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.

HLA-F polymorphisms in a Euro-Brazilian population from Southern Brazil.

HLA-F is a non-classical major histocompatibility complex (MHC) gene. It codes class Ib MHC molecules with restricted distribution and less nucleotide variations than MHC class Ia genes. Of the 22 alleles registered on the IMGT database only four alleles encode for proteins that differ in their primary structure. To estimate genotype and allele frequencies, this study targeted on known protein coding regions of the HLA-F gene. Genotyping was performed by Sequence Base Typing (SBT). The sample was composed by 199-unrelated bone marrow donors from the Brazilian Bone Marrow Donor Registry (REDOME), Euro-Brazilians, from Southern Brazil. About 1673 bp were analyzed. The most frequent allele was HLA-F*01:01 (87.19%), followed by HLA-F*01:03 (12.31%), HLA-F*01:02 (0.25%) and HLA-F*01:04 (0.25%). Significant linkage disequilibrium (LD) was verified between HLA-F and HLA classes I and II alleles. This is the first study regarding HLA-F polymorphisms in a Euro-Brazilian population contributing to the Southern Brazilian genetic characterization.

HLA-G 5' URR SNPs and 3' UTR 14-bp insertion/deletion polymorphism in an Afro-Brazilian population from Paraná State.

The present study investigated 23 SNPs in the 5'URR promoter region and the 14 bp ins/del polymorphism at the 3'UTR region of the HLA-G gene in 150 individuals with Afro-Brazilian ancestry. Three haplotypes were found to be the most frequent. Comparing these polymorphisms in other samples, our data suggest that Afro-Brazilians are more similar to the Euro-Brazilians than to Hutterite population.

Identification of a new HLA-DRB1*11 variant, HLA-DRB1*11:130, by sequence-based typing in a Brazilian individual.

HLA-DRB1*11:130, detected in a Euro-Brazilian female, presents a point mutation at codon 59.3 (GAG→GAC).

HLA class II polymorphisms and recurrent spontaneous abortion in a Southern Brazilian cohort.

A high proportion of human recurrent spontaneous abortions (RSA) remain unexplained. The possible association between RSA and different genetic polymorphisms within the human leucocyte antigen system (HLA system, the human major histocompatibility complex) has been investigated with conflicting results since many decades. Here, we describe a case-control study with 136 Southern Brazilian women of predominantly European ancestry (75 control and 61 cases with unexplained RSA). We investigated the relationship between unexplained RSA and alleles and genotypes from two classical loci of the HLA: HLA-DRB1 and HLA-DQB1, as well as three loci related to cytokine production and their serum levels: TNFA (-308G>A), IL10 (-1082G>A, -819T>C, -592A>C) and IFNG (+874A>T). Genotyping was performed by an allele-specific PCR method. While all results concerning cytokine-related genes turned out to be negative, we found the genotype HLA-DQB1*02:02, 03:01 to be significantly decreased and the allele HLA-DRB1*11:04 to be significantly increased among patients.

HLA-DRB1*08:48, a novel allele identified in a Brazilian donor.

The new allele presents a point mutation at codon 67.1 (ATC→CTC) resulting in a conservative change from isoleucine to leucine.

Recombinant DNA immunotherapy ameliorate established airway allergy in a IL-10 dependent pathway.

BACKGROUND: Previous studies have established that mycobacterial infections ameliorate allergic inflammation. However, a non-infectious approach that controls allergic responses might represent a safer and more promising strategy. The 60-65 kDa heat shock protein (Hsp) family is endowed with anti-inflammatory properties, but it is still unclear whether and how single mycobacterial Hsp control allergic disorders. OBJECTIVE: Therefore, in this study we determined whether the administration of Mycobacterial leprae Hsp65 expressed by recombinant a DNA plasmid could attenuate a previously established allergic response. METHODS: We used an experimental model of airway allergic inflammation to test the effects of immunotherapy with DNA encoding Hsp65. Allergic mice, previously sensitized and challenged with ovalbumin, were treated with tree intramuscular doses of recombinant DNA encoding Hsp65. After treatment, mice received a second allergen challenge and the allergic response was measured. RESULTS: We found that immunotherapy attenuated eosinophilia, pulmonary inflammation, Th2 cytokine and mucus production. Moreover, we showed that the inhibition of allergic response is dependent on IL-10 production. Both Hsp65 and allergen-specific IL-10-producing cells contributed to this effect. Cells transferred from DNA-immunized mice to allergic mice migrated to allergic sites and down-modulated the Th2 response. CONCLUSIONS AND CLINICAL RELEVANCE: Our findings clearly show that immunotherapy with DNA encoding Hsp65 can attenuate an established Th2 allergic inflammation through an IL-10-dependent mechanism; moreover, the migration of allergen- and Hsp65-specific cells to the allergic sites exerts a fundamental role. This work represents a novel contribution to the understanding of immune regulation by Hsp65 in allergic diseases.

A novel allele, HLA-A*66:14, identified in a Brazilian volunteer bone marrow donor.

The new allele might have arisen from HLA-A*66:01 through a point mutation at codon 182.1 (ACG→GCG) resulting in a non-conservative change from threonine to alanine.

Two novel alleles, HLA-B*40:125 and B*40:129, in the Brazilian population.

The new alleles, HLA-B*40:125 and HLA-B*40:129, present a mismatch at codon 103.1 (G → C) and three mismatches at codons 9.1 (C → T), 11.1 (T → G) and 12.1 (G → A).

A new allele, HLA-B*4212, identified in a Brazilian volunteer bone marrow donors by sequence-based typing.

Here we report the discovery of a novel HLA-B allele, named B*4212 in a Brazilian volunteer bone marrow donor. The new sequence has nucleotide variation at position 496 (T→G) as compared with B*4201. This variation results in a conservative amino acid substitution from valine to glycine at codon 165 of exon 3.

Leukotrienes are not essential for the efficacy of a heterologous vaccine against Mycobacterium tuberculosis infection

Leukotrienes are reported to be potent proinflammatory mediators that play a role in the development of several inflammatory diseases such as asthma, rheumatoid arthritis and periodontal disease. Leukotrienes have also been associated with protection against infectious diseases. However, the role of leukotrienes in Mycobacterium tuberculosis infection is not understood. To answer this question, we studied the role of leukotrienes in the protective immune response conferred by prime-boost heterologous immunization against tuberculosis. We immunized BALB/c mice (4-11/group) with subcutaneous BCG vaccine (1 x 10(5) M. bovis BCG) (prime) followed by intramuscular DNA-HSP65 vaccine (100 µg) (boost). During the 30 days following the challenge, the animals were treated by gavage daily with MK-886 (5 mg·kg-1·day-1) to inhibit leukotriene synthesis. We showed that MK-886-treated mice were more susceptible to M. tuberculosis infection by counting the number of M. tuberculosis colony-forming units in lungs. The histopathological analysis showed an impaired influx of leukocytes to the lungs of MK-886-treated mice after infection, confirming the involvement of leukotrienes in the protective immune response against experimental tuberculosis. However, prime-boost-immunized mice treated with MK-886 remained protected after challenge with M. tuberculosis, suggesting that leukotrienes are not required for the protective effect elicited by immunization. Protection against M. tuberculosis challenge achieved by prime-boost immunization in the absence of leukotrienes was accompanied by an increase in IL-17 production in the lungs of these animals, as measured by ELISA. Therefore, these data suggest that the production of IL-17 in MK-886-treated, immunized mice could contribute to the generation of a protective immune response after infection with M. tuberculosis.

HLA-B*18:35, a new HLA-B*18 allele identified by sequence-based typing in a Brazilian volunteer bone marrow donor.

A novel human leukocyte antigen-B allele named B*18:35 was identified in a Brazilian volunteer bone marrow donor.

Leukotrienes are not essential for the efficacy of a heterologous vaccine against Mycobacterium tuberculosis infection.

Leukotrienes are reported to be potent proinflammatory mediators that play a role in the development of several inflammatory diseases such as asthma, rheumatoid arthritis and periodontal disease. Leukotrienes have also been associated with protection against infectious diseases. However, the role of leukotrienes in Mycobacterium tuberculosis infection is not understood. To answer this question, we studied the role of leukotrienes in the protective immune response conferred by prime-boost heterologous immunization against tuberculosis. We immunized BALB/c mice (4-11/group) with subcutaneous BCG vaccine (1 x 10(5) M. bovis BCG) (prime) followed by intramuscular DNA-HSP65 vaccine (100 microg) (boost). During the 30 days following the challenge, the animals were treated by gavage daily with MK-886 (5 mg x kg(-1) x day(-1)) to inhibit leukotriene synthesis. We showed that MK-886-treated mice were more susceptible to M. tuberculosis infection by counting the number of M. tuberculosis colony-forming units in lungs. The histopathological analysis showed an impaired influx of leukocytes to the lungs of MK-886-treated mice after infection, confirming the involvement of leukotrienes in the protective immune response against experimental tuberculosis. However, prime-boost-immunized mice treated with MK-886 remained protected after challenge with M. tuberculosis, suggesting that leukotrienes are not required for the protective effect elicited by immunization. Protection against M. tuberculosis challenge achieved by prime-boost immunization in the absence of leukotrienes was accompanied by an increase in IL-17 production in the lungs of these animals, as measured by ELISA. Therefore, these data suggest that the production of IL-17 in MK-886-treated, immunized mice could contribute to the generation of a protective immune response after infection with M. tuberculosis.