Immunoinformatics and analysis of antigen distribution of Ureaplasma diversum strains isolated from different Brazilian states.

BACKGROUND: Ureaplasma diversum has numerous virulence factors that contribute to pathogenesis in cattle, including Lipid-associated membrane proteins (LAMPs). Therefore, the objectives of this study were to evaluate in silico important characteristics for immunobiological applications and for heterologous expression of 36 LAMPs of U. diversum (UdLAMPs) and, also, to verify by conventional PCR the distribution of these antigens in strains of Brazilian states (Bahia, Minas Gerais, São Paulo, and Mato Grosso do Sul). The Manatee database was used to obtain the gene and peptide sequences of the antigens. Similarity and identity studies were performed using BLASTp and direct antigenicity was evaluated by the VaxiJen v2.0 server. Epitope prediction for B lymphocytes was performed on the BepiPred v2.0 and CBTOPE v1.0 servers. NetBoLApan v1.0 was used to predict CD8+ T lymphocyte epitopes. Subcellular location and presence of transmembrane regions were verified by the software PSORTb v3.0.2 and TMHMM v2.2 respectively. SignalP v5.0, SecretomeP v2.0, and DOLOP servers were used to predict the extracellular excretion signal. Physico-chemical properties were evaluated by the web-software ProtParam, Solpro, and Protein-sol. RESULTS: In silico analysis revealed that many UdLAMPs have desirable properties for immunobiological applications and heterologous expression. The proteins gudiv_61, gudiv_103, gudiv_517, and gudiv_681 were most promising. Strains from the 4 states were PCR positive for antigens predicted with immunogenic and/or with good characteristics for expression in a heterologous system. CONCLUSION: These works contribute to a better understanding of the immunobiological properties of the UdLAMPs and provide a profile of the distribution of these antigens in different Brazilian states.

Immunization with Pseudomonas aeruginosa outer membrane vesicles stimulates protective immunity in mice.

Pseudomonas aeruginosa is an opportunistic pathogen responsible for a wide range of severe nosocomial and community acquired infections, these infections are major health problems for cystic fibrosis patients and immune-compromised individuals. The emergence of multidrug-resistant isolates highlights the need to develop alternative strategies for treatment of P. aeruginosa infections. Outer membrane vesicles (OMVs) are spherical nanometer-sized proteolipids that are secreted from numerous of pathogenic Gram-negative bacteria, and a number of studies have confirmed the protective efficacy for use of OMVs as candidate vaccines. In this study, OMVs from P. aeruginosa (PA_OMVs) were isolated, formulated with aluminum phosphate adjuvant and used as a vaccine in a mouse model of acute lung infection. The results confirmed that active immunization with PA_OMVs was able to reduce bacterial colonization, cytokine secretion and tissue damage in the lung tissue, thus protecting mice from lethal challenge of P. aeruginosa. Cytokines assay validated that immunization with PA_OMVs was efficient to induce a mixed cellular immune response in mice. Further, high level of specific antibodies was detected in mice immunized with PA_OMVs, and results from opsonophagocytic killing assay and passive immunization suggested that humoral immune response may be critical for PA_OMVs mediated protection. These findings demonstrated that PA_OMVs may be served as a novel candidate vaccine for the prevention of P. aeruginosa infection.

Nrf2 regulates the inflammatory response, including heme oxygenase-1 induction, by mycoplasma pneumoniae lipid-associated membrane proteins in THP-1 cells.

A series of inflammatory responses caused by Mycoplasma pneumoniae largely depend on the lipid-associated membrane proteins (LAMPs). Nuclear factor E2-related factor 2 (Nrf2), a transcription factor, is considered to be a critical modulator of inflammatory responses and cellular redox homeostasis. Monocytes play an important role in the invasion and immunity to resist pathogens. Here, we investigated the role of Nrf2 in the anti-inflammatory response stimulated by LAMPs using the human monocyte cell line THP-1. LAMPs were shown to affect the localization of Nrf2, and the levels of reactive oxygen species and inflammatory reactants, including nitric oxide (NO), prostaglandin E2 (PGE2) and cytokines (IL-6, IL-8), were highly elevated in LAMP-stimulated Nrf2-silenced THP-1 cells. Moreover, LAMPs induced the levels of mRNA and the expression of heme oxygenase-1 (HO-1). In summary, our results demonstrated that LAMPs cause nuclear translocation of Nrf2, which further suppresses the expression of inflammatory reactants in THP-1 cells.

PG1058 Is a Novel Multidomain Protein Component of the Bacterial Type IX Secretion System.

Porphyromonas gingivalis utilises the Bacteroidetes-specific type IX secretion system (T9SS) to export proteins across the outer membrane (OM), including virulence factors such as the gingipains. The secreted proteins have a conserved carboxy-terminal domain essential for type IX secretion that is cleaved upon export. In P. gingivalis the T9SS substrates undergo glycosylation with anionic lipopolysaccharide (A-LPS) and are attached to the OM. In this study, comparative analyses of 24 Bacteroidetes genomes identified ten putative novel components of the T9SS in P. gingivalis, one of which was PG1058. Computer modelling of the PG1058 structure predicted a novel N- to C-terminal architecture comprising a tetratricopeptide repeat (TPR) domain, a ß-propeller domain, a carboxypeptidase regulatory domain-like fold (CRD) and an OmpA_C-like putative peptidoglycan binding domain. Inactivation of pg1058 in P. gingivalis resulted in loss of both colonial pigmentation and surface-associated proteolytic activity; a phenotype common to T9SS mutants. Immunoblot and LC-MS/MS analyses of subcellular fractions revealed T9SS substrates accumulated within the pg1058 mutant periplasm whilst whole-cell ELISA showed the Kgp gingipain was absent from the cell surface, confirming perturbed T9SS function. Immunoblot, TEM and whole-cell ELISA analyses indicated A-LPS was produced and present on the pg1058 mutant cell surface although it was not linked to T9SS substrate proteins. This indicated that PG1058 is crucial for export of T9SS substrates but not for the translocation of A-LPS. PG1058 is a predicted lipoprotein and was localised to the periplasmic side of the OM using whole-cell ELISA, immunoblot and LC-MS/MS analyses of subcellular fractions. The structural prediction and localisation of PG1058 suggests that it may have a role as an essential scaffold linking the periplasmic and OM components of the T9SS.

A lipidated form of the extracellular domain of influenza M2 protein as a self-adjuvanting vaccine candidate.

The highly conserved extracellular domain of Matrix protein 2 (M2e) of influenza A virus has been previously investigated as a potential target for an universal influenza vaccine. In this study we prepared four lipopeptide influenza vaccine candidates in which the TLR2 agonist S-[2,3-bis(palmitoyloxy)propyl] cysteine, (Pam2Cys) was attached to either the N- or C-terminus of the M2e consensus sequence SLLTEVETPIRNEWGCRCNDSSDP and its analogue sequence with the two cysteine residues replaced with serine residues. The results of animal study show that each of these lipopeptides induced strong M2e-specific antibody responses in the absence of extraneous T helper cell epitope(s) which are normally incorporated in the previous studies or addition of extraneous adjuvant and that these antibodies are protective against lethal challenge with influenza virus. Comparison of different routes of inoculation demonstrated that intranasal administration of M2e lipopeptide induced higher titers of IgA and IgG2b antibodies in the bronchoalveolar lavage than did subcutaneous vaccination and was better at mitigating the severity of viral challenge. Finally, we show that anti-M2e antibody specificities absent from the antibody repertoire elicited by a commercially available influenza vaccine and by virus infection can be introduced by immunization with M2e-lipopeptide and boosted by viral challenge. Immunization with this lipidated form of the M2e epitope therefore offers a means of using a widely conserved epitope to generate protective antibodies which are not otherwise induced.

Hypersensitivity to tomato (Lycopersicon esculentum) in peach-allergic patients: rPrup3 and rPrup1 are predictive of symptom severity

Antecedentes: La relevancia de los diferentes alérgenos del tomate, en relación a la severidad de los síntomas producidos tras su ingesta, no ha sido aún establecida. Objetivos: Evaluar la relación entre las reacciones alérgicas graves inducidas por melocotón y tomate y entre los síntomas presentados tras ingesta de tomate, y el patrón de sensibilizaciones IgE mediadas frente a rPru p1, rPrup3, rPrup4, rBetv1, rBetv2, rBetv4, rPhlp1 y rPhlp12 con el fin de concretar la responsabilidad de cada uno de los alérgenos en la gravedad de las reacciones producidas por el tomate. Métodos: Dentro de una población de pacientes alérgicos a melocotón seleccionamos aquellos pacientes con antecedentes de reacciones a tomate mediante una provocación oral abierta (OFC), pruebas cutáneas (SPT) e IgE específica a tomate, a alérgenos recombinantes de melocotón y gramíneas. La gravedad de los síntomas producidos por el tomate estaba relacionada con la presencia de IgE frente a los diferentes alérgenos así como a los síntomas causados por la ingesta de melocotón. Resultados: Se halló una asociación significativa entre las reacciones alérgicas graves a tomate con las reacciones graves a melocotón (p = 0,017) así como con los valores de IgE específica a rPrup3 (p = 0,029), en tanto que los valores de IgE específica a rPrup1, rBetv1, rBetv2 y Phleum pratense se relacionaban con síntomas leves tras ingesta de tomate (p = 0,047, p = 0,0414, p = 0,0457, p = 0,0022 respectivamente). Conclusión: Existe una relación significativa entre los síntomas producidos por el melocotón y el tomate. La presencia de IgE específica frente a rPrup3 parece ser un marcador de síntomas graves por alergia a tomate, en tanto que la presencia de IgE específica anti rPrup1 parece ser un marcador de síntomas leves en los pacientes alérgicos a tomate (AU)

Background: The role of allergens in the severity of tomato allergy symptoms has not yet been studied. Objectives: To evaluate the relationship between severe allergic reactions to peach and tomato and between tomato allergy symptoms and the pattern of IgE positivity for rPrup1, rPrup3, rPrup4, rBetv1, rBetv2, rBetv4, rPhlp1, and rPhlp12 in order to identify the role of recombinant allergens in the severity of reactions to tomato. Methods: We studied peach-allergic patients with clinical reactions to tomato by performing an open food challenge, skin prick test, and determination of serum specific IgE to tomato and to recombinant peach, birch, and grass allergens. Statistical analysis was carried out to evaluate the relationship between the severity of tomato symptoms and IgE positivity to the different allergens and to peach-induced symptoms. Results: We found a significant association between severe reactions to tomato and severe reactions to peach (P=.017) and levels of IgE to rPrup3 (P=.029) and between mild tomato allergy symptoms and levels of IgE to rPrup1 (P=.047), anti-rBetv1 (P=.0414), anti-rBetv2 (P=.0457), and Phleum pratense (P=.0022). Conclusion: We observed a significant relationship between peach and symptoms of tomato allergy. IgE positivity for rPrup3 seems to be a surrogate biochemical marker for severe tomato allergy, whereas the presence of anti-rPrup1 IgE may be an indicator of mild tomato allergy (AU)

Do skin prick test and in vitro techniques diagnose sensitization to peach lipid transfer protein and profilin equally well in allergy to plant food and pollen?

Objetivo: Comparar las pruebas cutáneas prick (PC) con técnicas in vitro (fluoro enzimoinmunoensayo –FEIA- en detección única y múltiple) para detectar sensibilización a profilina y a LTP. Métodos: Se estudiaron retrospectivamente 181 pacientes con alergia a polen y a alimentos vegetales y 61 controles. Se realizaron PC frente a profilina de palmera (Pho d 2) y LTP de melocotón (Pru p 3) y se analizó la IgE específica a Phl p 12 y Pru p 3 por FEIA y por micromatriz de proteínas alergénicas. Resultados: Quince de los 201 sujetos con PC negativa a LTP mostraron ensibilización a este alérgeno mediante IgE específica sérica y en 18 de 41 con PC positivas a LTP no se observó esta sensibilización por otras técnicas. Diecisiete de los 186 sujetos con PC negativa a profilina detectaron IgE específica sérica frente a Phl p 12 y en 30 de los 56 con PC positiva a profilina no se objetivó sensibilización a Phl p 12 en suero. Se observó un acuerdo moderado entre las tres técnicas estudiadas. Conclusiones: La PC frente e a LTP y profilina es un método sensible detectando estas sensibilizaciones y muestra un acuerdo aceptable con las técnicas in vitro, especialmente en los pacientes con negatividad de la PC frente a LTP y a profilina (AU)

Objective: To compare the skin prick test (SPT) with in vitro techniques (single and multiplex fluorescence enzyme-immunoassay [FEIA]) for detecting sensitization to profilin and lipid transfer protein (LTP). Methods: We retrospectively studied 181 patients with pollen and/or plant food allergy and 61 controls. SPT was performed with date palm profilin (Pho d 2) and peach LTP (Pru p 3), and specific IgE (sIgE) to Phl p 12 and Pru p 3 was analyzed using single FEIA and microarray. Results: Fifteen of 201 patients with negative results for LTP in the SPT were sensitized to this allergen in the in vitro tests, and 18 of 41 patients with positive results for LTP in the SPT were not sensitized according to the in vitro tests. Seventeen of 186 patients with negative results for profilin in the SPT were sensitized to Phl p 12 by serum sIgE, and 30 out of 56 patients with positive results for profilin in SPT were not sensitized to Phl p 12 according to the other tests. Moderate agreement was observed between the 3 techniques studied. Conclusions: SPT is a sensitive technique for detecting sensitization to LTP and profilin. Its results are similar to those of in vitro techniques, especially in patients with negative SPT results for peach LTP and palm tree profiling (AU)

Are basophil activation and sulphidoleukotriene determination useful tests for monitoring patients with peach allergy receiving sublingual immunotherapy with a pru p 3-enriched peach extract?

Introduction: Treatment of food allergy essentially consists of food avoidance, but immunotherapy with food is emerging as a new therapeutic option. Objective: To evaluate clinical improvement and immunological changes in patients with peach allergy following sublingual immunotherapy (SLIT) with a Pru p 3 quantified peach extract. Methods: A randomized, double-blind, placebo-controlled clinical trial with peach SLIT was conducted. We assessed clinical efficacy after 6 months of treatment by means of double-blind, placebo-controlled oral challenges with peach and also evaluated immunological changes (basophil activation test [BAT] and determination of sulphidoleukotriene production) following stimulation with peach peel and pulp, rPru p 3, rMal d 1, and rMal d 4 stimulation. We also measured specific IgE and IgG4 to Pru p 3. Results: After 6 months of SLIT (T6), the active group showed a 3-fold improvement in tolerance to Pru p 3 and a significant increase in IgE to rPru p 3 and in sLT production following stimulation with peach peel and rPru p 3. There was also a significant increase in BAT results after stimulation with rPru p 3 at 1 month of SLIT (T1). Statistically significant between-group differences were only observed for BAT with peach peel and pulp at T1 and T6 and for BAT with rPru p 3 at T6. No changes were observed in BAT with rMal d 1 or rMal d 4 or in IgG4 levels to nPru p 3. Conclusions: SLIT with a Pru p 3 quantified peach extract is clinically effective and leads to an increase in basophil activation and sulphidoleukotriene production following stimulation with rPru p 3 and peach peel in the first months of treatment (AU)

Introducción: El tratamiento de la alergia alimentaria se basa en la evitación del alimento. La inmunoterapia con alimentos está emergiendo como una nueva opción terapéutica. Evaluar la mejoría clínica y los cambios inmunológicos de una inmunoterapia sublingual (ITSL) de melocotón (cuantificada en Pru p 3) en pacientes con alergia a melocotón. Métodos: Ensayo clínico doble-ciego controlado con placebo con una SLIT de melocotón. Valoramos la eficacia clínica a los 6 meses del tratamiento mediante provocaciones orales doble-ciego controladas con placebo (PODCCP) y los cambios inmunológicos (test de activación de basófilos -BAT- y liberación de sulfidoleucotrienos -sLT-) tras estimulación celular con piel y pulpa de melocotón, rPru p 3, rMal d 1 y rMal d 4, IgE e IgG4 a Pru p 3. Resultados: A los 6 meses del tratamiento (T6), la tolerancia a Pru p 3 mediante PODCCP en el grupo activo fue 3 veces superior a la basal (T0), se observó un incremento significativo en la IgE específica a rPru p 3 y en la liberación de sLT tras estimulación con piel de melocotón y rPru p 3, así como en el TAB tras estimulación con rPru p 3 al mes del tratamiento (T1). Se observaron diferencias intergrupo (activo-placebo) en T1 y T6 para piel y pulpa de melocotón y en T6 para rPru p 3 mediante TAB. No se observaron modificaciones en rMal d 1 y rMal d 4 o en los niveles de IgG4 a nPru p 3. Conclusiones: La ITSL con un extracto de melocotón cuantificado en Pru p 3, es clínicamente efectiva y provoca un incremento en la activación basófila y en la liberación de sLT tras estimulación celular con rPru p 3 y piel de melocotón en los primeros meses de tratamiento (AU)

Baseline Tryptase Levels Are Related to Age, Total IgE, and Anti-rPru p 3 IgE Levels in Peach-Allergic Patients

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Proteomics characterization of cytoplasmic and lipid-associated membrane proteins of human pathogen Mycoplasma fermentans M64.

Mycoplasma fermentans is a potent human pathogen which has been implicated in several diseases. Notably, its lipid-associated membrane proteins (LAMPs) play a role in immunomodulation and development of infection-associated inflammatory diseases. However, the systematic protein identification of pathogenic M. fermentans has not been reported. From our recent sequencing results of M. fermentans M64 isolated from human respiratory tract, its genome is around 1.1 Mb and encodes 1050 predicted protein-coding genes. In the present study, soluble proteome of M. fermentans was resolved and analyzed using two-dimensional gel electrophoresis. In addition, Triton X-114 extraction was carried out to enrich amphiphilic proteins including putative lipoproteins and membrane proteins. Subsequent mass spectrometric analyses of these proteins had identified a total of 181 M. fermentans ORFs. Further bioinformatics analysis of these ORFs encoding proteins with known or so far unknown orthologues among bacteria revealed that a total of 131 proteins are homologous to known proteins, 11 proteins are conserved hypothetical proteins, and the remaining 39 proteins are likely M. fermentans-specific proteins. Moreover, Triton X-114-enriched fraction was shown to activate NF-kB activity of raw264.7 macrophage and a total of 21 lipoproteins with predicted signal peptide were identified therefrom. Together, our work provides the first proteome reference map of M. fermentans as well as several putative virulence-associated proteins as diagnostic markers or vaccine candidates for further functional study of this human pathogen.