Peptide Immunization Research Articles

γ-Core Guided Antibiotic Design Based on Human Enteric Defensin 5.

An increase in the number of infections caused by resistant bacteria worldwide necessitates the development of alternatives to antibiotics. Human defensin (HD) 5 is an innate immune peptide with broad-spectrum antibacterial activity, but its complicated structure makes its preparation difficult. Herein, we truncated the HD5 structure by extracting the highly conserved γ-core motif. A structure-activity study showed that this motif was ineffective in killing bacteria in the absence of specific spatial conformation. Notably, after the introduction of two intramolecular disulfide bonds, its antibacterial activity was markedly improved. Glu and Ser residues were then replaced with Arg to create the derivative RC18, which exhibited stronger potency than HD5, particularly against methicillin-resistant S. aureus (MRSA). Mechanistically, RC18 bound to lipid A and lipoteichoic acid at higher affinities than HD5. Furthermore, RC18 was more efficient than HD5 in penetrating the bacterial membranes. Molecular dynamics simulation revealed that five Arg residues, Arg1, Arg7, Arg9, Arg15, and Arg18, mediated most of the polar interactions of RC18 with the phospholipid head groups during membrane penetration. In vivo experiments indicated that RC18 decreased MRSA colonization and dramatically improved the survival of infected mice, thus demonstrating that RC18 is a promising drug candidate to treat MRSA infections.

An Anti-TAZ Monoclonal Antibody Recognizing Cell Surface Expressed TAZ Protein in Human Tumor Cells.

WWTR1 or TAZ is a transcriptional co-activator protein expressed in cytoplasm which functions as the main downstream effector of the Hippo signaling pathway. This pathway is an evolutionally conserved signal cascade, which plays a pivotal role in organ size control and tumorigenesis. Ectopic expression of TAZ has already been observed in many malignancies, while the ectopic localization of TAZ is reported for the first time. The aim of this study was to produce a specific monoclonal antibody (mAb) against a synthetic peptide derived from WWTR1 protein to be used as a research tool in human carcinomas. A 21-mer synthetic peptide (derived from human TAZ protein) was used for immunization of BALB/c mice after conjugation with Keyhole Limpet Haemocyanin (KLH) using hybridoma technology. The generated mAb reacted with the immunizing peptide employing ELISA assay. The reactivity of the antibody with native TAZ protein was assessed through Western blot, immunocytochemistry, and flow cytometry using different cancer cell lines. The produced mAb could recognize the immunizing peptide in ELISA and Kaff was 0.6×10-9 M. The produced anti-TAZ mAb unlike available commercial anti-TAZ antibody, was capable of specifically recognizing cell surface TAZ in human carcinoma cell lines including MCF-7, Raji, and A431 in Western blot, immunocytochemistry, and flow cytometry assays. As expected, no reactivity was observed using normal Peripheral Blood Mononuclear Cell (PBMC) from healthy donors. Based on the results, TAZ is ectopically expressed on the surface of tumor cell lines which is not the case in normal cells. The generated mAb has a potential to be used as a research tool in studying the expression of TAZ in human carcinomas in different applications.

Establishment of a Sensitive Monoclonal Antibody Against Mouse CCR9 (C9Mab-24) for Flow Cytometry

The CC chemokine receptor 9 (CCR9), also known as CD199, is one of chemokine receptors. The CC chemokine ligand 25 (CCL25) is known to be the only ligand for CCR9. The CCR9-CCL25 interaction plays important roles in chemotaxis of lymphocytes and tumor cell migration. Therefore, CCR9-CCL25 axis is a promising target for tumor therapy and diagnosis. In this study, we established a sensitive and specific monoclonal antibody (mAb) against mouse CCR9 (mCCR9) using N-terminal peptide immunization method. The established anti-mCCR9 mAb, C9Mab-24 (rat immunoglobulin [IgG]2a, kappa), reacted with mCCR9-overexpressed Chinese hamster ovary-K1 (CHO/mCCR9) and mCCR9-endogenously expressed cell line, RL2, through flow cytometry. Kinetic analyses using flow cytometry showed that the dissociation constants (KD) of C9Mab-24 for CHO/mCCR9 and RL2 cell lines were 6.0 × 10-9 M and 4.7 × 10-10 M, respectively. Results indicated that C9Mab-24 is useful for detecting mCCR9 through flow cytometry, thereby providing a possibility for targeting mCCR9-expressing cells in vivo experiments.

Abstract IA015: Immunoprevention of triple negative breast cancer by TOP2A derived peptide vaccination

Abstract Top2A is a key enzyme involved in DNA replication and is a therapeutic target for several cancer types including breast cancer. Overexpression of Top2A has been observed in both human and mouse triple-negative breast cancer (TNBC). The present study evaluated both immunogenicity and antitumor efficacy of a newly formulated multi-peptide vaccine targeting multiple epitopes of the Top2A protein. Top2A-specific MHC II epitopes with optimal binding affinity were identified using a combined scoring system, which predicted their potential to elicit a Th1 immune response. The formulated vaccine contained top three Top2A peptides, which elicited the strongest immunologic response and showed 100% sequence homology between human and mouse. Anti-tumor efficacy of the Top2A vaccine was initially evaluated in a syngeneic TNBC mouse model, in which pre-graft preventive vaccination was associated with significantly decreased tumor growth as compared to the adjuvant controls. The Top2A peptide vaccine exhibited striking efficacy in a genetically engineered TNBC mouse model (C3(1)/Tag), reducing tumor burden by >90% when compared with adjuvant alone. Splenocytes collected from vaccinated animals showed a robust immunologic response to the immunizing peptides. There were no overt toxicities observed with the Top2A vaccination. To explore potential mechanisms underlying the anti-tumor response induced by Top2A vaccine treatment, scTCR-seq of tumors in both control and Top2A vaccine groups revealed new T cell clones as a consequence of Top2A vaccination. Furthermore, in vitro stimulation of these splenocytes by the vaccinated Top2A peptides resulted in the secretion of cytokines indicative of Th1 responses but with minimal secretion of Th2-related cytokines. Our data indicate that the newly developed multi-peptide Top2A vaccine is immunogenic and efficacious in the prevention of TNBC development and progression in vivo. Citation Format: Sang Beom Lee, Jing Pan, Donghai Xiong, Katie Palen, Bryon Johnson, Jeffrey E. Green, Shizuko Sei, Robert H. Shoemaker, Ronald A. Lubet, Yian Wang, Ming You. Immunoprevention of triple negative breast cancer by TOP2A derived peptide vaccination [abstract]. In: Proceedings of the Second Biennial NCI Meeting: Translational Advances in Cancer Prevention Agent Development (TACPAD); 2022 Sep 7-9. Philadelphia (PA): AACR; Can Prev Res 2022;15(12 Suppl_2): Abstract nr IA015.

The secreted immune response peptide 1 functions as a phytocytokine in rice immunity.

Small signalling peptides play important roles in various plant processes, but information regarding their involvement in plant immunity is limited. We previously identified a novel small secreted protein in rice, called immune response peptide 1 (IRP1). Here, we studied the function of IRP1 in rice immunity. Rice plants overexpressing IRP1 enhanced resistance to the virulent rice blast fungus. Application of synthetic IRP1 to rice suspension cells triggered the expression of IRP1 itself and the defence gene phenylalanine ammonia-lyase 1 (PAL1). RNA-seq results revealed that 84% of genes up-regulated by IRP1, including 13 OsWRKY transcription factors, were also induced by a microbe-associated molecular pattern (MAMP), chitin, indicating that IRP1 and chitin share a similar signalling pathway. Co-treatment with chitin and IRP1 elevated the expression level of PAL1 and OsWRKYs in an additive manner. The increased chitin concentration arrested the induction of IRP1 and PAL1 expression by IRP1, but did not affect IRP1-triggered mitogen-activated protein kinases (MAPKs) activation. Collectively, our findings indicate that IRP1 functions as a phytocytokine in rice immunity regulating MAPKs and OsWRKYs that can amplify chitin and other signalling pathways, and provide new insights into how MAMPs and phytocytokines cooperatively regulate rice immunity.

Development of a Novel Anti-Mouse CCR6 Monoclonal Antibody (C6Mab-13) by N-Terminal Peptide Immunization.

The CC chemokine receptor 6 (CCR6) is a G protein-coupled receptor family member that is highly expressed in B lymphocytes, certain subsets of effector and memory T cells, and immature dendritic cells. CCR6 has only one chemokine ligand, CCL20. The CCL20-CCR6 axis has been recognized as a therapeutic target for autoimmune diseases and tumor. This study developed specific monoclonal antibodies (mAbs) against mouse CCR6 (mCCR6) using the peptide immunization method. The established anti-mCCR6 mAb, C6Mab-13 (rat IgG1, kappa), reacted with mCCR6-overexpressed Chinese hamster ovary-K1 (CHO/mCCR6), and mCCR6-endogenously expressed P388 (mouse lymphoid neoplasma) and J774-1 (mouse macrophage-like) cells in flow cytometry. The dissociation constant (KD) of C6Mab-13 for CHO/mCCR6 cells was determined to be 2.8 × 10-9 M, indicating that C6Mab-13 binds to mCCR6 with high affinity. In summary, C6Mab-13 is useful for detecting mCCR6-expressing cells through flow cytometry.

Development of a Sensitive Anti-Human CCR9 Monoclonal Antibody (C9Mab-11) by N-Terminal Peptide Immunization.

The C-C chemokine receptor 9 (CCR9) belongs to the G-protein-coupled receptor superfamily, and is highly expressed on the T cells and intestinal cells. CCR9 regulates various immune responses by binding to the C-C chemokine ligand, CCL25, and is involved in inflammatory diseases and tumors. Therefore, the development of sensitive monoclonal antibodies (mAbs) for CCR9 is necessary for treatment and diagnosis. In this study, we established a specific anti-human CCR9 (hCCR9) mAb; C9Mab-11 (mouse IgG2a, kappa), using the synthetic peptide immunization method. C9Mab-11 reacted with hCCR9-overexpressed Chinese hamster ovary-K1 (CHO/hCCR9) and hCCR9-endogenously expressed MOLT-4 (human T-lymphoblastic leukemia) cells in flow cytometry. The dissociation constant (KD) of C9Mab-11 for CHO/hCCR9 and MOLT-4 cells were determined to be 1.2 × 10-9 M and 4.9 × 10-10 M, respectively, indicating that C9Mab-11 possesses a high affinity for both exogenously and endogenously hCCR9-expressing cells. Furthermore, C9Mab-11 clearly detected hCCR9 protein in CHO/hCCR9 cells using western blot analysis. In summary, C9Mab-11 can be a useful tool for analyzing hCCR9-related biological responses.

Screening of immunopeptides from Zhiciweipi (Corium Erinacei Praeparaum) and their immunity to RAW264.7 macrophages

Zhiciweipi (Corium Erinacei Praeparaum) is one of the traditional Chinese animal medicines used to treat hematochezia and hemorrhoids. At present, there is a lack of the workflow for ingredient identification of Zhiciweipi (Corium Erinacei Praeparaum) and the studies regarding immunocompetent ingredient screening and evaluation. This study aims to screen immunopeptides in Zhiciweipi (Corium Erinacei Praeparaum) and investigate their immunity to RAW264.7 macrophages. In this study, the workflow for protein polypeptide identification and analysis of Zhiciweipi (Corium Erinacei Praeparaum) was established based on LC-MS/MS. According to Gene ontology_Biological process (GO_BP) functional and signal pathway enrichment analysis results, the identified proteins are related with vascular development, wound healing, IL2 STAT5 signal, and so on. A total of 20 immune regulatory genes were identified from the Venn diagram of protein and immune regulatory genes in Zhiciweipi (Corium Erinacei Praeparaum). The PPI network analysis demonstrated that ALB protein ranks the top in term of significance. According to analysis on physical properties and structural and safety prediction of 61 polypeptide fragments related with serum albumin in Zhiciweipi (Corium Erinacei Praeparaum) which are corresponding to ALB protein, RHPYFYAPELL and RHPYFYAPELLYF (RHP-LYF) are the most optimal. The molecular docking showed that RHPYFYAPELL and RHP-LYF can make effective bonding with IL-2 and STAT5. The molecular dynamic experiment reflected that RHP-LYF has the strongest affinity with ALB. In the present study, CCK8 method was applied to test RHP-LYF treatment of RAW264.7 cells in the mice macrophage system at different concentrations. Results showed that such treatment may not inhibit cell growth when the RHP-LYF concentration is lower than 200 ng/mL. The polypeptide fluorescence marking experiment proved that polypeptides can enter into cells and can be detected in the whole cell. Effects of RHP-LYF on IL-2, TNFα, IFNγ and IL10 levels in RAW264.7 cells and mice serum were detected by ELISA kit. Results showed that polypeptide not only can inhibit secretion of inflammatory factors in vivo and in vitro, but also promote secretion of anti-inflammatory factors, and further test effects of RAW264.7 cells on expressions of p-STAT5 and STAT5 through Western Blot. Results showed that polypeptide treatment may inhibit level of p-STAT5 and has dosage-dependent effect. Polypeptides from Zhiciweipi (Corium Erinacei Praeparaum) are safe and have immunization to RAW264.7 macrophage. All of these results can provide a meaningful method to drug quality assessment, immune bioactive peptide screening and evaluation of Zhiciweipi (Corium Erinacei Praeparaum) and other animals rich of peptides.

Redeployment of Placental Gene Programming: Can Invasive Placentation Molecular Switches Complement the Hallmarks of Cancer?

Placentation arose in mammalian evolution some 150-200 M years ago and integrates in a rather ingenious manner a large number of previously evolved multicellular regulatory pathways which include: angiogenesis, inflammatory cytokines and interleukins, HLA expression, immune peptides, immune regulatory receptors (for NK decidual cells, T and B cells, including Tregs, macrophages, antigen-presenting cells ), endothelial cells and fibroblasts, immune checkpoints (including PD-L1), many paracrine or endocrine hormones and growth factors, tissue enzymes, extracellular vesicles, several different mechanisms for epithelial-mesenchymal transitions, several hypoxia adaptations, morphogenetic changes, and others. Speculating from real genomics and epigenetic data from a single clinical case of a pregnant young woman with breast cancer, it has been recently proposed that mammalian cancer cells do not have to invent “de novo” immune escape mechanisms, including so-called “immune editing”, but to redeploy–probably by epigenetic mechanisms–intrinsic or encrypted gene programmes physiologically used by the process of invasive placentation in mammals. Most of these programmes were not evolved specifically for the placenta–but there were probably some exceptions, such as those still poorly understood key pathways related to foeto-maternal tolerance or perhaps trophoblast differentiation and decidual invasion. In my opinion, invasive placentation molecular switches can complement the hallmarks of cancer, by re-using “placental gene programming” to the competitive advantage of cancer cells.

Epitope Mapping of an Anti-Mouse CXCR6 Monoclonal Antibody (Cx6Mab-1) Using the 2 × Alanine Scanning Method.

The CXC chemokine receptor 6 (CXCR6) is a member of the G protein-coupled receptor family that is highly expressed in helper T type 1 cells, cytotoxic T lymphocytes (CTLs), and natural killer cells. CXCR6 plays critical roles in local expansion of effector-like CTLs in tumor microenvironment to potentiate the antitumor response. Therefore, the development of anti-CXCR6 monoclonal antibodies (mAbs) is essential to evaluate the immune microenvironment of tumors. Using N-terminal peptide immunization, we previously developed an anti-mouse CXCR6 (mCXCR6) mAb, Cx6Mab-1 (rat IgG1, kappa) , which is useful for flow cytometry and western blotting. In this study, we determined the critical epitope of Cx6Mab-1 by enzyme-linked immunosorbent assay (ELISA) using the 1 × alanine scanning (1 × Ala-scan) method or the 2 × alanine scanning (2 × Ala-scan) method. Although we first performed ELISA by 1 × Ala-scan using one alanine-substituted peptides of mCXCR6 N-terminal domain (amino acids 1-20), we could not identify the Cx6Mab-1 epitope. We next performed ELISA by 2 × Ala-scan using two alanine (or glycine) residues-substituted peptides of mCXCR6 N-terminal domain, and found that Cx6Mab-1 did not recognize S8A-A9G, A9G-L10A, L10A-Y11A, and G13A-H14A of the mCXCR6 N-terminal peptide. The results indicate that the binding epitope of Cx6Mab-1 includes Ser8, Ala9, Leu10, Tyr11, Gly13, and His14 of mCXCR6. Therefore, we could demonstrate that the 2 × Ala scan method is useful for determining the critical epitope of mAbs.

COPD: Perspectives of Immune Peptide Therapy and Lung Cancer Prevention

Background: Chronic obstructive pulmonary disease (COPD) occurs due to chronic inflammation, which leads to thickening of the airway walls, increased mucus production and, ultimately, permanent changes in lung structure. Meta-analysis indicates an increased risk of lung cancer in patients with COPD, so timely and comprehensive cancer prevention is extremely important. Objective: Determine the mechanisms of interaction between immune peptides and immunocompetent cells, which lead to the elimination of pathogens and prevent the development of metaplasia on the background of chronic inflammation. Methods: Selection and analysis of open access scientific publications. Results: Restoring the ability of secretory cells to synthesize IgA and maintaining this synthesis at the appropriate level can provide the necessary protection of the respiratory system from infection. Strengthening immune surveillance over the mucous membrane promotes not only the elimination of pathogens, but also to the destruction and removal of disabled and infected cells and cells that have undergone metaplasia - this is how the immunity program is implemented to counteract infection and prevent cancer. Conclusion: The use of exogenous anti-infective peptides for the treatment and prevention of exacerbations of COPD in the context of antibiotic resistance, to stimulate airway immune function and to prevent cancer is currently considered a promising area in clinical pulmonology.

Development of an Anti-human CCR2 Monoclonal Antibody (C2Mab-9) by N-Terminal Peptide Immunization.

The CC chemokine receptor type-2 (CCR2) is one of the members of the G protein-coupled receptor superfamily, which are expressed on the cell surface of immune and tumor cells. CCR2 binds to the C-C motif chemokine ligand 2 (CCL2)/monocyte chemoattractant protein-1 (MCP-1), which is produced by various cells, including tumor and immune-related cells. Therefore, the development of sensitive monoclonal antibodies (mAbs) for CCR2 has been desired for treatment and diagnosis. In this study, we established a specific antihuman CCR2 (hCCR2) mAb, C2Mab-9 (mouse IgG1, kappa), using the synthetic peptide immunization method. Flow cytometric and immunocytochemical results showed that C2Mab-9 reacted with hCCR2-expressing U937 (human histiocytic lymphoma) and natural killer cells. Furthermore, C2Mab-9 showed the moderate binding affinity for both cells. Conclusively, C2Mab-9 can be a useful tool for analyzing hCCR2-related biological responses.

Epitope Mapping of an Anti-elephant Podoplanin Monoclonal Antibody (PMab-295) Using Enzyme-Linked Immunosorbent Assay

Podoplanin (PDPN) is a marker of lung type I alveolar cells, kidney podocytes, and lymphatic endothelial cells. The overexpression of PDPN contributes to the malignant progression of tumors. Therefore, the development of anti-PDPN monoclonal antibodies (mAbs) to animals is essential to evaluate the pathogenesis and cellular functions. Using peptide immunization, we previously developed an anti-elephant PDPN (elePDPN) mAb, PMab-295, which is useful for flow cytometry, Western blotting, and immunohistochemistry. In this study, we determined the critical epitope of PMab-295 by enzyme-linked immunosorbent assay (ELISA). We performed ELISA with the alanine-substituted peptides of elePDPN extracellular domain (amino acids 38-51), and found that PMab-295 did not recognize the alanine-substituted peptides of M41A, P44A, and E47A. Furthermore, these peptides could not inhibit the recognition of PMab-295 to elePDPN-expressing cells by flow cytometry and immunohistochemistry. The results indicate that the binding epitope of PMab-295 includes Met41, Pro44, and Glu47 of elePDPN.

Development of a Monoclonal Antibody PMab-295 Against Elephant Podoplanin.

Podoplanin (PDPN) is an essential marker of lung type I alveolar cells, kidney podocytes, and lymphatic endothelial cells. Monoclonal antibodies (mAbs) that can specifically recognize PDPN in immunohistochemistry are important to analyze the development of tissues and the pathogenesis of diseases, including cancers. We have developed anti-PDPN mAbs against many animal species; however, mAbs that can recognize elephant-derived membrane proteins and distinguish the specific cell types in immunohistochemistry are limited. In this study, a novel anti-elephant PDPN (elePDPN) mAb, PMab-295 (IgG1, kappa), was established using the peptide immunization method. PMab-295 recognized both elePDPN-overexpressed Chinese hamster ovary (CHO)-K1 cells and endogenous elePDPN-expressed LACF-NaNaI cells by flow cytometry and western blotting. Kinetic analyses using flow cytometry showed that the KD of PMab-295 for CHO/elePDPN was 1.5 × 10-8 M. Furthermore, PMab-295 detected elePDPN-expressing cells using immunohistochemistry. These results showed the usefulness of PMab-295 to investigate the molecular function of elePDPN and the pathogenesis of diseases.

Identification of the Binding Epitope of an Anti-mouse CCR4 Monoclonal Antibody, C4Mab-1.

C-C chemokine receptor 4 (CCR4) is one of G protein-coupled receptors, and interacts with chemokines, CCL17 and CCL22. CCR4 is expressed on T cells such as helper T type 2 cells, regulatory T cells, and interleukin 17-producing T helper cells. CCR4 is associated with T cells trafficking into the tumor microenvironment, and is associated with tumor progression or metastasis. Therefore, CCR4 may be a potential therapeutic option for T cell malignancies. C4Mab-1 is a novel anti-mouse CCR4 (mCCR4) monoclonal antibody produced by mCCR4 N-terminal peptide immunization. C4Mab-1 is useful for flow cytometric analysis. In this study, we conducted the epitope mapping of C4Mab-1 using enzyme-linked immunosorbent assay (ELISA) and peptide blocking assay. The result of ELISA indicated that Thr7, Asp8, and Gln11 of mCCR4 are the critical amino acids for the C4Mab-1 binding. Furthermore, peptide blocking assay by flow cytometry showed that Thr7, Asp8, and Gln11 of mCCR4 are essential for C4Mab-1 binding to mCCR4-overexpressed Chinese hamster ovary-K1 (CHO/mCCR4) cells, and Val6, Thr9, and Thr10 are involved in the C4Mab-1 binding to CHO/mCCR4 cells. These results indicate that the critical binding epitope of C4Mab-1 includes Thr7, Asp8, and Gln11 of mCCR4.

Targeting cholesteryl ester accumulation in the heart improves cardiac insulin response.

Antibodies against the P3 sequence (Gly1127-Cys1140) of LRP1 (anti-P3 Abs) specifically block cholesteryl ester (CE) accumulation in vascular cells. LRP1 is a key regulator of insulin receptor (InsR) trafficking in different cell types. The link between CE accumulation and the insulin response are largely unknown. Here, the effects of P3 peptide immunization on the alterations induced by a high-fat diet (HFD) in cardiac insulin response were evaluated. Irrelevant (IrP)- or P3 peptide-immunized rabbits were randomized into groups fed either HFD or normal chow. Cardiac lipid content was characterized by thin-layer chromatography, confocal microscopy, and electron microscopy. LRP1, InsR and glucose transporter type 4 (GLUT4) levels were determined in membranes and total lysates from rabbit heart. The interaction between InsR and LRP1 was analyzed by immunoprecipitation and confocal microscopy. Insulin signaling activity and glucose uptake were evaluated in HL-1 cells exposed to rabbit serum from the different groups. HFD reduces cardiac InsR and GLUT4 membrane levels and the interactions between LRP1/InsR. Targeting the P3 sequence on LRP1 through anti-P3 Abs specifically reduces CE accumulation in the heart independently of changes in the circulating lipid profile. This restores InsR and GLUT4 levels in cardiac membranes as well as the LRP1/InsR interactions of HFD-fed rabbits. In addition, anti-P3 Abs restores the insulin signaling cascade and glucose uptake in HL-1 cells exposed to hypercholesterolemic rabbit serum. LRP1-immunotargeting can block CE accumulation within the heart with specificity, selectivity, and efficacy, thereby improving the cardiac insulin response; this has important therapeutic implications for a wide range of cardiac diseases. Fundació MARATÓ TV3: grant 101521-10, Instiuto de Salud Carlos III (ISCIII) and ERDFPI18/01584, Fundación BBVA Ayudas a Equipos de Investigación 2019. SECyT-UNC grants PROYECTOS CONSOLIDAR 2018-2021; FONCyT, Préstamo BID PICT grant 2015-0807 and grant 2017-4497.

Mass Spectrometry-Based Peptide Profiling of Haemolymph from Pterostichus melas Exposed to Pendimethalin Herbicide

Pendimethalin-based herbicides are used worldwide for pre-emergence selective control of annual grasses and weeds in croplands. The endurance of herbicides residues in the environment has an impact on the soil biodiversity and fertility, also affecting non-target species, including terrestrial invertebrates. Carabid beetles are known as natural pest control agents in the soil food web of agroecosystems, and feed on invertebrates and weed seeds. Here, a mass spectrometry untargeted profiling of haemolymph is used to investigate Pterostichus melas metabolic response after to pendimethalin-based herbicide exposure. Mass spectrometric data are examined with statistical approaches, such as principal component analysis, for possible correlation with biological effects. Those signals with high correlation are submitted to tandem mass spectrometry to identify the associated biomarker. The time course exposure showed many interesting findings, including a significant downregulation of related to immune and defense peptides (M-lycotoxin-Ls4a, Peptide hormone 1, Paralytic peptide 2, and Serine protease inhibitor 2). Overall, the observed peptide deregulations concur with the general mechanism of uptake and elimination of toxicants reported for Arthropods.

Optimization of Enzymatic Hydrolysis Process of Immune Active Peptide in Chub Mackerel

Optimization of Enzymatic Hydrolysis Process of Immune Active Peptide in Chub Mackerel

Chicken-Specific Kinome Analysis of Early Host Immune Signaling Pathways in the Cecum of Newly Hatched Chickens Infected With Salmonella enterica Serovar Enteritidis.

Poultry is a major source of human foodborne illness caused by broad host range Salmonella serovars (paratyphoid), and developing cost-effective, pre-harvest interventions to reduce these pathogens would be valuable to the industry and consumer. Host responses to infectious agents are often regulated through phosphorylation. However, proteomic mechanisms of Salmonella acute infection biology and host responses to the bacteria have been limited concentrating predominately on the genomic responses of the host to infection. Our recent development of chicken-specific peptide arrays for kinome analysis of host phosphorylation-based cellular signaling responses provided us with the opportunity to develop a more detailed understanding of the early (4-24 h post-infection) host-pathogen interactions during the initial colonization of the cecum by Salmonella. Using the chicken-specific kinomic immune peptide array, biological pathway analysis showed infection with S. Enteritidis increased signaling related to the innate immune response, relative to the non-infected control ceca. Notably, the acute innate immune signaling pathways were characterized by increased peptide phosphorylation (activation) of the Toll-like receptor and NOD-like receptor signaling pathways, the activation of the chemokine signaling pathway, and the activation of the apoptosis signaling pathways. In addition, Salmonella infection induced a dramatic alteration in the phosphorylation events of the JAK-STAT signaling pathway. Lastly, there is also significant activation of the T cell receptor signaling pathway demonstrating the initiation of the acquired immune response to Salmonella infection. Based on the individual phosphorylation events altered by the early Salmonella infection of the cecum, certain conclusions can be drawn: (1) Salmonella was recognized by both TLR and NOD receptors that initiated the innate immune response; (2) activation of the PPRs induced the production of chemokines CXCLi2 (IL-8) and cytokines IL-2, IL-6, IFN-α, and IFN-γ; (3) Salmonella infection targeted the JAK-STAT pathway as a means of evading the host response by targeting the dephosphorylation of JAK1 and TYK2 and STAT1,2,3,4, and 6; (4) apoptosis appears to be a host defense mechanism where the infection with Salmonella induced both the intrinsic and extrinsic apoptotic pathways; and (5) the T cell receptor signaling pathway activates the AP-1 and NF-κB transcription factor cascades, but not NFAT.

Development of a Monoclonal Antibody to Pig CD69 Reveals Early Activation of T Cells in Pig after PRRSV and ASFV Infection.

The CD69 molecule, as an early activation marker of lymphocytes, is often used to assess the activation of cellular immunity. However, for pigs, an anti-pig CD69 antibody is not yet available for this purpose after infection or vaccination. In this study, a monoclonal antibody (mAb) against pig CD69 was produced by peptide immunization and hybridoma technique. One mAb (5F12) showed good reactivity with pig CD69 that was expressed in transfected-HEK-293T cells and on mitogen-activated porcine peripheral blood mononuclear cells (PBMCs) by indirect immunofluorescence assay and flow cytometry. This mAb did not cross-react with activated lymphocytes from mouse, bovine, and chicken. Epitope mapping showed that the epitope recognized by this mAb was located at amino acid residues 147–161 of pig CD69. By conjugating with fluorochrome, this mAb was used to detect the early activation of lymphocytes in PRRSV- and ASFV-infected pigs by flow cytometry. The results showed that PRRSV infection induced the dominant activation of CD4 T cells in mediastinal lymph nodes and CD8 T cells in the spleen at 14 days post-infection, in terms of CD69 expression. In an experiment on ASFV infection, we found that ASFV infection resulted in the early activation of NK cells, B cells, and distinct T cell subsets with variable magnitude in PBMCs, spleen, and submandibular lymph nodes. Our study revealed an early event of lymphocyte and T cell activation after PRRSV and ASFV infections and provides an important immunological tool for the in-depth analysis of cellular immune response in pigs after infection or vaccination.