication scores with modulation of mucosal/systemic antibody responses were seen only in active groups (DPT and DPT+MRB).

Structural and immunological characterization of sulphatides: relevance of sulphate moieties in Trypanosoma cruzi glycoconjugates.

Sulphoglycosphingolipids, present on the surface of diverse cells, participate in the regulation of various cellular events. However, little is known about the structure and the role of sulphoglycosphingolipids in trypanosomatids. Herein, sulphated dihexosylceramide structures - composed mainly of sphingosine as the long chain base acylated with stearic acid - have been determined for the first time in Trypanosoma cruzi epimastigotes by UV-MALDI-TOF-MS analysis. Interestingly, inhibition ELISA assays using cruzipain as antigen and polyclonal rabbit antibodies specific for cruzipain, the major cysteine proteinase of T. cruzi, or for its C-terminal domain, have demonstrated (i) that sulphate epitopes are shared between cruzipain and sulphatides of T. cruzi, (ii) that cross-reactivity maps to the C-terminal domain and (iii) the existence of other antigenic determinants in the glycolipidic structures. These features provide evidence that sulphate groups are antigenic in sulphate-containing parasite glycoconjugates. Furthermore, IgG2 antibody levels inversely correlate with disease severity in chronic Chagas disease patients, suggesting that IgG2 antibodies specific for sulphated epitopes might be associated with protective immunity and might be considered as potential surrogates of the course of chronic Chagas disease.