The effect of infection with the entomopathogenic fungus Conidiobolus coronatus (Entomopthorales) on eighteen cytokine-like proteins in Galleria mellonella (Lepidoptera) larvae.

Background: In response to the replace mammal research models with insects in preliminary immunological studies, interest has grown in invertebrate defense systems. The immunological response is regulated by cytokines; however, while their role in mammals is well understood, little is known of their function in insects. A suitable target for studies into insect immunology is Galleria mellonella (Lepidoptera), the wax moth: a common host for human fungal and bacterial pathogens. G. mellonella is also a perfect subject for studies into the presence of cytokine-like proteins. Specific objectives: The main goal of present research was detection in insect immunocompetent cells the 18 mammalian cytokines (IL-1α, IL-1ß, IL-2, IL-3, IL-6, IL-7, IL-8, IL-12, IL-13, IL-15, IL-17, IL-19, IFN-γ, TNF-α, TNF-ß, GM-CSF, M-CSF, G-CSF), which play important role in immunological response and indication how their level change after fungal infection. Methodology: The changes of cytokine-like proteins level were detected in hemocytes taken from G. mellonella larvae infected with entomopathogenic fungus, C. coronatus. The presence of cytokine-proteins was confirmed with using fluorescence microscopy (in cultured hemocytes) and flow cytometry (in freshly collected hemolymph). The ELISA test was used to detect changes in concentration of examined cytokine-like proteins. Results: Our findings indicated the presence of eighteen cytokine-like molecules in G. mellonella hemocytes during infection with C. coronatus. The hemocytes taken from infected larvae demonstrated higher fluorescence intensity for six cytokine-like proteins (GM-CSF, M-CSF, IL-3, IL-15, IL-1ß and IL-19) compared to untreated controls. ELISA test indicated significantly higher IL-3 and IL-15. M-CSF, IL-1α and IL-19 concentration in the hemolymph after fungal infection, and significantly lower TNF-ß and G-CSF. Conclusions: Our findings confirm that the selected cytokine-like molecules are present in insect hemocytes and that their concentrations change after fungal infection, which might suggest that they play a role in the anti-fungal immunological response.

Antibodies to Aedes aegypti D7L salivary proteins as a new serological tool to estimate human exposure to Aedes mosquitoes.

Introduction: Aedes spp. are the most prolific mosquito vectors in the world. Found on every continent, they can effectively transmit various arboviruses, including the dengue virus which continues to cause outbreaks worldwide and is spreading into previously non-endemic areas. The lack of widely available dengue vaccines accentuates the importance of targeted vector control strategies to reduce the dengue burden. High-throughput tools to estimate human-mosquito contact and evaluate vector control interventions are lacking. We propose a novel serological tool that allows rapid screening of human cohorts for exposure to potentially infectious mosquitoes. Methods: We tested 563 serum samples from a longitudinal pediatric cohort study previously conducted in Cambodia. Children enrolled in the study were dengue-naive at baseline and were followed biannually for dengue incidence for two years. We used Western blotting and enzyme-linked immunosorbent assays to identify immunogenic Aedes aegypti salivary proteins and measure total anti-Ae. aegypti IgG. Results: We found a correlation (rs=0.86) between IgG responses against AeD7L1 and AeD7L2 recombinant proteins and those to whole salivary gland homogenate. We observed seasonal fluctuations of AeD7L1+2 IgG responses and no cross-reactivity with Culex quinquefasciatus and Anopheles dirus mosquitoes. The baseline median AeD7L1+2 IgG responses for young children were higher in those who developed asymptomatic versus symptomatic dengue. Discussion: The IgG response against AeD7L1+2 recombinant proteins is a highly sensitive and Aedes specific marker of human exposure to Aedes bites that can facilitate standardization of future serosurveys and epidemiological studies by its ability to provide a robust estimation of human-mosquito contact in a high-throughput fashion.

Structure-based epitope prediction and assessment of cross-reactivity of Myrmecia pilosula venom-specific IgE and recombinant Sol g proteins (Solenopsis geminata).

The global distribution of tropical fire ants (Solenopsis geminata) raises concerns about anaphylaxis and serious medical issues in numerous countries. This investigation focused on the cross-reactivity of allergen-specific IgE antibodies between S. geminata and Myrmecia pilosula (Jack Jumper ant) venom proteins due to the potential emergence of cross-reactive allergies in the future. Antibody epitope analysis unveiled one predominant conformational epitope on Sol g 1.1 (PI score of 0.989), followed by Sol g 2.2, Sol g 4.1, and Sol g 3.1. Additionally, Pilosulin 1 showed high allergenic potential (PI score of 0.94), with Pilosulin 5a (PI score of 0.797) leading in B-cell epitopes. The sequence analysis indicated that Sol g 2.2 and Sol g 4.1 pose a high risk of cross-reactivity with Pilosulins 4.1a and 5a. Furthermore, the cross-reactivity of recombinant Sol g proteins with M. pilosula-specific IgE antibodies from 41 patients revealed high cross-reactivity for r-Sol g 3.1 (58.53%) and r-Sol g 4.1 (43.90%), followed by r-Sol g 2.2 (26.82%), and r-Sol g 1.1 (9.75%). Therefore, this study demonstrates cross-reactivity (85.36%) between S. geminata and M. pilosula, highlighting the allergenic risk. Understanding these reactions is vital for the prevention of severe allergic reactions, especially in individuals with pre-existing Jumper Jack ant allergy, informing future management strategies.

Immune Gene Repertoire of Soft Scale Insects (Hemiptera: Coccidae).

Insects possess an effective immune system, which has been extensively characterized in several model species, revealing a plethora of conserved genes involved in recognition, signaling, and responses to pathogens and parasites. However, some taxonomic groups, characterized by peculiar trophic niches, such as plant-sap feeders, which are often important pests of crops and forestry ecosystems, have been largely overlooked regarding their immune gene repertoire. Here we annotated the immune genes of soft scale insects (Hemiptera: Coccidae) for which omics data are publicly available. By using immune genes of aphids and Drosophila to query the genome of Ericerus pela, as well as the transcriptomes of Ceroplastes cirripediformis and Coccus sp., we highlight the lack of peptidoglycan recognition proteins, galectins, thaumatins, and antimicrobial peptides in Coccidae. This work contributes to expanding our knowledge about the evolutionary trajectories of immune genes and offers a list of promising candidates for developing new control strategies based on the suppression of pests' immunity through RNAi technologies.

Wolbachia infection-responsive immune genes suppress Plasmodium falciparum infection in Anopheles stephensi.

Wolbachia, a maternally transmitted symbiotic bacterium of insects, can suppress a variety of human pathogens in mosquitoes, including malaria-causing Plasmodium in the Anopheles vector. However, the mechanistic basis of Wolbachia-mediated Plasmodium suppression in mosquitoes is not well understood. In this study, we compared the midgut and carcass transcriptomes of stably infected Anopheles stephensi with Wolbachia wAlbB to uninfected mosquitoes in order to discover Wolbachia infection-responsive immune genes that may play a role in Wolbachia-mediated anti-Plasmodium activity. We show that wAlbB infection upregulates 10 putative immune genes and downregulates 14 in midguts, while it upregulates 31 putative immune genes and downregulates 15 in carcasses at 24 h after blood-fed feeding, the time at which the Plasmodium ookinetes are traversing the midgut tissue. Only a few of these regulated immune genes were also significantly differentially expressed between Wolbachia-infected and non-infected midguts and carcasses of sugar-fed mosquitoes. Silencing of the Wolbachia infection-responsive immune genes TEP 4, TEP 15, lysozyme C2, CLIPB2, CLIPB4, PGRP-LD and two novel genes (a peritrophin-44-like gene and a macro domain-encoding gene) resulted in a significantly greater permissiveness to P. falciparum infection. These results indicate that Wolbachia infection modulates mosquito immunity and other processes that are likely to decrease Anopheles permissiveness to Plasmodium infection.

Molecular Characterization of the Allergenic Arginine Kinase from the Edible Insect Hermetia illucens (Black Soldier Fly).

SCOPE: Arginine kinase (AK) is an important enzyme for energy metabolism of invertebrate cells by participating in the maintenance of constant levels of ATP. However, AK is also recognized as a major allergen in insects and crustaceans capable of cross-reactivity with sera of patients sensitized to orthologous proteins. In the perspective of introducing insects or their derivatives in the human diet in Western world, it is of primary importance to evaluate possible risks for allergic consumers. METHODS AND RESULTS: This work reports the identification and characterization of AK from Hermetia illucens commonly known as the black soldier fly, a promising insect for human consumption. To evaluate allergenicity of AK from H. illucens, putative linear and conformational epitopes are identified by bioinformatics analyses, and Dot-Blot assays are carried out by using sera of patients allergic to shrimp or mites to validate the cross-reactivity. Gastrointestinal digestion reduces significantly the linear epitopes resulting in lower allergenicity, while the secondary structure is altered at increasing temperatures supporting the possible loss or reduction of conformational epitopes. CONCLUSION: The results indicate that the possible allergenicity of AK should be taken in consideration when dealing with novel foods containing H. illucens or its derivatives.

Human IgG responses to Aedes mosquito salivary peptide Nterm-34kDa and its comparison to Anopheles salivary antigen (gSG6-P1) IgG responses measured among individuals living in Lower Moshi, Tanzania.

BACKGROUND: The level of human exposure to arbovirus vectors, the Aedes mosquitoes, is mainly assessed by entomological methods which are labour intensive, difficult to sustain at a large scale and are affected if transmission and exposure levels are low. Alternatively, serological biomarkers which detect levels of human exposure to mosquito bites may complement the existing epidemiologic tools as they seem cost-effective, simple, rapid, and sensitive. This study explored human IgG responses to an Aedes mosquito salivary gland peptide Nterm-34kDa in Lower Moshi, a highland area with evidence of circulating arboviruses and compared the Aedes IgG responses to Anopheles mosquitoes' salivary antigen (GSG6-P1) IgG responses. METHODS: Three cross-sectional surveys were conducted in 2019: during the first dry season in March, at the end of the rainy season in June and during the second dry season in September in five villages located in Lower Moshi. Blood samples were collected from enrolled participants above six months of age (age span: 7 months to 94 years) and analysed for the presence of anti-Nterm-34kDa IgG antibodies. Possible associations between Nterm-34kDa seroprevalence and participants' characteristics were determined. Levels of IgG responses and seroprevalence were correlated and compared to the already measured IgG responses and seroprevalence of Anopheles mosquitoes' salivary antigen, GSG6-P1. RESULTS: During the first dry season, Nterm-34kDa seroprevalence was 34.1% and significantly increased at the end of the rainy season to 45.3% (Chi square (χ2) = 6.42 p = 0.011). During the second dry season, the seroprevalence significantly declined to 26.5% (χ2 = 15.12 p<0.001). During the rainy season, seroprevalence was significantly higher among residents of Oria village (adjusted odds ratio (AOR) = 2.86; 95% CI = 1.0-7.8; p = 0.041) compared to Newland. Moreover, during the rainy season, the risk of exposure was significantly lower among individuals aged between 16 and 30 years (AOR = 0.25; 95% CI = 0.1 = 0.9; p = 0.036) compared to individuals aged between 0 and 5 years. There was weak to moderate negative correlation between N-term 34kDa IgG and gSG6-P1 antigens. N-term 34kDa seroprevalence were higher compared to gSG6-P1 seroprevalence. CONCLUSION: The findings of this study support that IgG antibody responses towards the Aedes mosquito salivary peptide Nterm-34kDa are detectable among individuals living in lower Moshi and vary with season and geographical area. More individuals are exposed to Aedes mosquito bites than Anopheles mosquito and those exposed to Aedes bites are not necessarily exposed to Anopheles mosquitoes.

Allergenic characterization of Bomb m 4, a 30-kDa Bombyx mori lipoprotein 6 from silkworm pupa.

BACKGROUND: Silkworm pupa (SWP) food anaphylaxis has been described frequently in Asian countries. However, false-positive reactions by skin pricks and serum IgE (sIgE) tests to the extract complicate diagnosis, requiring identification of clinically relevant major allergens. OBJECTIVES: In this study, we characterized a novel SWP allergen, Bomb m 4, a 30-kDa lipoprotein, and evaluated its diagnostic sensitivity. METHODS: Bomb m 4 was identified by a proteomic analysis. This recombinant (r)Bomb m 4 was overexpressed in Escherichia coli, and the IgE reactivity by ELISA was compared with other reported allergenic proteins: Bomb m 1 (arginine kinase), 27-kDa glycoprotein, Bomb m 3 (tropomyosin) using the serum samples from 17 SWP allergic patients and 11 asymptomatic sensitized subjects. RESULTS: rBomb m 4-specific IgE was recognized by all 17 SWP allergic patients. The 27-kDa glycoprotein and Bomb m 1 sIgE were found in 35.3% and 0%, respectively, in the SWP allergic patients. ELISA sIgE reactivity increased significantly, when 4 M urea was added in serum samples. However, only 16% inhibition of sIgE reactivity to the whole SWP extract was exhibited by rBomb m 4, whereas more than 93% of self-inhibition of rBomb m 4 sIgE was obtained, possibly due to the low abundance of Bomb m 4 in the extract. Three linear epitopes (81-95, 191-205 and 224-238 residues) of rBomb m 4 were identified. These epitopes are shown to be released by pepsin digestion. Receiver operator characteristic (ROC) analysis showed the highest diagnostic value of Bomb m 4 followed by Bomb m 1, 27-kDa glycoprotein and Bomb m 3. CONCLUSION: Bomb m 4 is the major allergen of SWP allergic patients. It has cryptic epitopes which are exposed to IgE antibodies with digestive enzymes. This recombinant Bomb m 4 allergen permits exact diagnosis of SWP allergy.

Identification of Per a 13 as a novel allergen in American cockroach.

BACKGROUND: Cockroaches are an important source of indoor allergens. Environmental exposure to cockroach allergens is closely associated with the development of immunoglobulin E (IgE)-mediated allergic diseases. However, the allergenic components in the American cockroaches are not fully studied yet. In order to develop novel diagnostic and therapeutic strategies for cockroach allergy, it is necessary to comprehensively investigate this undescribed allergen in the American cockroach. METHODS: The full-length cDNA of the potential allergen was isolated from the cDNA library of the American cockroach by PCR cloning. Both the recombinant and natural protein molecules were purified and characterized. The allergenicity was further analyzed by enzyme linked immunosorbent assay, immunoblot, and basophil activation test using sera from cockroach allergic patients. RESULTS: A novel allergen belonging to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was firstly identified in the American cockroach and named as Per a 13. The cDNA of this allergen is 1255 base pairs in length and contains an open reading frame of 999 base pairs, encoding 332 amino acids. The purified Per a 13 was fully characterized and assessed to react with IgEs from 49.3 % of cockroach allergic patients, and patients with allergic rhinitis were more sensitized to it. Moreover, the allergenicity was further confirmed by immunoblot and basophil activation test. CONCLUSIONS: We firstly identified GAPDH (Per a 13) in the American cockroach, which is a novel type of inhalant allergen derived from animal species. These findings could be useful in developing novel diagnostic and therapeutic strategies for cockroach allergy.

A Mosquito AgTRIO Monoclonal Antibody Reduces Early Plasmodium Infection of Mice.

Malaria begins when an infected mosquito injects saliva containing Plasmodium sporozoites into the skin of a vertebrate host. Passive immunization of mice with antiserum against the Anopheles gambiae mosquito saliva protein TRIO (AgTRIO) offers significant protection against Plasmodium infection of mice. Furthermore, passive transfer of both AgTRIO antiserum and an anti-circumsporozoite protein monoclonal antibody provides synergistic protection. In this study, we generated monoclonal antibodies against AgTRIO to delineate the regions of AgTRIO associated with protective immunity. Monoclonal antibody 13F-1 markedly reduced Plasmodium infection in mice and recognized a region (VDDLMAKFN) in the carboxyl terminus of AgTRIO. 13F-1 is an IgG2a isotype monoclonal antibody, and the Fc region is required for protection. These data will aid in the generation of future malaria vaccines that may include both pathogen and vector antigens.

Immunomodulation by Mosquito Salivary Protein AgSAP Contributes to Early Host Infection by Plasmodium.

Malaria is caused when Plasmodium sporozoites are injected along with saliva by an anopheline mosquito into the dermis of a vertebrate host. Arthropod saliva has pleiotropic effects that can influence local host responses, pathogen transmission, and exacerbation of the disease. A mass spectrometry screen identified mosquito salivary proteins that are associated with Plasmodium sporozoites during saliva secretions. In this study, we demonstrate that one of these salivary antigens, Anopheles gambiae sporozoite-associated protein (AgSAP), interacts directly with Plasmodium falciparum and Plasmodium berghei sporozoites. AgSAP binds to heparan sulfate and inhibits local inflammatory responses in the skin. The silencing of AgSAP in mosquitoes reduces their ability to effectively transmit sporozoites to mice. Moreover, immunization with AgSAP decreases the Plasmodium burden in mice that are bitten by Plasmodium-infected mosquitoes. These data suggest that AgSAP facilitates early Plasmodium infection in the vertebrate host and serves as a target for the prevention of malaria. IMPORTANCE Malaria is a vector-borne disease caused by Plasmodium sporozoites. When an anopheline mosquito bites its host, it releases Plasmodium sporozoites as well as saliva components. Mosquito proteins have the potential to serve as antigens to prevent or influence malaria without directly targeting the pathogen. This may help set a new paradigm for vaccine development. In this study, we have elucidated the role of a novel salivary antigen, named Anopheles gambiae sporozoite-associated protein (AgSAP). The results presented here show that AgSAP interacts with Plasmodium falciparum and Plasmodium berghei sporozoites and modulates local inflammatory responses in the skin. Furthermore, our results show that AgSAP is a novel mosquito salivary antigen that influences the early stages of Plasmodium infection in the vertebrate host. Individuals living in countries where malaria is endemic generate antibodies against AgSAP, which indicates that AgSAP can serve as a biomarker for disease prevalence and epidemiological analysis.

Anopheles salivary antigens as serological biomarkers of vector exposure and malaria transmission: A systematic review with multilevel modelling.

Background: Entomological surveillance for malaria is inherently resource-intensive and produces crude population-level measures of vector exposure which are insensitive in low-transmission settings. Antibodies against Anopheles salivary proteins measured at the individual level may serve as proxy biomarkers for vector exposure and malaria transmission, but their relationship is yet to be quantified. Methods: A systematic review of studies measuring antibodies against Anopheles salivary antigens (PROSPERO: CRD42020185449). Multilevel modelling (to account for multiple study-specific observations [level 1], nested within study [level 2], and study nested within country [level 3]) estimated associations between seroprevalence with Anopheles human biting rate (HBR) and malaria transmission measures. Results: From 3981 studies identified in literature searches, 42 studies across 16 countries were included contributing 393 study-specific observations of anti-Anopheles salivary antibodies determined in 42,764 samples. A positive association between HBR (log transformed) and seroprevalence was found; overall a twofold (100% relative) increase in HBR was associated with a 23% increase in odds of seropositivity (OR: 1.23, 95% CI: 1.10-1.37; p<0.001). The association between HBR and Anopheles salivary antibodies was strongest with concordant, rather than discordant, Anopheles species. Seroprevalence was also significantly positively associated with established epidemiological measures of malaria transmission: entomological inoculation rate, Plasmodium spp. prevalence, and malarial endemicity class. Conclusions: Anopheles salivary antibody biomarkers can serve as a proxy measure for HBR and malaria transmission, and could monitor malaria receptivity of a population to sustain malaria transmission. Validation of Anopheles species-specific biomarkers is important given the global heterogeneity in the distribution of Anopheles species. Salivary biomarkers have the potential to transform surveillance by replacing impractical, inaccurate entomological investigations, especially in areas progressing towards malaria elimination. Funding: Australian National Health and Medical Research Council, Wellcome Trust.

Transcriptome of the Maize Leafhopper (Dalbulus maidis) and Its Transcriptional Response to Maize Rayado Fino Virus (MRFV), Which It Transmits in a Persistent, Propagative Manner.

The corn leafhopper (Dalbulus maidis) is an important vector of maize rayado fino virus (MRFV), a positive-strand RNA (+ssRNA) marafivirus which it transmits in a persistent propagative manner. The interaction of D. maidis with MRFV, including infection of the insect and subsequent transmission to new plants, is not well understood at the molecular level. To examine the leafhopper-virus interaction, a D. maidis transcriptome was assembled and differences in transcript abundance between virus-exposed and naive D. maidis were examined at two time points (4 h and 7 days) post exposure to MRFV. The D. maidis transcriptome contained 56,116 transcripts generated from 1,727,369,026 100-nt paired-end reads from whole adult insects. The transcriptome of D. maidis shared highest identity and most orthologs with the leafhopper Graminella nigrifrons (65% of transcripts had matches with E values of <10-5) versus planthoppers Sogatella furcifera (with 23% of transcript matches below the E value cutoff) and Peregrinus maidis (with 21% transcript matches below the E value cutoff), as expected based on taxonomy. D. maidis expressed genes in the Toll, Imd, and Jak/Stat insect immune signaling pathways, RNA interference (RNAi) pathway genes, prophenoloxidase-activating system pathways, and immune recognition protein-encoding genes such as peptidoglycan recognition proteins (PGRPs), antimicrobial peptides, and other effectors. Statistical analysis (performed by R package DESeq2) identified 72 transcripts at 4 h and 67 at 7 days that were significantly responsive to MRFV exposure. Genes expected to be favorable for virus propagation, such as protein synthesis-related genes and genes encoding superoxide dismutase, were significantly upregulated after MRFV exposure. IMPORTANCE The transcriptome of the corn leafhopper, D. maidis, revealed conserved biochemical pathways for immunity and discovered transcripts responsive to MRFV-infected plants at two time points, providing a basis for functional identification of genes that either limit or promote the virus-vector interaction. Compared to other hopper species and the propagative plant viruses they transmit, D. maidis shared 15 responsive transcripts with S. furcifera (to southern rice black-streaked dwarf virus [SRBSDV]), one with G. nigrifrons (to maize fine streak virus [MFSV]), and one with P. maidis (to maize mosaic virus [MMV]), but no virus-responsive transcripts identified were shared among all four hopper vector species.

Use of anti-gSG6-P1 IgG as a serological biomarker to assess temporal exposure to Anopheles' mosquito bites in Lower Moshi.

BACKGROUND: Malaria prevalence in the highlands of Northern Tanzania is currently below 1% making this an elimination prone setting. As climate changes may facilitate increasing distribution of Anopheles mosquitoes in such settings, there is a need to monitor changes in risks of exposure to ensure that established control tools meet the required needs. This study explored the use of human antibodies against gambiae salivary gland protein 6 peptide 1 (gSG6-P1) as a biomarker of Anopheles exposure and assessed temporal exposure to mosquito bites in populations living in Lower Moshi, Northern Tanzania. METHODS: Three cross-sectional surveys were conducted in 2019: during the dry season in March, at the end of the rainy season in June and during the dry season in September. Blood samples were collected from enrolled participants and analysed for the presence of anti-gSG6-P1 IgG. Mosquitoes were sampled from 10% of the participants' households, quantified and identified to species level. Possible associations between gSG6-P1 seroprevalence and participants' characteristics were determined. RESULTS: The total number of Anopheles mosquitoes collected was highest during the rainy season (n = 1364) when compared to the two dry seasons (n = 360 and n = 1075, respectively). The gSG6-P1 seroprevalence increased from 18.8% during the dry season to 25.0% during the rainy season (χ2 = 2.66; p = 0.103) followed by a significant decline to 11.0% during the next dry season (χ2 = 12.56; p = 0.001). The largest number of mosquitoes were collected in one village (Oria), but the seroprevalence was significantly lower among the residents as compared to the rest of the villages (p = 0.039), explained by Oria having the highest number of participants owning and using bed nets. Both individual and household gSG6-P1 IgG levels had no correlation with numbers of Anopheles mosquitoes collected. CONCLUSION: Anti-gSG6-P1 IgG is a potential tool in detecting and distinguishing temporal and spatial variations in exposure to Anopheles mosquito bites in settings of extremely low malaria transmission where entomological tools may be obsolete. However studies with larger sample size and extensive mosquito sampling are warranted to further explore the association between this serological marker and abundance of Anopheles mosquito.

Identification and the immunological role of two Nimrod family genes in the silkworm, Bombyx mori.

In animals, immune signaling pathways and effector molecules participate in attenuating microbial infection. Recent work has shown that the Nimrod family proteins can directly bind to bacteria, and this binding leads to bacterial phagocytosis. Although the Nimrod gene family has been reported in many non-drosophilids, their functions remain unexplored in most insect species. Here, we report two members (Nimrod-B and Draper) of the Nimrod gene family from Bombyx mori and analyzed their role in immunity. The two genes were ubiquitously expressed in the tested tissues; but, they transcribed preferentially in immune tissues. The developmental profiles showed that BmNimrod-B and BmDraper transcription levels were highest in the pupal stages. Challenge with microbial pathogens induced the transcription levels of all two genes at different time points. Knockdown of BmDraper decreased the bacterial clearance and increased their replication relative to the control group, whereas, BmNimrod-B suppression had a non-significant effect on them. Furthermore, the mortality rate was increased after BmDraper silencing. The knockdown of these genes did not significantly affect the production of antimicrobial peptides following E. coli infection. Taken together, the Nimrod family genes play a crucial role in host defense by positively regulating the antibacterial immune response in silkworm B. mori.

Identification of B cell epitopes of Per a 5 allergen using bioinformatic approach.

BACKGROUND: Immune epitopes of allergens are pivotal for development of novel diagnostic and therapeutic modalities. Present study aims to identify antigenic determinants of Per a 5, a clinically relevant cross reactive cockroach allergen. METHODS: The three dimensional structure of Per a 5 was modelled using Modeller 9v11 software. A combination of sequence and structure based computational tools were employed for predicting B cell epitopes. Epitopes were synthesized and immunoreactivity was assessed by ELISA using cockroach hypersensitive patient's sera. Cross-reactivity potential of predicted epitopes was assessed with SDAP and ConSurf and validated by IgE ELISA with fungal and mite hypersensitive patient's sera. RESULTS: Per a 5 structure exhibited good quality factor in ERRAT and high stereochemical stability. In silico analysis revealed six B cell epitopes (BC-P1 to P6). BC-P3 demonstrated significant IgE binding followed by BC-P2 and BC-P1 with cockroach hypersensitive patient's sera. Per a 5 epitopes demonstrate considerable similarity with broad spectrum of allergens from fungal, mites, helminths, fruits and nuts. Analysis of PD values indicate BC-P4 to be well conserved among dust mite and helminth GSTs (8.89, 10.63 and 10.69 with D. pteronyssinus, W. bancrofti and F. hepatica respectively). ConSurf analysis of Per a 5 revealed specific enrichment of evolutionarily similar amino acid residues in BC-P2 (with fungal and mite GSTs) and BC-P4 (with mite and helminth GSTs). Further, IgE binding analysis of epitopes demonstrate BC-P2, BC-P3 and BC-P5 as high IgE binders in fungal hypersensitive sera while BC-P1, BC-P2, BC-P4 and BC-P5 demonstrated significant IgE binding with mite hypersensitive sera. CONCLUSIONS: Among the predicted epitopes, BC-P3 demonstrates maximal IgE binding ability. Computational analysis suggests strong evolutionary conservation and cross reactive potential of BC-P4 with allergens in dust mite and helminths. ELISA highlights predictive potential of analysing evolutionarily conserved residues for uncovering potentially cross reactive antigenic determinants. GENERAL SIGNIFICANCE: Immune epitopes of Per a 5 were identified for aiding molecular diagnosis and potential cross reactivity.

Antagonism of Protease Activated Receptor-2 by GB88 Reduces Inflammation Triggered by Protease Allergen Tyr-p3.

The occurrence of allergic diseases induced by aeroallergens has increased in the past decades. Among inhalant allergens, mites remain the important causal agent of allergic diseases. Storage mites- Tyrophagus putrescentiae are found in stored products or domestic environments. Major allergen Tyr-p3 plays a significant role in triggering IgE-mediated hypersensitivity. However, its effects on pulmonary inflammation, internalization, and activation in human epithelium remain elusive. Protease-activated receptors (PARs) are activated upon cleavage by proteases. A549 cells were used as an epithelial model to examine the PAR activation by Tyr-p3 and therapeutic potential of PAR-2 antagonist (GB88) in allergic responses. Enzymatic properties and allergen localization of Tyr-p3 were performed. The release of inflammatory mediators, phosphorylation of mitogen-activated protein kinase (MAPK), and cell junction disruptions were evaluated after Tyr-p3 challenge. Enzymatic properties determined by substrate digestion and protease inhibitors indicated that Tyr-p3 processes a trypsin-like serine protease activity. The PAR-2 mRNA levels were significantly increased by nTyr-p3 but inhibited by protease inhibitors or GB88. Protease allergen of nTyr-p3 significantly increased the levels of pro-inflammatory cytokines (IL-6 and TNF-α), chemokine (IL-8), and IL-1ß in epithelial cells. nTyr-p3 markedly increased phosphorylation of extracellular signal-regulated kinase (ERK)1/2 and MAP kinase. When cells were pretreated with GB88 then added nTyr-p3, the phosphorylated ERK1/2 did not inhibit by GB88. GB88 increased ERK1/2 phosphorylation in human epithelium cells. GB88 is able to block PAR-2-mediated calcium signaling which inhibits the nTyr-p3-induced Ca2+ release. Among the pharmacologic inhibitors, the most effective inhibitor of the nTyr-p3 in the induction of IL-8 or IL-1ß levels was GB88 followed by SBTI, MAPK/ERK, ERK, and p38 inhibitors. Levels of inflammatory mediators, including GM-CSF, VEGF, COX-2, TSLP, and IL-33 were reduced by treatment of GB88 or SBTI. Further, GB88 treatment down-regulated the nTyr-p3-induced PAR-2 expression in allergic patients with asthma or rhinitis. Tight junction and adherens junction were disrupted in epithelial cells by nTyr-p3 exposure; however, this effect was avoided by GB88. Immunostaining with frozen sections of the mite body showed the presence of Tyr-p3 throughout the intestinal digestive system, especially in the hindgut around the excretion site. In conclusion, our findings suggest that Tyr-p3 from domestic mites leads to disruption of the airway epithelial barrier after inhalation. Proteolytic activity of Tyr-p3 causes the PAR-2 mRNA expression, thus leading to the release of numerous inflammatory mediators. Antagonism of PAR2 activity suggests GB88 as the therapeutic potential for anti-inflammation medicine, especially in allergy development triggered by protease allergens.

Characterization of New Allergens from the Venom of the European Paper Wasp Polistes dominula.

Discriminating Polistes dominula and Vespula spp. venom allergy is of growing importance worldwide, as systemic reactions to either species' sting can lead to severe outcomes. Administering the correct allergen-specific immunotherapy is therefore a prerequisite to ensure the safety and health of venom-allergic patients. Component-resolved diagnostics of Hymenoptera venom allergy might be improved by adding additional allergens to the diagnostic allergen panel. Therefore, three potential new allergens from P. dominula venom-immune responsive protein 30 (IRP30), vascular endothelial growth factor C (VEGF C) and phospholipase A2 (PLA2)-were cloned, recombinantly produced and biochemically characterized. Sera sIgE titers of Hymenoptera venom-allergic patients were measured in vitro to assess the allergenicity and potential cross-reactivity of the venom proteins. IRP30 and VEGF C were classified as minor allergens, as sensitization rates lay around 20-40%. About 50% of P. dominula venom-allergic patients had measurable sIgE titers directed against PLA2 from P. dominula venom. Interestingly, PLA2 was unable to activate basophils of allergic patients, questioning its role in the context of clinically relevant sensitization. Although the obtained results hint to a questionable benefit of the characterized P. dominula venom proteins for improved diagnosis of venom-allergic patients, they can contribute to a deeper understanding of the molecular mechanisms of Hymenoptera venoms and to the identification of factors that determine the allergenic potential of proteins.

The identification of nuclear factor Akirin with immune defense role in silkworm, Bombyx mori.

Akirins, highly conserved nuclear factors, regulate diverse physiological processes such as innate immunity. The biological functions of Akirins have extensively been studied in vertebrates and many invertebrates; however, there is no report so far on lepidopteran insects. In the present study, we identified and characterized a novel Akirin from the silkworm, Bombyx mori (designated as BmAkirin), and explored its potential roles in innate immunity. The expression analysis revealed the unequal mRNA levels of BmAkirin in all the tested tissues; however, the gene's transcription level was highest in testis, followed by ovaries and hemocytes. It also had significant expression levels at the early stages of embryonic development. Expression of BmAkirin in fat bodies and hemocytes exhibited an increase in various degrees when challenged with virus, fungus, Gram-negative bacteria, and Gram-positive bacteria. Immunofluorescence analysis showed BmAkirin protein was prominently localized in the nucleus. Knockdown of BmAkirin strongly reduced the expression of AMPs and decreased the survival ability of larva upon immune stimulation. Moreover, the bacterial clearance ability of larvae was also decreased following the depletion of BmAkirin. Collectively, our results demonstrate that BmAkirin plays an indispensable role in the innate immunity of Bombyx mori (B. mori) by positively modulating AMPs expression in vivo.

A Review of Honeybee Venom Allergens and Allergenicity.

Honeybee venom is a source of proteins with allergenic properties which can result in in various symptoms, ranging from local reactions through to systematic life-threatening anaphylaxis, or even death. According to the World Allergy Organization (WAO), honeybee venom allergy is one of the most common causes of anaphylaxis. Among the proteins present in honeybee venom, 12 protein fractions were registered by the World Health Organization's Allergen Nomenclature Sub-Committee (WHO/IUIS) as allergenic. Most of them are highly immunogenic glycoproteins that cross-react with IgE and, as a consequence, may give false positive results in allergy diagnosis. Allergenic fractions are different in terms of molecular weight and biological activity. Eight of these allergenic fractions have also been identified in honey. This explains frequent adverse reactions after consuming honey in people allergic to venom and sheds new light on the causes of allergic symptoms in some individuals after honey consumption. At the same time, it also indicates the possibility of using honey as a natural source of allergen in specific immunotherapy.