A Component-Resolved Therapeutic Vaccine for Cockroach Allergy Made of Per a 9 and Transforming Growth Factor-ß Homologue, an Immunosuppressive Protein of Brugia malayi.

Allergen-specific-immunotherapy (ASIT) can cause long-term resolution of allergic diseases, reduces drug use and chances of new allergen sensitization. Nevertheless, therapeutic vaccine and data on ASIT efficacy for cockroach (CR) allergy are relatively scarce. In this study, efficacy and mechanism of a novel intranasal vaccine consisting of liposome (L)-entrapped mixture of American CR (Periplaneta americana) major allergen (Per a 9) and immunosuppressive protein of Brugia malayi nematode named transforming growth factor-beta homologue (TGH) in treatment of CR allergy were investigated along with two other vaccines (L-Per a 9 alone and L-TGH alone). All three vaccines could reduce pathogenic type 2 response and lung immunopathology in the vaccines-treated CR-allergic mice, but by different mechanisms. L-Per a 9 caused a deviation of the pathogenic type 2 to type 1 response (IFN-γ-upregulation), whereas the L-(TGH + Per a 9) and L-TGH generated regulatory immune responses including up-expression of immunosuppressive cytokine genes and increment of serum adenosine and lung indoleamine-2,3-dioxygenase-1 which are signatures of regulatory T cells (Tregs) and tolerogenic dendritic cells, respectively. The L-(TGH + Per a 9) should be further evaluated towards clinical application, as this vaccine has a propensity to induce broadly effective therapeutic effects for inhalant allergies.

Bioinformatic and immunological investigation of Per a 5 (delta class GST) allergen from Periplaneta americana.

INTRODUCTION: GSTs are multifunctional enzymes involved in cellular detoxification and present as potent allergens in several sources. Present study investigates allergenic relevance of GST from P. americana and determine its cross reactive potential with other indoor allergen sources. METHODS: Computational analysis with FASTA and ConSurf webserver was performed to determine potentially cross reactive allergens. Further, Per a 5 gene was cloned in pET 22b+ vector and expressed in E.coli BL21 cells and the rPer a 5 protein was purified using Ni-NTA affinity chromatography. Enzymatic activity of rPer a 5 was assessed using CDNB and cumene hydroperoxide. ELISA and immunoblot were performed using cockroach hypersensitive patient's sera. Functional activity of rPer a 5 was evaluated by basophil activation test. Inhibition studies were carried out with D. pteronyssinus, A. alternata and C. lunata extracts. RESULTS: Per a 5 demonstrates highest sequence similarity with delta class GST of Blattella germanica (94.9%). It also exhibits significant sequence similarity (50-58%) with mite, fungal and helminth allergenic GSTs. ConSurf analysis reveals high degree of evolutionary similarity in N terminal region of Per a 5, especially at GST dimerization interface. The purified rPer a 5 protein resolved at 27 kDa on SDS-PAGE. The rPer a 5 protein exhibits GST activity and possess upto 65% immunoreactivity with cockroach hypersensitive patient's sera in ELISA and immunoblot. It upregulates expression of CD203c on basophils signifying its biological ability to activate effector cells. rPer a 5 significantly inhibits corresponding GSTs in P. americana, D. pteronyssinus, A. alternata and C. lunata with EC50 values of 15.5 ng. 38.38 ng, 41.4 ng and 61.66 ng, respectively. CONCLUSION: Recombinant delta class GST of P. americana is a clinically relevant allergen showing upto 65% immunoreactivity with hypersensitive patient's sera. Per a 5 GST allergen showed phylogenetic similarity with dust mite, fungal and birch allergens thereby demonstrating allergen cross reactivity.

Insect Body Defence Reactions against Bee Venom: Do Adipokinetic Hormones Play a Role?

Bees originally developed their stinging apparatus and venom against members of their own species from other hives or against predatory insects. Nevertheless, the biological and biochemical response of arthropods to bee venom is not well studied. Thus, in this study, the physiological responses of a model insect species (American cockroach, Periplaneta americana) to honeybee venom were investigated. Bee venom toxins elicited severe stress (LD50 = 1.063 uL venom) resulting in a significant increase in adipokinetic hormones (AKHs) in the cockroach central nervous system and haemolymph. Venom treatment induced a large destruction of muscle cell ultrastructure, especially myofibrils and sarcomeres. Interestingly, co-application of venom with cockroach Peram-CAH-II AKH eliminated this effect. Envenomation modulated the levels of carbohydrates, lipids, and proteins in the haemolymph and the activity of digestive amylases, lipases, and proteases in the midgut. Bee venom significantly reduced vitellogenin levels in females. Dopamine and glutathione (GSH and GSSG) insignificantly increased after venom treatment. However, dopamine levels significantly increased after Peram-CAH-II application and after co-application with bee venom, while GSH and GSSG levels immediately increased after co-application. The results suggest a general reaction of the cockroach body to bee venom and at least a partial involvement of AKHs.

Association between skin prick test and serum specific immunoglobulin E to American cockroach allergens in allergic rhinitis patients.

INTRODUCTION AND OBJECTIVES: American cockroach is a common aeroallergen sensitization in allergic rhinitis (AR) patients. Association between skin prick test (SPT) and specific immunoglobulin E (sIgE) to American cockroach allergen remains uncertain. This study aimed to evaluate the association between SPT and sIgE to American cockroach allergen in patients with AR. MATERIALS AND METHODS: This cross-sectional study was conducted in Thai AR patients aged 6-25 years from September 2013 to October 2014. SPT and sIgE to American cockroach allergen were performed and the correlation was calculated using SPSS Statistics version 18. RESULTS: Sixty-seven AR patients, with median age of 15 years were included in this study. SPT and sIgE to American cockroach allergen showed a positive result in 68.7% and 58.2% cases, respectively. Positive SPT or positive sIgE to American cockroach was found in 79.1%. Thirty-two patients (47.8%) tested positive for both SPT and sIgE to American cockroach allergen. Fourteen from a total of 67 cases (20.9%) with negative sIgE had positive SPT to American cockroach, while seven cases (10.4%) with negative SPT had positive sIgE to American cockroach. Moderate correlation was observed between mean wheal diameter (MWD) and sIgE level to American cockroach (r=0.465, p=0.001). No significant correlation was found between MWD of SPT or sIgE level to American cockroach and AR severity. CONCLUSION: A moderate correlation was observed between MWD of SPT and sIgE level to American cockroach. If SPT is negative in allergic rhinitis patients highly suspected of having American cockroach allergy, serum sIgE should be considered and vice versa.

Concordance of skin prick test and serum-specific IgE to locally produced component-resolved diagnostics for cockroach allergy.

BACKGROUND: Diagnosis of Periplaneta americana (American cockroach, ACR) allergy is commonly performed based on clinical history and skin prick test (SPT) or specific serum IgE (sIgE) measurement. The concordance of the findings with the SPT and sIgE results has never been investigated. OBJECTIVE: To compare the results of SPT with commercial ACR-extract (C-ACE) and sIgE measurement, using commercial kit and in-house enzyme-linked immunosorbent assay (ELISA) to the locally produced ACR extract (L-ACE) and native Per a 1, Per a 5, Per a 7, and Per a 9. METHODS: Sera from 66 individuals clinically diagnosed with chronic allergic rhinitis were included; 46 were positive SPT to C-ACE, and 20 were negative. Specific serum IgE levels were established by using a commercial test kit (ImmunoCap) and an in-house IgE-ELISA RESULTS: The percentage the C-ACE SPT-positive cases that were positive by the ImmunoCap-sIgE was 32.6%, indicating low concordance of the 2 assays. With the in-house ELISA, Per a 9 gave the highest sensitivity (98.00%), positive predictive value (PPV; 95.74%), and negative predictive value (NPV; 94.74%) of the sIgE quantification. The correlation coefficients (R) of the L-ACE-SPT and sIgE to L-ACE, Per a 1, Per a 5, Per a 7, and Per a 9 and ImmunoCap sIgE were 0.133, 0.278, 0.419, 0.280, and 0.432, and 0.256, respectively. CONCLUSION: Skin prick test and sIgE measurement using commercial reagents have low concordance. Data of this study showed that sIgE to the native Per a 9 should be considered as an adjunct to the clinical history in diagnosis of ACR sensitization/allergy, particularly when the SPT and the nasal challenge, which is the gold standard method, cannot be performed.

Cloning and Purification of Per a 4, a Gene Encoding a Periplaneta Americana Allergen, and Preparation of Its Monoclonal Antibodies.

OBJECTIVE: To clone, express, purify the Per a 4 gene encoding an allergen of Periplaneta Americana and prepare monoclonal antibodies against the recombinant allergen. METHODS: The total RNA was extracted from P. Americana, and the target gene was amplified by RT-PCR and cloned into pMD18-T vector. After being confirmed by nucleotide sequencing, the gene was then inserted into pGEX-3X to construct the express vector pGEX-3X-Per a 4. Further, the pGEX-3X-Per a 4 was transformed into E. coli BL21 (DE3), and induced for expression by IPTG. By affinity chromatography, the recombinant allergen was purified and identified by SDS-PAGE and Western blotting. The BALB/c mouse was immunized with the recombinant allergen to prepare the specific monoclonal antibodies, which was then identified by co-immunoprecipitatin and western blotting. RESULTS: The full-length cDNA encoding Per a 4 of P. Americana was obtained with 552 bp in length, which had 99.4% similarity with the reference sequence (GenBank AY792948). The constructed vector pGEX-3X-Per a 4 was transformed in E. coli BL21 (DE3), expressed with the induction of IPTG. By SDS-PAGE, a band of about 49 KD was present. Further, the western-blotting showed that the prepared monoclonal antibodies can bind the serum antibodies in patients allergic to P. Americana. CONCLUSIONS: Both the recombinant allergen Per a 4 and its monoclonal antibodies were obtained.

DNA extracellular traps are part of the immune repertoire of Periplaneta americana.

Extracellular traps (ETs), web-like structures composed of DNA and histones, are released by innate immune cells in a wide range of organisms. ETs capture microorganisms, thereby avoiding their spread, and also concentrate antimicrobial molecules, which helps to kill microbes. Although vertebrate innate immune systems share homology with the insect immune system, ETosis have yet to be characterized in insects. Here, we report that the hemocytes of the hemimetabolous insect Periplaneta americana release ETs upon in vitro stimulation. We further discuss the relationship between ETs and nodulation and in controlling bacterial spread in vivo.

Serine protease allergen favours Th2 responses via PAR-2 and STAT3 activation in murine model.

BACKGROUND: Protease activity of Per a 10 favours Th2 responses by differential regulation of IL-12p70 and IL-23 cytokine subunits. This study aimed to elucidate the underlying mechanism of differential regulation of IL-12p70 and IL-23. METHODS: PAR-2 activation was blocked in murine model by administering SAM11 before each sensitization. CD11c+ p-STAT3+ cells were measured in lungs by flow cytometry. BMDCs were pretreated with SAM11 or isotype control or stattic and stimulated with Per a 10. p-STAT3 levels were measured using Western blot. Transcript levels of IL-12p35, IL-12/23p40 and IL-23p19 were measured using RT-PCR. Cytokine levels were analysed using ELISA. RESULTS: Protease activity of Per a 10 increased p-STAT3 levels in mouse lungs, which was reduced upon PAR-2 blockage. Percentage of p-STAT3+ CD11c+ cells was higher in Per a 10-administered mice and was reduced upon PAR-2 blockage. IL-12p35 and IL-12p70 levels were higher, and IL-23p19 and IL-23 levels were lower in both SAM11-treated mice and BMDCs indicating a role of PAR-2-mediated signalling. IL-4, TSLP, IL-17A, EPO activity, total cell count and specific IgE and IgG1 levels were lower in SAM11-administered mice. Inhibiting STAT3 activation via stattic also leads to lower levels of IL-23p19 and IL-23 and higher levels of IL-12p35. CONCLUSIONS: Per a 10 leads to PAR-2 activation on BMDCs resulting in downstream activation of STAT3 to regulate the balance between IL-12/IL-23 subunits causing a cytokine milieu rich in IL-23 to favour Th2 polarization.

In silico epitope prediction, expression and functional analysis of Per a 10 allergen from the American cockroach.

Cockroach (CR) allergies caused by the American cockroach hyave been recognized to be repsonsible for IgE-mediated type I hypersensitivity worldwide. Per a 10 is one of the recognized main allergens of the American CR. In a previous study, we examined another American CR allergen, Per a 9 in patients with CR allergies and examined epitope sequences in this allergen. In the present study, we aimed to examine epitope sequences in the Per a 10 allergen. for this purpose, the Per a 10 gene was cloned and expressed in Escherichia coli (E. coli) systems. Our results revealed that 9 out of 16 (56.3%) sera from patients with American CR allergies reacted to Per a10, as assessed by ELISA, confirming that Per a 10 is a major allergen of the American CR. Our results also revealed that the expression of CD63 and CCR3 on passively sensitized basophils (obtained sera of patients with American CR allergies) was increased by approximately 2.3-fold, indicating that recombinant Per a 10 is functionally active. In addition, 3 immunoinformatics tools, namely the DNAStar Protean system, the Bioinformatics Predicted Antigenic Peptides (BPAP) system and the BepiPred 1.0 server were used to predict the peptides and the results revealed 8 peptides (2-12, 55-67, 98-120, 125-133, 149-160, 170-182, 201-208 and 223-227) as potential B cell epitopes of the Per a 10 allergen. Moreover, Per a 10 was predicted to have 3 T cell epitope sequences, namely 83-92, 139-147 and 162-170. The findings of our study on the CR allergen may prove to be useful in the development of peptide-based vaccine for the prevention and/or treatment of CR allergies.

Molecular cloning, expression, IgE binding activities and in silico epitope prediction of Per a 9 allergens of the American cockroach.

Per a 9 is a major allergen of the American cockroach (CR), which has been recognized as an important cause of imunoglobulin E-mediated type I hypersensitivity worldwide. However, it is not neasy to obtain a substantial quantity of this allergen for use in functional studies. In the present study, the Per a 9 gene was cloned and expressed in Escherichia coli (E. coli) systems. It was found that 13/16 (81.3%) of the sera from patients with allergies caused by the American CR reacted to Per a 9, as assessed by enzyme-linked immunosorbent assay, confirming that Per a 9 is a major allergen of CR. The induction of the expression of CD63 and CCR3 in passively sensitized basophils (from sera of patients with allergies caused by the American CR) by approximately 4.2-fold indicated that recombinant Per a 9 was functionally active. Three immunoinformatics tools, including the DNAStar Protean system, Bioinformatics Predicted Antigenic Peptides (BPAP) system and the BepiPred 1.0 server were used to predict the potential B cell epitopes, while Net-MHCIIpan-2.0 and NetMHCII-2.2 were used to predict the T cell epitopes of Per a 9. As a result, we predicted 11 peptides (23-28, 39-46, 58-64, 91-118, 131-136, 145-154, 159-165, 176-183, 290-299, 309-320 and 338-344) as potential B cell linear epitopes. In T cell prediction, the Per a 9 allergen was predicted to have 5 potential T cell epitope sequences, 119-127, 194-202, 210-218, 239-250 and 279-290. The findings of our study may prove to be useful in the development of peptide-based vaccines to combat CR-induced allergies.

Immunogenic peptides: B & T Cell Epitopes of Per a 10 Allergen of Periplaneta americana.

Mapping of B and T cell epitopes of an allergen can be utilised in the development of alternative therapeutic modalities and diagnostics. The present study was aimed to identify B and T cell epitopes of Per a 10, a major cockroach allergen, by computational tools and subsequent validation by in vitro experiments. Per a 10 three-dimensional structure was homology modelled using structure of anionic trypsin from pacific chum salmon as a template. Seven B cell epitopes (B-P1 to B-P7) were predicted by sequence and structure based methods. Three T cell epitopes (T-P8 to T-P10) were predicted by binding score and inhibitory concentration dependent prediction tools. Predicted epitopes were synthesized and biological activity was assessed by ELISA, ELISA inhibition and PBMC proliferation assays. B cell peptides B-P5, B-P6 and B-P7 showed significantly high IgE binding with pooled and individual cockroach hypersensitive patients' sera while the T cell peptides did not show IgE binding. ELISA inhibition was performed to determine the potency of the predicted peptides. Fifty nanogram of peptide B-P7 was required for 50% IgE binding inhibition of surface bound Per a 10 whereas seventy five nanogram and ninety nanogram of B-P5 and B-P6 were required for the same respectively. Upon stimulation with T-P8 and T-P10 peptides, PBMCs from cockroach allergic patients' (n = 15) showed significant lymphocyte proliferation and induced IL-4 and IL-5 cytokine release in the culture supernatant demonstrating Th2 dominant cell mediated response of predicted T cell peptides. In conclusion, Per a 10 3-D structure obtained by homology modelling was used to identify B and T cell epitopes, followed by in vitro validation. The identified peptides can be potentially used in designing diagnostics and therapies for cockroach allergy.

[Influence of serum levels of vitamin D on IgE response in schoolchildren with asthma in poor communities]. / Influencia de los niveles séricos de vitamina D sobre la respuesta IgE en niños escolares con asma en comunidades pobres.

BACKGROUND: Asthma is a common disease in the world and vitamin D (Vit-D) has been associated with the presence and severity of this disease. OBJECTIVE: To establish the association between levels of Vit-D and IgE response in schoolchildren with asthma living in four cities in Colombia. METHODS: Case-control study in 1340 schoolchildren (687 asthmatic and 653 controls) from communities in extreme poverty in Barranquilla, Cartagena, Santa Marta, and Montería. Serum concentrations of Vit-D, total IgE, and anti-Dermatophagoides farinae, Periplaneta americana, and Ascaris lumbricoides (AL) specific IgE were measured. RESULTS: Controls reported higher concentrations of Vit-D [61.9 ± 28.4 ng/mL] than cases [53 ± 23.3 ng / mL] (p < 0.05). Total IgE was higher in cases (p < 0.05). Only anti-AL IgE showed a clear difference: in controls, optical density was 0.27 ± 0.25; in cases, 0.22 ± 0.24 (p < 0.05). Vit-D showed differences between cases and controls in each population. CONCLUSIONS: An association could not be demonstrated between Vit-D deficiency and asthma, as total IgE was elevated in patients and controls. The results suggest that Vit-D influences the specif IgE response in poor asthmatic children in areas endemic for helminthiasis.

Antecedentes: El asma es una enfermedad frecuente en el mundo y la vitamina D (Vit-D) se ha asociado con la presencia y severidad de esta enfermedad. Objetivo: Establecer la asociación entre los niveles de Vit-D y la respuesta IgE en escolares con asma residentes de cuatro ciudades colombiananas. Métodos: Estudio de casos y controles en 1340 escolares (687 asmáticos y 653 controles) de comunidades en extrema pobreza de Barranquilla, Cartagena, Santa Marta y Montería. Se midieron las concentraciones séricas de Vit-D, IgE total e IgE específica anti Dermatofagoides farinae, Periplaneta americana y Ascaris lumbricoides (AL). Resultados: Los controles reportaron concentraciones mayores de Vit-D [61.9 ± 28.4 ng/mL] que los casos [53 ± 23.3 ng/mL] (p<0.05). La IgE total fue mayor en los casos (p<0.05). Solo IgE anti-AL mostró una diferencia clara: controles, densidad óptica 0.27 ± 0.25; casos 0.22 ± 0.24 (p<0.05). La Vit-D presentó diferencias entre casos y controles en cada población. Conclusiones: No se pudo demostrar la asociación entre deficiencia de Vit-D y asma, dado que la IgE total estuvo elevada en los pacientes y en los controles. Los resultados sugieren que la Vit-D influye en la respuesta IgE específica en niños asmáticos pobres en zonas endémicas para helmintiasis.

De Novo Transcriptome Analysis and Detection of Antimicrobial Peptides of the American Cockroach Periplaneta americana (Linnaeus).

Cockroaches are surrogate hosts for microbes that cause many human diseases. In spite of their generally destructive nature, cockroaches have recently been found to harbor potentially beneficial and medically useful substances such as drugs and allergens. However, genomic information for the American cockroach (Periplaneta americana) is currently unavailable; therefore, transcriptome and gene expression profiling is needed as an important resource to better understand the fundamental biological mechanisms of this species, which would be particularly useful for the selection of novel antimicrobial peptides. Thus, we performed de novo transcriptome analysis of P. americana that were or were not immunized with Escherichia coli. Using an Illumina HiSeq sequencer, we generated a total of 9.5 Gb of sequences, which were assembled into 85,984 contigs and functionally annotated using Basic Local Alignment Search Tool (BLAST), Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) database terms. Finally, using an in silico antimicrobial peptide prediction method, 86 antimicrobial peptide candidates were predicted from the transcriptome, and 21 of these peptides were experimentally validated for their antimicrobial activity against yeast and gram positive and -negative bacteria by a radial diffusion assay. Notably, 11 peptides showed strong antimicrobial activities against these organisms and displayed little or no cytotoxic effects in the hemolysis and cell viability assay. This work provides prerequisite baseline data for the identification and development of novel antimicrobial peptides, which is expected to provide a better understanding of the phenomenon of innate immunity in similar species.

Two new types of allergens from the cockroach, Periplaneta americana.

Periplaneta americana cockroach is an important source of inhalant indoor allergen resource, and there are more than twenty IgE-binding components identified in P. americana, but only nine allergens were characterized. Our knowledge about cockroach allergens remains poor. In this work, two novel allergen proteins Per a 11 (alpha-amylase) and Per a 12 (chitinase) with molecular weight around 55 and 45 kDa, respectively, were purified and characterized from the midgut of cockroaches. Their primary sequences were determined by Edman degradation, mass spectrometry, and cDNA cloning. Sera from 39 and 30 of 47 (83.0% and 63.8%) patients reacted to Per a 11 and Per a 12 on immunoblots, respectively. The allergenicity of Per a 11 and Per a 12 was further confirmed by competitive ELISA, basophil activation test (BAT), and skin prick test (SPT). They appear to be of importance for the allergic reactions induced by cockroach and have a potential for component-based diagnosis of allergy.

Protease activity of Per a 10 potentiates Th2 polarization by increasing IL-23 and OX40L.

Proteases are implicated in exacerbation of allergic diseases. In this study, the role of proteolytic activity of Per a 10 was evaluated on Th2 polarization. Intranasal administration of Per a 10 in mice led to allergic airway inflammation as seen by higher IgE levels, cellular infiltration, IL-17A, and Th2 cytokines, whereas, inactive (Δ)Per a 10 showed attenuated response. There was an increased OX40L expression on lung and lymph node dendritic cells in Per a 10 immunized group and on Per a 10 stimulated BMDCs. Reduction in CD40 expression without any change at transcript level in lungs of Per a 10 immunized mice suggested CD40 cleavage. BMDCs pulsed with Per a 10 showed reduced CD40 expression with lower IL-12p70 secretion as compared to heat inactivated Per a 10. IL-23, TNF-α, and IL-6 levels were significantly higher in Per a 10 stimulated BMDCs supernatant. In DC-T cell coculture studies, Per a 10 pulsed BMDCs showed higher levels of IL-4 and IL-13 that were reduced on blocking of either IL-23 or OX40L. In conclusion, the data suggests a critical role of protease activity of Per a 10 in promoting Th2 polarization by increasing IL-23 secretion and OX40L expression on dendritic cells.

DC type 2 polarization depends on both the allergic status of the individual and protease activity of Per a 10.

Cockroach proteases are important risk factors for asthma development in predisposed individuals. In the present study, effect of allergic status of patients on DCs polarization in response to protease allergen Per a 10 was investigated. Cockroach-allergic, other-allergic patients and healthy individuals were selected following the guidelines of ATS/ARIA. Monocyte-derived dendritic cells (DCs) were generated from the selected individuals and stimulated with Per a 10. Flow cytometric analysis showed a significantly high expression of CD80 and CD86 on DCs from cockroach-allergic patients after Per a 10 stimulation as compared to healthy individuals or other-allergic patients (P<0.05). Per a 10 induced comparable level of CD83 expression on DCs from all the 3 groups, showing it was irrespective of the allergic status. CD40 expression was significantly low (P<0.05) on the DCs from cockroach-allergic patients as compared to healthy individuals or other-allergic patients. Further, proteolytically active Per a 10 induced lower CD40 expression on DCs than the heat-inactivated Per a 10 (P<0.05) indicating role of protease activity in the generation of an immune response. The sCD40 level in active Per a 10 stimulated DC cultures was significantly higher than in heat-inactivated Per a 10 (P<0.05). There was two-fold decrease (P<0.05) in IL-12 production by active Per a 10-stimulated DCs than heat-inactivated Per a 10-stimulated DCs. Per a 10-stimulated DCs from cockroach-allergic patients secreted high levels of IL-5, IL-6, TNF-α than that from healthy individuals or other-allergic patients (P<0.05). Furthermore, Per a 10-stimulated DCs from cockroach-allergic patients induced increased secretions of IL-4, IL-5, IL-6, TNF-α and low IL-12 by T cells as compared to those from other groups (P<0.05). Thus, in presence of Per a 10 allergen, polarization of DCs shifts toward type 2 in cockroach-allergic patients but not in the healthy individuals or other-allergic patients. In conclusion, both allergic status of the individual and protease activity of Per a 10 are important parameters that participate in DCs polarization.

Mixing compatibilities of Aspergillus and American cockroach allergens with other high-protease fungal and insect extracts.

BACKGROUND: Recent studies have shown that Alternaria and German cockroach allergens can be degraded by endogenous proteases from other insect and fungal extracts when combined for immunotherapy, but data supporting the compatibilities of other high-protease products in comparable mixtures have not been reported. OBJECTIVE: To assess the stabilities and compatibilities of Aspergillus fumigatus and American cockroach allergens after mixing with protease-rich extracts from other insects or fungi at concentrations similar to those recommended for subcutaneous immunotherapy. METHODS: Mixtures containing A fumigatus, American cockroach, and other fungal or insect extracts were evaluated by quantitative (enzyme-linked immunosorbent assays) and qualitative (immunoblotting) methods. Test mixtures and control samples at 10% to 50% glycerin concentrations were analyzed after storage for up to 12 months at 2°C to 8°C. RESULTS: Moderate to high recoveries of Aspergillus extract activities were retained in control samples and extract mixtures under all conditions examined. American cockroach extract controls were partly degraded at 10% to 25% glycerin, and cockroach allergen compatibilities were decreased significantly in mixtures with several fungal extracts at 25% glycerin. Mixing with other insects did not compromise the stability of American cockroach allergens at 25% to 50% glycerin. CONCLUSION: Aspergillus extracts exhibited favorable stabilities after mixing with other high-protease products. American cockroach extract potencies were unstable in less than 50% glycerin, even in the absence of other protease-containing allergens, and were destabilized in mixtures with several fungal extracts. Addition of fungal and insect extracts to separate treatment vials or preparation of fungal-insect mixtures at elevated glycerin concentrations might be necessary to produce compatible patient formulations for allergen immunotherapy injections.

Per a 10 protease activity modulates CD40 expression on dendritic cell surface by nuclear factor-kappaB pathway.

Serine protease activity of Per a 10 from Periplaneta americana modulates dendritic cell (DC) functions by a mechanism(s) that remains unclear. In the present study, Per a 10 protease activity on CD40 expression and downstream signalling was evaluated in DCs. Monocyte-derived DCs from cockroach-allergic patients were treated with proteolytically active/heat-inactivated Per a 10. Stimulation with active Per a 10 demonstrated low CD40 expression on DCs surface (P < 0·05), while enhanced soluble CD40 level in the culture supernatant (P < 0·05) compared to the heat-inactivated Per a 10, suggesting cleavage of CD40. Per a 10 activity reduced the interleukin (IL)-12 and interferon (IFN)-γ secretion by DCs (P < 0·05) compared to heat-inactivated Per a 10, indicating that low CD40 expression is associated with low levels of IL-12 secretion. Active Per a 10 stimulation caused low nuclear factor-kappa B (NF-κB) activation in DCs compared to heat-inactivated Per a 10. Inhibition of the NF-κB pathway suppressed the CD40 expression and IL-12 secretion by DCs, further indicating that NF-κB is required for CD40 up-regulation. CD40 expression activated the tumour necrosis factor (TNF) receptor-associated factor 6 (TRAF6), thereby suggesting its involvement in NF-κB activation. Protease activity of Per a 10 induced p38 mitogen-activated protein kinase (MAPK) activation that showed no significant effect on CD40 expression by DCs. However, inhibiting p38 MAPK or NF-κB suppressed the secretion of IL-12, IFN-γ, IL-6 and TNF-α by DCs. Such DCs further reduced the secretion of IL-4, IL-6, IL-12 and TNF-α by CD4(+) T cells. In conclusion, protease activity of Per a 10 reduces CD40 expression on DCs. CD40 down-regulation leads to low NF-κB levels, thereby modulating DC-mediated immune responses.