Salivary Gland Proteins Research Articles

Improvement of primary Sjögren's syndrome salivary gland function by Xinfeng capsule and its effect on EGR1-STAT3 signaling pathway.

The study was aimed to investigate the effects of Xinfeng capsule (XFC) on tissue morphology, and gland function of the salivary gland (SG) in a primary Sjögren's syndrome (pSS) mouse model. An animal model of pSS was established by inducing SG protein in C57BL/6 mice. SG tissues were collected for tissue sequencing and subsequent experiments to detect the expression of cholinergic receptor muscarinic 3(M3R), early growth response factor 1 (EGR1) and target genes in the SG before and after XFC intervention, with in vitro validation. Downstream targets of the EGR1 gene were predicted and analyzed using data analysis. EGR1 showed high expression and was selected for subsequent experiments. Administration of XFC significantly increased saliva production (P < 0.001) and reduced the extent of lymphatic infiltration observed in SG. Furthermore, the expression of EGR1 was increased in the model group with statistical significance in contrast with the control group but decreased after administration of XFC (P < 0.05). Data analysis predicted the downstream target of EGR1 as signal transducer and activator of transcription 3 (STAT3), which was validated in SG tissues of mice (P < .05). XFC demonstrated a significant improvement in the salivary secretion function of the SG in pSS mice. EGR1 can serve as a biomarker and therapeutic target for pSS.

Explorative study of stimulated saliva proteome in head and neck cancer patients pre- and post-treatment

Objectivesto compare saliva proteome of patients before treatment of head and neck cancer and six months post-treatment with controls. DesignFive dentate patients and five age and gender-matched controls were included. The stimulated salivary secretion rate was determined, and saliva was stored at −80 °C. After thawing, 30 mg of each sample and a reference (aliqouts of all samples) was trypsin digested. The digested peptides were analyzed by mass spectrometry. The relative abundances were transformed to log2 and significant differences determined. Relative abundances of mucins were compared with patient's problems with dry mouth, sticky saliva and swallowing. Data are available via ProteomeXchange with identifier PXD047500. Results966 proteins with ≥2 unique peptides were found. Compared with controls, 30 proteins were found in significantly lower relative abundances and 65 in higher at pre-treatment and 38 proteins in significantly lower relative abundances and 34 proteins in higher post-treatment. Regarding proteins from the salivary glands, a significantly lower relative abundance of Cystatins was detected pre-treatment and significantly lower relative abundances of Cystatin, Cysteine-rich secretory protein 3, Lactoperoxidase, Prolactin-inducible protein and Proline-rich protein 4 post-treatment. No clear relation between relative abundance of mucins and dry mouth, sticky saliva and problems with swallowing was detected. ConclusionDecreases in several salivary gland proteins post cancer treatment might lead to a reduced defense against oral disorders. Knowledge about changes in saliva proteins in connection with oral cancer treatment is important for planning dental care for these patients.

Iguratimod suppresses plasma cell differentiation and ameliorates experimental Sjögren's syndrome in mice by promoting TEC kinase degradation.

Primary Sjögren's syndrome (pSS) is a chronic inflammatory autoimmune disease with an unclear pathogenesis, and there is currently no approved drug for the treatment of this disease. Iguratimod, as a novel clinical anti-rheumatic drug in China and Japan, has shown remarkable efficacy in improving the symptoms of patients with pSS in clinical studies. In this study we investigated the mechanisms underlying the therapeutic effect of iguratimod in the treatment of pSS. Experimental Sjögren's syndrome (ESS) model was established in female mice by immunizing with salivary gland protein. After immunization, ESS mice were orally treated with iguratimod (10, 30, 100 mg·kg-1·d-1) or hydroxychloroquine (50 mg·kg-1·d-1) for 70 days. We showed that iguratimod administration dose-dependently increased saliva secretion, and ameliorated ESS development by predominantly inhibiting B cells activation and plasma cell differentiation. Iguratimod (30 and 100 mg·kg-1·d-1) was more effective than hydroxychloroquine (50 mg·kg-1·d-1). When the potential target of iguratimod was searched, we found that iguratimod bound to TEC kinase and promoted its degradation through the autophagy-lysosome pathway in BAFF-activated B cells, thereby directly inhibiting TEC-regulated B cells function, suggesting that the action mode of iguratimod on TEC was different from that of conventional kinase inhibitors. In addition, we found a crucial role of TEC overexpression in plasma cells of patients with pSS. Together, we demonstrate that iguratimod effectively ameliorates ESS via its unique suppression of TEC function, which will be helpful for its clinical application. Targeting TEC kinase, a new regulatory factor for B cells, may be a promising therapeutic option.

Transcriptomic analysis of Anopheles gambiae from Benin reveals overexpression of salivary and cuticular proteins associated with cross-resistance to pyrethroids and organophosphates

BackgroundInsecticide resistance (IR) is one of the major threats to malaria vector control programs in endemic countries. However, the mechanisms underlying IR are poorly understood. Thus, investigating gene expression patterns related to IR can offer important insights into the molecular basis of IR in mosquitoes. In this study, RNA-Seq was used to characterize gene expression in Anopheles gambiae surviving exposure to pyrethroids (deltamethrin, alphacypermethrin) and an organophosphate (pirimiphos-methyl).ResultsLarvae of An. gambiae s.s. collected from Bassila and Djougou in Benin were reared to adulthood and phenotyped for IR using a modified CDC intensity bottle bioassay. The results showed that mosquitoes from Djougou were more resistant to pyrethroids (5X deltamethrin: 51.7% mortality; 2X alphacypermethrin: 47.4%) than Bassila (1X deltamethrin: 70.7%; 1X alphacypermethrin: 77.7%), while the latter were more resistant to pirimiphos-methyl (1.5X: 48.3% in Bassila and 1X: 21.5% in Djougou). RNA-seq was then conducted on resistant mosquitoes, non-exposed mosquitoes from the same locations and the laboratory-susceptible An. gambiae s.s. Kisumu strain. The results showed overexpression of detoxification genes, including cytochrome P450s (CYP12F2, CYP12F3, CYP4H15, CYP4H17, CYP6Z3, CYP9K1, CYP4G16, and CYP4D17), carboxylesterase genes (COEJHE5E, COE22933) and glutathione S-transferases (GSTE2 and GSTMS3) in all three resistant mosquito groups analyzed. Genes encoding cuticular proteins (CPR130, CPR10, CPR15, CPR16, CPR127, CPAP3-C, CPAP3-B, and CPR76) were also overexpressed in all the resistant groups, indicating their potential role in cross resistance in An. gambiae. Salivary gland protein genes related to ‘salivary cysteine-rich peptide’ and ‘salivary secreted mucin 3’ were also over-expressed and shared across all resistant groups.ConclusionOur results suggest that in addition to metabolic enzymes, cuticular and salivary gland proteins could play an important role in cross-resistance to multiple classes of insecticides in Benin. These genes warrant further investigation to validate their functional role in An. gambiae resistance to insecticides.

Whole transcriptomic analysis reveals overexpression of salivary gland and cuticular proteins genes in insecticide-resistant Anopheles arabiensis from Western Kenya

BackgroundEffective vector control is key to malaria prevention. However, this is now compromised by increased insecticide resistance due to continued reliance on insecticide-based control interventions. In Kenya, we have observed heterogenous resistance to pyrethroids and organophosphates in Anopheles arabiensis which is one of the most widespread malaria vectors in the country. We investigated the gene expression profiles of insecticide resistant An. arabiensis populations from Migori and Siaya counties in Western Kenya using RNA-Sequencing. Centers for Disease Control and Prevention (CDC) bottle assays were conducted using deltamethrin (DELTA), alphacypermethrin (ACYP) and pirimiphos-methyl (PMM) to determine the resistance status in both sites.ResultsMosquitoes from Migori had average mortalities of 91%, 92% and 58% while those from Siaya had 85%, 86%, and 30% when exposed to DELTA, ACYP and PMM, respectively. RNA-Seq analysis was done on pools of mosquitoes which survived exposure (‘resistant’), mosquitoes that were not exposed, and the insecticide-susceptible An. arabiensis Dongola strain. Gene expression profiles of resistant mosquitoes from both Migori and Siaya showed an overexpression mainly of salivary gland proteins belonging to both the short and long form D7 genes, and cuticular proteins (including CPR9, CPR10, CPR15, CPR16). Additionally, the overexpression of detoxification genes including cytochrome P450s (CYP9M1, CYP325H1, CYP4C27, CYP9L1 and CYP307A1), 2 carboxylesterases and a glutathione-S-transferase (GSTE4) were also shared between DELTA, ACYP, and PMM survivors, pointing to potential contribution to cross resistance to both pyrethroid and organophosphate insecticides.ConclusionThis study provides novel insights into the molecular basis of insecticide resistance in An. arabiensis in Western Kenya and suggests that salivary gland proteins and cuticular proteins are associated with resistance to multiple classes of insecticides.

Identification and characterization of calcium binding protein, spermatid-associated 1 (CABS1)# in selected human tissues and fluids.

Calcium binding protein, spermatid associated 1 (CABS1) is a protein most widely studied in spermatogenesis. However, mRNA for CABS1 has been found in numerous tissues, albeit with little information about the protein. Previously, we identified CABS1 mRNA and protein in human salivary glands and provided evidence that in humans CABS1 contains a heptapeptide near its carboxyl terminus that has anti-inflammatory activities. Moreover, levels of an immunoreactive form of CABS1 were elevated in psychological stress. To more fully characterize human CABS1 we developed additional polyclonal and monoclonal antibodies to different sections of the protein and used these antibodies to characterize CABS1 in an overexpression cell lysate, human salivary glands, saliva, serum and testes using western blot, immunohistochemistry and bioinformatics approaches exploiting the Gene Expression Omnibus (GEO) database. CABS1 appears to have multiple molecular weight forms, consistent with its recognition as a structurally disordered protein, a protein with structural plasticity. Interestingly, in human testes, its cellular distribution differs from that in rodents and pigs, and includes Leydig cells, primary spermatogonia, Sertoli cells and developing spermatocytes and spermatids, Geodata suggests that CABS1 is much more widely distributed than previously recognized, including in the urogenital, gastrointestinal and respiratory tracts, as well as in the nervous system, immune system and other tissues. Much remains to be learned about this intriguing protein.

Integrative analysis of transcriptome and proteome in primary Sjögren syndrome

ObjectivePrimary Sjögren's syndrome (pSS) is a intricate autoimmune disease mainly characterized of immune-mediated destruction of exocrine tissues, such as salivary and lacrimal glands, occurring dry mouth and eyes. Although some breakthroughs in understanding pSS have been uncovered, many questions remain about its pathogenesis, especially the internal relations between exocrine glands and secretions. MethodTranscriptomic and proteomic analyses were conducted on salivary tissues and saliva in experimental Sjögren syndrome (ESS). The ESS model was established by immunization with salivary gland protein. The expression of mRNAs and proteins in salivary tissues and saliva were determined by high-throughput sequencing transcriptomic analysis and LC-MS/MS-based proteome, respectively. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were used to recognize dysregulated genes and proteins. The association between RNA and protein abundance was investigated to provides a comprehensive understanding of RNA-protein correlations in the pathogenesis of pSS. ResultsAs a result, we successfully established the ESS model. We recognized 3221 differentially expressed genes (DEGs) and 253 differentially expressed proteins (DEPs). The sample analysis showed that 61 proteins overlapped through the integrative analysis of transcriptomics and proteomics data. The enrichment pathway analysis of DEGs and DEPs in samples showed alterations in renin-angiotensin-system (RAS), lysosome, and apoptosis. Notably, we found that some genes, such as AGT, FN1, Klk1b26, Klk1, Klk1b5, Klk1b3 had a consistent trend in the regulation at the RNA and protein levels and might be potential diagnostic biomarkers of pSS. ConclusionHerein, we found critical processes and potential biomakers that may contribute to pSS pathogenesis by analyzing dysregulated genes and pathways. Additionally, the integrative multi-omics datasets provided additional insight into understanding complicated disease mechanisms.

Hong Kong Guangdong Rheumatology Meeting

Sjögren’s syndrome is a chronic autoimmune disease that is estimated to affect 35 million people worldwide. Hallmarks of the disease are a loss of salivary and lacrimal gland function as well as lymphocytic infiltration, elevated proinflammatory cytokines, and circulating autoantibodies. The effective treatments are limited. However, new therapies targeting specific immune pathways associated with Sjögren’s syndrome are being developed. Gene transfer to the salivary glands has been considered a promising approach to treating the dysfunction. The application of this treatment on salivary gland injuries has been studied for decades, yet its clinical progress is delayed. This chapter provides a coup d’oeil into gene transfer methods and various gene/vector types for salivary glands. AAV2-LAMP3-treated mice developed an SS-like phenotype with progressive salivary hypofunction and autoantibody production. To confirm the role of TLR4 in the induction of BMP6 following stimulation, AAV2-LAMP3–treated mice with established salivary hypofunction were treated i.p. with the TLR4 antagonist TAK242 for 10 days, and the effect of the drug on salivary gland protein expression and function was tested. We observed that treatment with TAK242 significantly decreased BMP6 expression and increased AQP5 expression in the salivary gland tissues. In agreement with this observation, the saliva flow rate of AAV2-LAMP3–treated mice also increased compared with that in vehicle control–treated mice. Gene therapy using recombinant viruses as gene transfer vectors to deliver a water channel (aquaporin 1, AQP1) to restore membrane water permeability in the salivary gland was shown to be safe and resulted in some patients with radiation-induced xerostomia having a sustained increase in saliva flow. A clinical trial using AAV2 to transfer AQP1 to the salivary glands of radiation-induced xerostomia patients is ongoing (NCT02446249) and results from this study may support application of this treatment in Sjögren’s syndrome. Gene therapy could also be used to deliver immunomodulators locally to the salivary gland resulting in a higher local concentration within the gland that could deliver therapeutics levels of recombinant protein, while minimizing the side effects associated with the drugs. Another study investigated whether a neurturin-expressing adenovirus could be used for gene therapy in vivo to protect parasympathetic neurons and prevent gland hypofunction after irradiation. The results suggest that in vivo neurturin gene therapy prior to irradiation protects parasympathetic function and prevents irradiation-induced hypofunction.

The Ixodes ricinus salivary gland proteome during feeding and B. Afzelii infection: New avenues for an anti-tick vaccine.

Borrelia burgdorferi sensu lato, the causative agents of Lyme borreliosis, are transmitted by Ixodes ticks. Tick saliva proteins are instrumental for survival of both the vector and spirochete and have been investigated as targets for vaccine targeting the vector. In Europe, the main vector for Lyme borreliosis is Ixodes ricinus, which predominantly transmits Borrelia afzelii. We here investigated the differential production of I. ricinus tick saliva proteins in response to feeding and B. afzelii infection. Label-free Quantitative Proteomics and Progenesis QI software was used to identify, compare, and select tick salivary gland proteins differentially produced during tick feeding and in response to B. afzelii infection. Tick saliva proteins were selected for validation, recombinantly expressed and used in both mouse and guinea pig vaccination and tick-challenge studies. We identified 870 I. ricinus proteins from which 68 were overrepresented upon 24-hours of feeding and B. afzelii infection. Selected tick proteins were successfully validated by confirming their expression at the RNA and native protein level in independent tick pools. When used in a recombinant vaccine formulation, these tick proteins significantly reduced the post-engorgement weights of I. ricinus nymphs in two experimental animal models. Despite the reduced ability of ticks to feed on vaccinated animals, we observed efficient transmission of B. afzelii to the murine host. Using quantitative proteomics, we identified differential protein production in I. ricinus salivary glands in response to B. afzelii infection and different feeding conditions. These results provide novel insights into the process of I. ricinus feeding and B. afzelii transmission and revealed novel candidates for an anti-tick vaccine.

Effect of chlorpyrifos on the expression and regulation of salivary gland protein of Haemaphysalis longicornis

ABSTRACT The aim of this study is to explore the detoxification and homoeostasis functions of salivary glands, and to look for possible target proteins of new drugs for tick prevention and treatment. Different concentrations of chlorpyrifos to deal with the semi-engorged female Haemaphysalis longicornis, through the DIA quantitative proteomics method to quantitatively determine of all the proteins in the salivary glands, and through a variety of the depth of the bioinformatics methods for protein analysis and function mining, explain the molecular mechanism of detoxification function of salivary glands. Quantitative information from 5 normal groups and 8 different treatment groups showed that a total of 3152 proteins were identified. The expression levels of these proteins changed with the chlorpyrifos different concentration and treatment times. However, after the overall analysis, it was found that after the action of chlorpyrifos, the proteins involved in detoxification, such as glutathione S-transferase and glutamine synthetase, the heat shock proteins involved in stress, and the acid sphingomyelinase involved in maintaining the body’s homoeostasis had corresponding changes. We found and explained the functions of detoxification, stress, and homoeostasis of salivary glands. The regulatory strategies of proteins that played a key role in these functions were explored.

Immune infiltration analysis reveals immune cell signatures in salivary gland tissue of primary Sjögren's syndrome.

Mouse models are the basis for primary Sjögren's syndrome (pSS) research. However, the depth of comparisons between mice and humans in salivary gland (SG) immune cells remains limited. The gene expression profiles of SGs from normal subjects and pSS patients were downloaded from the Gene Expression Comprehensive Database. The proportion of infiltrating immune cell subsets was then assessed by cell type identification by estimating relative subsets of RNA transcripts (CIBERSORT). An experimental Sjögren's syndrome (ESS) mouse model was successfully constructed using SG protein. Based on mouse SG tissue RNA-Seq data, the seq-ImmuCC model was used to quantitatively analyze the compositional ratios of 10 immune cells in pSS patients and mouse model SG tissues. Computed and obtained 31 human data samples using the CIBERSORT deconvolution method. The immune cell infiltration results showed that, compared to normal human SG tissue, the content of gamma delta T cells was significantly different from naive CD4+ T cells and significantly increased, while the plasma cell content decreased. Principal component analysis indicated differences in immune cell infiltration between pSS patients and normal subjects. Meanwhile, for ESS model mouse data analysis, we found that the proportion of macrophages increased, while the proportion of CD4+ T cells, B cells, and monocytes decreased. Furthermore, we found that the proportion of monocytes was decreased, while the proportion of macrophages was increased in the SG tissues of pSS patients and model mice. The infiltration of CD4+ T, CD8+ T, and B cells also showed some differences. We comprehensively analyzed SG immune infiltration in pSS patients and model mice. We demonstrated conserved and nonconserved aspects of the immune system in mice and humans at the level of immune cells to help explain the primary regulation of immune mechanisms during the development of Sjögren's syndrome.

LAMB3 and TACSTD2, both highly expressed in salivary gland mucoepidermoid carcinoma, represent potential diagnostic biomarkers and therapeutic targets

Salivary mucoepidermoid carcinoma (MEC) is the most common malignant tumor in major and minor salivary glands. The CRTC1-MAML2 or CRTC3-MAML2 gene arrangements, which occur in up to 80% of primary salivary glands with MEC, are the major oncogenic drivers of MEC carcinogenesis. However, the molecular consequences of these fusions remain unknown. We sought to identify the key functional genes involved in MEC, then characterize biomarkers with potential therapeutic relevance. We analyzed the gene expression profiles of two primary tumors (fusion-positive HCM-MEC020T and fusion-negative HCM-MEC030T); we compared them with the profiles of the established cell line HCM-MEC010 (derived from CRTC1-MAML2 fusion-positive MEC) and fibroblast cells derived from an HCM-MEC010 patient (non-tumor control cells). Fifty-three genes were identified as commonly expressed genes among the top 500 highly expressed genes in MEC but not in fibroblasts. Among these 53 genes, we focused on LAMB3 and TACSTD2, which are not predicted to be CRTC1/CRTC3-MAML2 target genes. LAMB3 (laminin subunit beta-3) is the basement membrane protein in normal salivary glands. TACSTD2 encodes a membrane protein associated with calcium signaling. To investigate the relationship between gene expression patterns and MEC pathological grade, we performed immunohistochemical staining of 13 MEC samples. LAMB3 and TACSTD2 proteins were expressed in the tumor cells of MEC samples (LAMB3, 12/13; TACSTD2, 13/13) but not in surrounding stromal cells. Although there were no statistically significant differences in LAMB3 or TACSTD2 expression among pathological grades, further studies are needed to elucidate the value of LAMB3 and TACSTD2 in clinical applications.

Olfactory Ecto-mesenchymal Stem Cell-derived Exosomes Ameliorate Murine Sjögren's Syndrome via Suppressing Tfh Cell Response.

To investigate the effect of olfactory ecto-mesenchymal stem cell-derived exosomes (OE-MSC-Exos) on T follicular helper (Tfh) cell response and their implication in treating experimental Sjögrens syndrome (ESS). C57BL/6 mice were immunized with salivary glands (SG) proteins to induce ESS mouse model. OE-MSC-Exos were added to the Tfh cell polarization condition, and the proportion of Tfh cells was detected by FCM. The PD-L1 of OE-MSCs was silenced with small interfering RNA to extract siPD-L1-OE-MSC-Exos. We found that transfer of OE-MSC-Exos markedly attenuated disease progression and reduced Tfh cell response in mice with ESS. In culture, OE-MSC-Exos potently inhibited the differentiation of Tfh cells from naïve T cells. Moreover, OE-MSC-Exos expressed high level of the ligand for the programmed cell death protein 1 (PD-L1), knocking down PD-L1 expression in OE-MSC-Exos significantly decreased their capacity to suppress Tfh cell differentiation in vitro. Consistently, transfer of OE-MSC-Exos with PD-L1 knockdown exhibited profoundly diminished therapeutic effect in ESS mice, accompanied with sustained Tfh cell response and high levels of autoantibody production. Our results suggest that OE-MSC-Exos may exert their therapeutic effect in ameliorating ESS progression via suppressing Tfh cell response in a PD-L1-dependent manner.

Immunofluorescence as a Method to Study Golgi Organization in Larval Salivary Glands of Drosophila melanogaster.

Immunofluorescence is an important research tool in cell biology that reveals structural organization of subcellular organelles by detecting their associated constituents. Here, we describe an antibody staining method to detect Golgi-associated proteins in Drosophila larval salivary glands, using the cis-Golgi protein Lava lamp and the clathrin adaptor AP-1 as a suitable example. Golgi bodies immunostained using this protocol can be visualized using confocal or structured illumination microscopy.

Identification of novel conserved Ixodes vaccine candidates; a promising role for non-secreted salivary gland proteins

Ixodes ricinus and Ixodes scapularis are the main vectors for the causative agents of Lyme borreliosis and a wide range of other pathogens. Repeated tick-bites are known to lead to tick rejection; a phenomenon designated as tick immunity. Tick immunity is mainly directed against tick salivary gland proteins (TSGPs) and has been shown to partially protect against experimental Lyme borreliosis. TSGPs recognized by antibodies from tick immune animals could therefore be interesting candidates for an anti-tick vaccine, which might also block pathogen transmission. To identify conserved Ixodes TSGPs that could serve as a universal anti-tick vaccine in both Europe and the US, a Yeast Surface Display containing salivary gland genes of nymphal I. ricinus expressed at 24, 48 and 72 h into tick feeding was probed with either sera from rabbits repeatedly exposed for 24 h to I. ricinus nymphal ticks and/or sera from rabbits immune to I. scapularis. Thus, we identified thirteen TSGP vaccine candidates, of which ten were secreted. For vaccination studies in rabbits, we selected six secreted TSGPs, five full length and one conserved peptide. None of these proteins hampered tick feeding. In contrast, vaccination of guinea pigs with four non-secreted TSGPs – two from the current and two from a previous human immunoscreening - did significantly reduce tick attachment and feeding. Therefore, non-secreted TSGPs appear to be involved in the development of tick immunity and are interesting candidates for an anti-tick vaccine.

The Extracts of Dendrobium Alleviate Dry Eye Disease in Rat Model by Regulating Aquaporin Expression and MAPKs/NF-κB Signalling.

Dry eye is one of the most common ocular surface diseases caused by tear film instability and ocular surface damage due to an abnormal quality or quantity of tears. Inflammatory factors can initiate relevant transduction signalling pathways and trigger the inflammatory cascade response, resulting in ocular surface inflammation. It has been shown that the active ingredients in Dendrobium, such as polysaccharides, alkaloids and phenols, have anti-inflammatory, anti-tumour and immunity-boosting effects, and Dendrobium officinale extract can improve glandular secretion function, increase salivary secretion and increase the expression level of water channel protein in salivary glands in patients with dry eye syndromes. We investigated the in vitro cytoprotective effect of Dendrobium extracts in sodium chloride induced hyperosmotic conditions in human cornea keratocytes (HKs). Results showed that Dendrobium officinale Kimura et Migo water extract (DOW) and Dendrobium loddigesii Rolfe water extract (DLW) could upregulate the expression of aquaporins (AQP)5 protein, thus exerting a repairing effect by promoting cell migration. Furthermore, oral administration of DOW and DLW enhanced tear production in rats and exerted a protective effect on ocular surface damage. DOW and DLW could upregulate the expression of AQP5 and mucin (muc)5ac proteins in the lacrimal gland and reduce the inflammatory response. DOW and DLW inhibited the activation of the corresponding mitogen-activated protein kinases (MAPK) and NF-KB pathway, thereby playing a role in improving dry eye symptoms. This study provides a new perspective on dry eye treatment, and DOW and DLW may be potential therapeutic agents for dry eye.

Sex-biased proteomic response to tomato spotted wilt virus infection of the salivary glands of Frankliniella occidentalis, the western flower thrips

Successful transmission of tomato spotted wilt virus (TSWV) by Frankliniella occidentalis requires robust infection of the salivary glands (SGs) and virus delivery to plants during salivation. Feeding behavior and transmission efficiency are sexually-dimorphic traits of this thrips vector species. Proteins secreted from male and female SG tissues, and the effect of TSWV infection on the thrips SG proteome are unknown. To begin to discern thrips factors that facilitate virus infection of SGs and transmission by F. occidentalis, we used gel- and label-free quantitative and qualitative proteomics to address two hypotheses: (i) TSWV infection modifies the composition and/or abundance of SG-expressed proteins in adults; and (ii) TSWV has a differential effect on the male and female SG proteome and secreted saliva. Our study revealed a sex-biased SG proteome for F. occidentalis, and TSWV infection modulated the SG proteome in a sex-dependent manner as evident by the number, differential abundance, identities and generalized roles of the proteins. Male SGs exhibited a larger proteomic response to the virus than female SGs. Intracellular processes modulated by TSWV in males indicated perturbation of SG cytoskeletal networks and cell-cell interactions, i.e., basement membrane (BM) and extracellular matrix (ECM) proteins, and subcellular processes consistent with a metabolic slow-down under infection. Several differentially-abundant proteins in infected male SGs play critical roles in viral life cycles of other host-virus pathosystems. In females, TSWV modulated processes consistent with tissue integrity and active translational and transcriptional regulation. A core set of proteins known for their roles in plant cell-wall degradation and protein metabolism were identified in saliva of both sexes, regardless of virus infection status. Saliva proteins secreted by TSWV-infected adults indicated energy generation, consumption and protein turnover, with an enrichment of cytoskeletal/BM/ECM proteins and tricarboxylic acid cycle proteins in male and female saliva, respectively. The nonstructural TSWV protein NSs - a multifunctional viral effector protein reported to target plant defenses against TSWV and thrips - was identified in female saliva. This study represents the first description of the SG proteome and secretome of a thysanopteran and provides many candidate proteins to further unravel the complex interplay between the virus, insect vector, and plant host.

Identification of Aedes aegypti salivary gland proteins interacting with human immune receptor proteins.

Mosquito saliva proteins modulate the human immune and hemostatic systems and control mosquito-borne pathogenic infections. One mechanism through which mosquito proteins may influence host immunity and hemostasis is their interactions with key human receptor proteins that may act as receptors for or coordinate attacks against invading pathogens. Here, using pull-down assays and proteomics-based mass spectrometry, we identified 11 Ae. aegypti salivary gland proteins (SGPs) (e.g., apyrase, Ae. aegypti venom allergen-1 [AaVA-1], neutrophil stimulating protein 1 [NeSt1], and D7 proteins), that interact with one or more of five human receptor proteins (cluster of differentiation 4 [CD4], CD14, CD86, dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin [DC-SIGN], and Toll-like receptor 4 [TLR4]). We focused on CD4- and DC-SIGN-interacting proteins and confirmed that CD4 directly interacts with AaVA-1, D7, and NeST1 recombinant proteins and that AaVA-1 showed a moderate interaction with DC-SIGN using ELISA. Bacteria responsive protein 1 (AgBR1), an Ae. aegypti saliva protein reported to enhance ZIKV infection in humans but that was not identified in our pull-down assay moderately interacts with CD4 in the ELISA assay. Functionally, we showed that AaVA-1 and NeST1 proteins promoted activation of CD4+ T cells. We propose the possible impact of these interactions and effects on mosquito-borne viral infections such as dengue, Zika, and chikungunya viruses. Overall, this study provides key insight into the vector-host (protein-protein) interaction network and suggests roles for these interactions in mosquito-borne viral infections.

Equine keratinocytes in the pathogenesis of insect bite hypersensitivity: Just another brick in the wall?

Equine insect bite hypersensitivity (IBH) is the most common skin disease affecting horses. It is described as an IgE-mediated, Type I hypersensitivity reaction to salivary gland proteins of Culicoides insects. Together with Th2 cells, epithelial barrier cells play an important role in development of Type I hypersensitivities. In order to elucidate the role of equine keratinocytes in development of IBH, we stimulated keratinocytes derived from IBH-affected (IBH-KER) (n = 9) and healthy horses (H-KER) (n = 9) with Culicoides recombinant allergens and extract, allergic cytokine milieu (ACM) and a Toll like receptor ligand 1/2 (TLR-1/2-L) and investigated their transcriptomes. Stimulation of keratinocytes with Culicoides allergens did not induce transcriptional changes. However, when stimulated with allergic cytokine milieu, their gene expression significantly changed. We found upregulation of genes encoding for CCL5, -11, -20, -27 and interleukins such as IL31. We also found a strong downregulation of genes such as SCEL and KRT16 involved in the formation of epithelial barrier. Following stimulation with TLR-1/2-L, keratinocytes significantly upregulated expression of genes affecting Toll like receptor and NOD-receptor signaling pathway as well as NF-kappa B signaling pathway, among others. The transcriptomes of IBH-KER and H-KER were very similar: without stimulations they only differed in one gene (CTSL); following stimulation with allergic cytokine milieu we found only 23 differentially expressed genes (e.g. CXCL10 and 11) and following stimulation with TLR-1/2-L they only differed by expression of seven genes. Our data suggests that keratinocytes contribute to the innate immune response and are able to elicit responses to different stimuli, possibly playing a role in the pathogenesis of IBH.

Leishmania tarentolae as Potential Live Vaccine Co-Expressing Distinct Salivary Gland Proteins Against Experimental Cutaneous Leishmaniasis in BALB/c Mice Model.

Leishmaniasis is a neglected vector-borne disease caused by Leishmania parasites transmitted through the infected sand flies bite. Current treatments are limited, partly due to their high cost and significant adverse effects, and no human vaccine is yet available. Sand flies saliva has been examined for their potential application as an anti-Leishmania vaccine. The salivary protein, PpSP15, was the first protective vaccine candidate against L. major. Additionally, PsSP9 was already introduced as a highly immunogenic salivary protein against L. tropica. Herein, we aimed to develop an effective multivalent live vaccine to control Cutaneous Leishmaniasis induced by two main species, L. major and L. tropica. Hence, the two above-mentioned salivary proteins using T2A linker were incorporated inside the L. tarentolae genome as a safe live vector. Then, the immunogenicity and protective effects of recombinant L. tarentolae co-expressing PpSP15 and PsSP9 were evaluated in pre-treated BALB/c mice with CpG against L. major and L. tropica. Following the cytokine assays, parasite burden and antibody assessment at different time-points at pre and post-infection, promising protective Th1 immunity was obtained in vaccinated mice with recombinant L. tarentolae co-expressing PpSP15 and PsSP9. This is the first study demonstrating the potency of a safe live vaccine based on the combination of different salivary proteins against the infectious challenge with two different species of Leishmania.