Research is revealing the complex coordination between cell signaling systems as they adapt to genetic and epigenetic changes. Tools to uncover these highly complex functional linkages will play an important role in advancing more efficacious disease treatments. Current tumor cell signal transduction research is identifying coordination between receptor types, receptor families, and transduction pathways to maintain tumor cell viability despite challenging tumor microenvironment conditions.In this report, coactivated abnormal levels of signaling activity for c-Met and HER family receptors in live tumor cells were measured by a new clinical test to identify a subpopulation of breast cancer patients that could be responsive to combined targeted therapies. The CELsignia Multi-Pathway Signaling Function (CELsignia) Test uses an impedance biosensor to quantify an individual patient’s ex vivo live tumor cell signaling response in real-time to specific HER family and c-Met co-stimulation and targeted therapies.The test identified breast tumors with hyperactive HER1, HER2, HER3/4, and c-Met coordinated signaling that express otherwise normal amounts of these receptors. The supporting data of the pre-clinical verification of this test included analyses of 79 breast cancer patients’ cell response to HER and c-Met agonists. The signaling results were confirmed using clinically approved matching targeted drugs, and combinations of targeted drugs in addition to correlative mouse xenograft tumor response to HER and c-Met targeted therapies.The results of this study demonstrated the potential benefit of a functional test for identifying a subpopulation of breast cancer patients with coordinated abnormal HER and c-Met signaling for a clinical trial testing combination targeted therapy.Video AbstractThe online version contains supplementary material available at 10.1186/s12964-021-00798-9. The concept of coordinated c-Met (or hepatocyte growth factor receptor) and human epidermal growth factor receptor (HER) tyrosine kinase family signaling in cancer progression is well-established , 2. InteHER2 gene (ERBB2) amplification and/or HER2 protein overexpression is detected in approximately 15–20% of breast cancers and is associated with more aggressive disease, progression, metastasis, and poor prognosis –16. AgenAn individual patient’s tumor cells adaptation to genomic or proteomic aberrations and how a patient may respond to further challenges by individual or combinations of targeted therapies may be more complex than previously suggested –22. The We previously reported the development of a novel assay to identify patients with abnormal HER2 signaling using an impedance biosensor assay measured from ex vivo cultured patient tumor cells , 24. To The test for HER2 and c-Met signaling was performed in a multiplex format known as the CELsignia Multi-Pathway Signaling Function (CELsignia) Test. The CELsignia Test measured ex vivo real-time live cell response to specific HER family and c-Met agonists to diagnose breast tumors with hyperactive HER1, HER2, HER3, HER4, and c-Met signaling activity. We report test results from breast tumor specimens obtained from 79 histopathologically HER2-negative breast cancer patients. From the resulting dataset, we estimated a c-Met signaling score cut-off to identify abnormal signaling patients and estimated the prevalence of HER2-negative breast cancer patients that have both hyperactive c-Met signaling and hyperactive signaling from at least one of the four HER-family receptors. Additionally, we report the responses to different c-Met inhibitors, HER1 inhibitor, different pan-HER inhibitors, and combinations of these therapies. We elucidate the potential synergistic involvement of c-Met signaling with HER family signaling and we report comparison data to traditional markers of cell stress that would be impractical to apply in a clinical setting. Finally, we verified the utility of the dysfunctional signaling test in a mouse xenograft model.Recombinant human epidermal growth factor (EGF) and neuregulin 1b (NRG1b) were purchased from R&D Systems . Recombinant human hepatocyte growth factor (HGF) was purchased from Peprotech . Collagen was obtained from Advanced Biomatrix  and fibronectin was obtained from Sigma . Neratinib, erlotinib, tepotinib were purchased from SelleckChem  and prepared at stock concentrations in fresh 100% DMSO (Amresco) before final dilution into assay medium. The monoclonal anti-HER2 antibody, 2C4, was expressed and purified from a mouse hybridoma cell line obtained from ATCC . Drugs for in vivo studies were prepared in 10% captisol.HER2-negative tumor specimens were obtained from excess, de-identified, surgically resected human breast cancer tissue. Tissues were obtained from multiple clinical sites located across the United States. IRB exemption was granted by Liberty IRB  after determining that the proposed research did not involve human subjects as defined under 45 CFR 46.102(f); Liberty IRB has full accreditation with the Association for the Accreditation of Human Research Protection Programs (AAHRPP). For inclusion in this study, each specimen was required to meet the following criteria: (1) a minimum specimen weight of 10–20 mg; (2) derived from a confirmed breast cancer  patient with identifiable tumor mass; and (3) obtained from female patients > 18 years old. The breast tumor samples were confirmed for no HER2 amplification using clinical standard procedures by the accredited clinical pathology lab of the institutions supplying the specimen. Additional File l-glutamine  according to ATCC specifications [HCC1954 cells were obtained from ATCC . The cells were cultured and maintained in the laboratory in RPMI medium  supplemented with 10% FBS  and ications . Tissue ications , 25. BriHER2 receptor expression data was confirmed by flow cytometry analysis. HER2 and c-Met expression was evaluated in primary cells isolated from the tumors at the time of CELsignia testing, using anti-HER2-PE  and anti-HGF-c-Met-Ax488  antibodies (Table S2). Fluorescence data was captured on a BD FACSCalibur  equipped with a 488-nm and 637-nm laser and data analysis was done using the FlowJo 2 software .For analysis of cell toxicity, single cell suspensions were stained with TMRE (200 nM for 30 min), washed with FACS buffer, and stained with Annexin V-AlexaFluor647  following the manufacturers’ instructions. The TMRE/Annexin V stained cells were resuspended in FACS buffer containing Sytox Blue  and analyzed on a Novocyte 3000 FACS instrument and NovoExpress software . For analysis of intracellular phosphoproteins by FACS, cells were stained using a Live/Dead fixable dye , fixed using 1.6% paraformaldehyde, permeabilized with cold-methanol, and then stained using antibodies towards AKT-pS473  and FAK-pS910 (BD Biosciences) (Table S2) prior to FACS analysis.Zero passaged cells were counted using an NC-250 and seeded into collagen-fibronectin coated 96-well E-plates and prepared for analysis on an Agilent xCELLigence RTCA instrument. Real-time live cell responses to specific HER3, HER1, and c-Met agonists  alone and in combination with and without antagonists were measured and quantified using an xCELLigence RTCA impedance biosensor (Agilent) as described previously . Cells wC (patient cells only with control media)CF1 (Patient cells + pathway factor NRG1b)CDF1 (Patient cells + anti-HER2 dimerizing antibody + pathway factor NRG1b)CF2 (Patient cells + pathway factor EGF)CDF2 (Patient cells + defining anti-HER2 antibody + pathway factor EGF)CF3 (Patient cells + pathway factor HGF)For the signaling function test, the 96-well biosensor E-plate was set up as follows: Test wells included:i = steps for each minute the cell attachment signal is recorded during the test. Ci = Control, no perturbation factor added to test cells. CF3i = Cells with HGF (c-Met pathway factor).HER2 signaling score was calculated as described previously where differences between the CF and CDF, indicating loss of HER2 dimer contribution  to HER1 and HER3 ligands, were applied to determine homodimer signaling from HER2 involved heterodimer signaling. HGF activated c-Met signaling, the c-Met signaling score, was defined as CF3-C using the equation below:In brief, the CELsignia test score is calculated by summing the individual minute readings over a 240-min time period where each reading reflects the change in impedance signal at that time point  compared to the same time point of a well containing cells with control media only Fig. .Fig. 1C50 values were derived using non-linear regression.Owing to limited numbers of non-passaged or low passaged primary cells, 1000-fold, five-point dose response for antagonists to HER-family and c-Met receptors was performed for each corresponding agonist stimulus (i.e. NRG1b/EGF for HER-family activation and HGF for c-Met activation) in a live-cell CELsignia assay and the observed values were averaged. Drug additions were made 18 h prior to agonist addition. The measurand recording period of four hours began after the agonist addition. Response was plotted as a percent of the maximum response to the agonist observed in the absence of any antagonist using GraphPad Prism software and ICp value of 0.7146, indicating that there was no significant difference between the HER2 signaling scores of these two study groups.The distribution of HER2 signaling scores from the 79-patient data set was first evaluated for compatibility with the distribution of HER2 signaling scores obtained from a previous study of 114 patient samples test (24) using the Kolmogorov–Smirnov two-sample test for identity of distributions with respect to HER2 signaling scores. The test statistics obtained was D = 0.10256, with a The c-Met signaling scores for the 79-patient data set were analyzed to determine whether the population was comprised of a single group or multiple sub-groups of patients, using the normal mix EM procedure in the R package “mixtools’ to fit a normal mixture model. Two and three component fit runs were subsequently performed, along with a baseline single-component model.p = 0.04; Fig. The two-component fit separates the bottom one-third of patients from the top two-thirds of the distribution. The three-component fit essentially separates the upper population of the two-component fit into a smaller intermediate population and a larger upper population. The three-component fit superimposed on a histogram of the data is shown in Fig. For the three-component fit, we plotted sensitivity versus the false positive rate for different cutoff points in a receiver operator characteristic curve  mice were procured from Jackson Labs . The mice were injected in their fat pad with HCC1954 cells dissolved in 150 µL of a 50% Matrigel solution. Tumors were allowed to form until they reached a minimum average size of 150 mm3. At this time, the tumor bearing cohort was divided up into 10-mouse treatment groups of uniform distribution of tumor sizes, just prior to initiating treatment. Tumor size and body weights were measured a minimum of twice per week. Upon completion of the study period, the mice were euthanized for necropsy and sample/tissue collection at the study endpoint or when tumor size reached ≥ 2 cm3.A selection of an appropriate cell line was necessary owing greatly to the limited commercially available tumorigenic breast cancer mouse models with the abnormal signaling function of interest to this study. Female NSG . The CELsignia test was performed on cell samples cultured from each patient tumor specimen to evaluate HER-family and c-Met signaling activity as described previously , applyinWe confirmed that the HER family signals arising from EGF and Neuregulin (NRG) agonism for this set of 79 patients were consistent with previous studies .Cutoff Determination for Abnormal c-Met Signal Function: The c-Met signaling scores for the 79-patient data set were analyzed using mixtools in the R statistical package and determined to be comprised of multiple sub-groups of patients, with a three-component fit best describing the population test score distribution. The highest value fitted component comprising 48% of the population, had a mean of 446 and a standard deviation of 195 .The ICThe CELsignia test was next used to determine the extent of signaling adaptation to drug targeting one receptor type occurring with signal initiation at a different target receptor. Primary tumor cells derived from three HER2-negative exemplary breast cancer patients were treated with combinations of agonists and inhibitors for HER1 , HER2 , or c-Met . A 3 × 3 grid of different wells each containing the patient cells treated with one of the inhibitors were then individually stimulated with agonists EGF (0.3 nM), NRG1b (3 nM), or HGF (50 pM). Percent-inhibition of each growth factor signal over a four-hour period by the three antagonists applied individually was determined and is illustrated in Fig. The observation of linkage between receptors when applying unmatched targeted antagonists and agonists prompted us to better define potential antagonism, additivity, or synergies by combining different growth factors with pan-HER inhibitors and c-Met inhibitors. Because extensive analysis by median effects methods requires a much larger number of cells not readily available with zero passage primary tumor cells from patients, the HCC1954 cell line was chosen for this study due to its abnormal c-Met signaling in addition to abnormal EGFR (HER1) signaling determined by the CELsignia test. The HCC1954 cell line overexpresses HER2, but does not have abnormal levels of CELsignia HER2 score or demonstrate HER2 targeting drug sensitivity associated with abnormal levels of HER2 specific signaling  as shownThe CELsignia test was performed to obtain median effects data using HCC1954 cells by the Chou and Talalay method . Since cWe next used median effects data to analyze combinations of drugs on the growth factor cocktail. The combination index data  to simultaneously initiate signaling via the HER-family and c-Met receptors. Neratinib , tepotinib , or a combination of the two receptor antagonists were added to the cells prior to combined growth factor addition. Cell index signaling units were calculated relative to the baseline values obtained from cells that were not treated with either agonists or antagonists.Data obtained from a representative patient cell sample shows that the combination treatment using the pan-HER and c-Met inhibitors was most efficient in blocking the simultaneous stimulation of pathways by the growth factor cocktail compared to the treatment with individual inhibitors Fig. . Tables During the combined drug testing, we investigated the benefit of targeting HER1 specifically in combination with c-Met. The results showed that tepotinib (c-Met inhibitor), erlotinib (HER1 inhibitor), and neratinib (pan-HER inhibitor) have limited effectiveness as single drugs when HER and c-Met dysfunctional signaling pathways are co-activated Table .Table 7CWe next examined the correlation of our CELsignia based real-time live cell signaling responses with commonly used research markers of drug-induced cell stress using single-cell flow cytometry analysis. Assays that require higher drug levels for longer periods of time run the risk of off-target and less specifically determinative effects. For these experiments, HCC1954   cell resNext, we extended the sampling time for flow cytometry marker experiments for prolonged treatment of HCC1954 cells with tepotinib and neratinib  to determine if significant marker changes occurred for the HCC1954 sensitivity to these drugs that is detected sooner by CELsignia in < 24 h. Under the extended time point conditions, enhanced cell death, decreased mitochondrial potential and increased annexin V positivity were detected at least 48 h after drug addition in the presence of both neratinib and more significantly for the neratinib/tepotinib cocktail, but not following tepotinib treatment alone Fig. b. This rhigh cells occurs with neratinib, when used singly and in combination with tepotinib  .In order to look for biocorrelative insight underlying CELsignia impedance-based dysfunctional signaling phenotypes for at least one specific cell sample, we analyzed the phosphorylation status of two canonical intracellular signaling markers related to cell signaling and survival AKT-pS473) and one example of adhesion complex-regulated adhesion/migration FAK-pS910) , 32 in H3 and one0 , 32 inhighpAKTlow population distribution increased). This change to pAKT is likely entirely due to 5 nM neratinib and this flow cytometry analysis of these two markers is not demonstrating any synergy of combining the two drugs as was easily demonstrated and quantified with the CELsignia test. These findings indicate that the pattern of HER-dependent differential sensitivity of HCC1954 cells to c-Met inhibitors identified by CELsignia correlates with unique effects of neratinib and combinations with tepotinib on both the AKT and FAK signaling pathways for this particular cell line.In contrast, tepotinib, when applied as a single agent, exerted no detectable dose-dependent effect on pFAK and pAKT population distributions. At higher doses of tepotinib, in the presence of a synergistic dose (5 nM) of neratinib . Each of the experimental mice were dosed by oral gavage with 100 µL dosing solution daily (QD) for 21 days (21 doses) [3) was measured and plotted as shown in Fig. To verify the results obtained from our in vitro analysis using both primary cells and the HCC1954 cell line, we next carried out an in vivo experiment using the HCC1954-xenograft model. Female NSG mice, 4–5 weeks old, were injected subcutaneously in the left mammary fat pad with two million HCC1954 cells. After the average tumor volume reached 150 mm1 doses) –35. The The CELsignia test is focused on reporting a clear and clinically actionable result—the abnormal coordinated dysfunctional signaling problem in cancer patients and the potential for successful combined targeted therapy intervention. The test can assess coincidental agonisms and antagonisms from different simultaneous sources.One remarkable distinction is that the test encompasses the effects of important factors that are not considered in other clinical tests including signaling time, local intra-cellular organization of proteins and their specific activities and contributions to signaling complexity , 38. We Apparently, good molecular tools for clinical application do not yet exist to quantify the detailed inner workings  of all the proteins in tumor cells at the level required to predict how to treat the patient. We developed the live cell CELsignia clinical test for quantifying dysfunctional signaling and then applied basic scientific process to pre-clinically verify and identify an estimated cutoff of signaling amount and the prevalence in a statistically significant population of 79 histopathologically HER2-negative breast cancer patients with concomitant c-Met and HER family-driven signaling activity.Many studies have sought to explain and treat the co-involvement of ErbB family and c-Met receptors in driving cancer . The proOveractive HER2 and HER2 heterodimerization with many other receptor types is a well-known problem in cancer and there are many drugs that can be clinically effective when the signaling dysfunction is actually present and occurring at relevant levels to drive the disease. Determining in which patients this is happening and determining the most efficacious points to inhibit the abnormal signaling with targeted therapies has been elusive. This is highlighted by reports that show in some cases effective targeted therapies such as pertuzumab can act as an artificial ligand to promote activation of ERK signaling . UnintenWatson and Gray demonstrated the importance of independent HER and c-Met involvement in individual drug resistance for some patients potentially due to the presence of agonists in the tissue microenvironment .Engelman et al. linked increased c-Met signaling to resistance to gefitinib via activated ERBB3  where c-In vitro analyses such as those described above, have prompted several clinical trials to test combinations of targeted therapeutics to treat ErbB co-involved c-Met cancer to little benefit. Several studies in the literature demonstrate the challenges with using RNA or protein levels as predictive markers for clinical utility in individual patient drug assignment , 42.The adaptive potential of signaling complexity is one of the most likely explanations why previous trials to identify subpopulations of responding patients before treatment with HER/c-Met associated disease have not led to significant positive outcomes. Various RTK and, in particular the ErbB family and the c-Met receptor, comprise a nexus of two important cellular function control pathways, MAPK and PI3K. These pathways have multiple layers of control circuitry that enable cells to adapt to various microenvironment or chemical perturbations and still continue to supply energetic and anabolic function. The control mechanisms rely on specific activities of different forms of these proteins and other complexities such as kinetics of differential phosphorylation, and regio-spatial and temporal contexts within an individual patient’s tumor cells . For exaStudies have reported on the linkage of cell signaling to morphological changes and changes at the cell surface involving different adhesion complexes that are the strongest contributors to impedance changes that can be detected by a biosensor that is applied for the CELsignia test results reported in this study –48. Usin50 values of each of the six pan-HER inhibitors and five c-Met inhibitors are reported indicating many efficacious inhibitors when applied to cell samples actually having combined signaling dysfunction.we confirmed the specificity of the agonist response by applying c-Met inhibitors , a HER1 inhibitor (erlotinib), pan-HER inhibitors , and combinations of these therapies using the CELsignia test on patient live tumor cell samples. ICWe characterized the extent of signaling complexity and cross-talk between simultaneous treatments with multiple HER family and c-Met agonists and antagonists in live tumor cell samples obtained from three different primary breast cancer patient tumor specimens and show very strong synergy between the simultaneous combined pan-HER and c-Met inhibition. These results suggest that disruption of one receptor type signaling must leave some fraction of the other type of receptor available for significant dysfunctional signaling.In this study, to further elucidate the role of c-Met signaling and its co-involvement with HER family signaling as a cancer driver, we demonstrated how growth factor receptor signaling is surprisingly strongly antagonistic between the HER family and c-Met when studying the combined stimulations. Our data show the strong synergy of the combination of pan-HER inhibition and c-Met inhibition against simultaneous abnormal receptor signaling between these receptors. Furthermore, the Chou and Talalay analyses using cell impedance testing demonstrate that the combination of growth factor antagonisms were mutually exclusive as was recently published in another report . Thus, wFinally, we provide evidence that targeted drugs disrupting the abnormal levels of HER/cMET signaling in tumor cells detected quantitatively in 4 h by CELsignia leads to mouse xenograft tumor reduction and other orthogonal biomarkers indicative of declining function and programmed cell death Fig.  at 48 h The findings of this report support identification of a significant sub-group of breast cancer patients with cooperating pan-HER and c-Met signaling dysfunction that may respond to treatment with a combination of pan-HER and c-Met inhibitors. Current tools provide a static assessment of pathway components at specific timepoints, but they have not provided a dynamic analysis quantifying dysfunctional signal transduction or accurately identifying sensitive disruption nodes for individual patients. The CELsignia, functional live tumor cell test described herein provides a dynamic assessment of cooperative signaling pathway activity. Using this test, a sub-set of HER2 negative breast cancer patients with cooperative and dysregulated HER family and c-Met pathways was identified. Two clinical trials to evaluate treatment response of this breast cancer patient sub-set to these combined pathway inhibitors identified by the CELsignia test are underway.Additional file 1. Patient characteristics based on age, stage of cancer, tumor histology, and expression of estrogen receptor in tumor cells. All patients enrolled in this study expressed normal levels of HER2 receptor.


The present study tested the hypothesis that sympathetic-mediated hypertrophy of neonatal rat ventricular cardiomyocytes (NRVMs) translated to an increase in calcium influx secondary to the upregulation of CaV1.2 channel subunits. Confocal imaging of norepinephrine (NE)-treated NRVMs revealed a hypertrophic response compared to untreated NRVMs. L-type CaV1.2 peak current density was increased 4-fold following a 24-h stimulation with NE. NE-treated NRVMs exhibited a significant upregulation of CaVα2δ1 and CaVβ3 protein levels without significant changes of CaVα1C and CaVβ2 protein levels. Pre-treatment with the β1-blocker metoprolol failed to inhibit hypertrophy or CaVβ3 upregulation whereas CaVα2δ1 protein levels were significantly reduced. NE promoted the phosphorylation of ERK 1/2, and the response was attenuated by the β1-blocker. U0126 pre-treatment suppressed NE-induced ERK1/2 phosphorylation but failed to attenuate hypertrophy. U0126 inhibition of ERK1/2 phosphorylation prevented NE-mediated upregulation of CaVα2δ1, whereas CaVβ3 protein levels remained elevated. Thus, β1-adrenergic receptor-mediated recruitment of the ERK1/2 plays a seminal role in the upregulation of CaVα2δ1 in NRVMs independent of the concomitant hypertrophic response. However, the upregulation of CaVβ3 protein levels may be directly dependent on the hypertrophic response of NRVMs.Intracellular Ca Severpression ,4,5. Carpression ,7,8. In pression ,7,8. Durpression ,7,8. Morpression ,7,8.2+ channels (LTCCs) mediate extracellular Ca2+ entry initiating Ca2+-induced Ca2+ release and triggering cardiac excitation-contraction coupling [Vα1 bound to the auxiliary subunits CaVβ, calmodulin, and CaVα2δ [Vα1C [Vβ2 [Vα2δ1 [Vα1C is the pore-forming subunit responsible for Ca2+ selectivity, activation and inactivation kinetics [Vβ is a cytoplasmic chaperone protein that supports the trafficking of CaV1.2 channels to the plasma membrane [Vα2δ1 is a large extracellular Glycosylphosphatidylinositol (GPI)-anchored protein that facilitates channel activation at physiological voltages and stimulates (≈5-fold) peak current density [Vα1C and CaVα2δ1 increased the peak current density of CaV1.2 [In ventricular cardiomyocytes, voltage-gated L-type Cacoupling ,9,10. LTd CaVα2δ ,12. In v2δ [Vα1C ,14, CaVβα1C [Vβ2 ,16, and 2 [Vα2δ1 ,18. CaVαkinetics . CaVβ ismembrane ,21,22. C density ,24,25. If CaV1.2 .1-adrenergic receptor activation increases voltage-gated L-type Ca2+ currents via protein kinase A phosphorylation which releases the Rad-mediated inhibition through a likely reduction in the affinity of Rad with the membrane and the CaVβ-subunit of the channel [2+ overload remains unresolved. Therefore, the present study tested the hypothesis that sympathetic-mediated hypertrophy of neonatal rat ventricular cardiomyocytes (NRVMs) translated to an increase in Ca2+ influx secondary to the upregulation in the protein expression of CaV1.2 channel subunits.Numerous studies have identified the sympathetic system as an essential homeostatic mechanism providing inotropic support, whereas chronic activation contributes in part to the progression of cardiac hypertrophy. Acute sympathetic stimulation of ventricular cardiomyocytes via the β channel . The relThe use and care of laboratory rats were according to the Canadian Council for Animal Care and approved by the Animal Care Committee of the Montreal Heart Institute.2 in DMEM-low glucose  supplemented with 7% heat-inactivated FBS and 1% penicillin-streptomycin for 24 h, subsequently washed and maintained in DMEM-low glucose containing insulin (5 μg/mL), transferrin (5 μg/mL), and selenium (5 ng/mL)  for 24 h prior to the experimental protocol. In all experiments, neonatal rat ventricular cardiomyocytes were plated at a density of 400–500 cells/mm2.Neonatal rat ventricular cardiomyocytes (NRVMs) were isolated from 1-day old Sprague-Dawley rat pups (sacrificed by decapitation)  as previously described ,27,28. VCardiomyocytes were treated with 1 µM norepinephrine (NE) for 1, 4, 16, 24, or 48 h. The untreated cardiomyocytes were subjected to the same procedure and plating duration as norepinephrine-treated cardiomyocytes. In parallel experiments, neonatal cardiomyocytes were pre-treated with 10 µM U0126  or 100 nM metoprolol tartrate  for 30 min to 1 h prior to the addition of 1 µM NE for 24 h.Vα2δ1 , and rabbit anti-CaVα1C . Secondary antibodies employed include goat anti-mouse coupled to Alexa 555  and donkey anti-rabbit coupled to Alexa 488 . The nucleus was identified with 4′,6′-diamidino-2-phenylindole  staining, and 4′,6-diamidino-2-phenylindole DAPI (1:800). Images were captured by confocal microscope with 20× or 63× objective. Images were analyzed using Zeiss LSM image software. The cross-sectional area (µm2) of 250 Troponin-T+ mononucleated cardiomyocytes from 5 different images per condition was determined using Zeiss LSM image software. For the colocalization analysis, wheat germ agglutinin (WGA) antibody  was added to cardiomyocytes prior to fixation.Neonatal rat ventricular cardiomyocytes (NRVMs) were plated on glass coverslips coated with poly-D-lysine in 12-well plates. Cardiomyocytes were fixed with 2% paraformaldehyde for 20 min with 2% paraformaldehyde. Immunofluorescence was performed as previously described . PrimaryVα2δ1 , rabbit CaVα1C , rabbit CaVβ2 , rabbit CaVβ3 , rabbit Phospho-ERK 1/2 , Total ERK 1/2  and rabbit GAPDH . Membranes were incubated with primary antibodies overnight at 4 °C followed by incubation with secondary anti-rabbit antibody  for 2 h at room temperature. Signal was detected using enhanced chemiluminescence (ECL) substrate.Protein lysates of neonatal rat ventricular cells were prepared and subjected to SDS-electrophoresis, as previously described . Lysates2, 1 MgCl2, and 10 HEPES, titrated to pH 7.4 with HCI. The measurements were performed at room temperature (22–25 °C).Whole-cell voltage-clamp recordings were performed in isolated NRVMs. Patch-clamp experiments were carried out with the Axopatch 200-B amplifier . PClamp software Clampex 10.4 coupled to a Digidata 1440A acquisition system  was used for on-line data acquisition and analysis. Electrodes were filled with a solution containing (in mM) 140 CsCl, 0.6 NaGTP, 3 MgATP, and 10 EGTA, 10 HEPES, titrated to pH 7.4 with KOH. Pipette resistance ranged from 3 to 5 megohms. The bath solution contained (in mM) 135 NMDG, 20 tetraethylammonium chloride, 2 CaCl+ channels, Ca2+ currents were elicited from a holding potential of −80 mV and were depolarized to potentials ranging from −80 to 50 mV in 5 mV increments lasting 450 ms for each step (protocol shown in the inset above the current traces). Ca2+ current densities (pA/pF) were obtained by dividing the peak currents by the cell capacitance. Average I-V curves were obtained by plotting peak current densities as a function of applied voltage. The I-V relationships were fitted to a Boltzmann equation. Patch-clamp data were analyzed using Clamp fit software 10.4 (Molecular Devices), Microsoft Excel 2016 , and Origin 2020 .Following a 40 ms prepulse to −40 mV to inactivate Nap < 0.05 was considered statistically significant.Data were presented as the mean ± S.E.M., and n represents the number of rat litters employed. Data were evaluated by one-way ANOVA analysis followed by Dunnett’s multiple comparison test or Tukey’s multiple comparison test (GraphPad). A value of V1.2 peak current density. Whole-cell Ca2+ current traces were detected in untreated NRVMs  (p < 0.01) four fold increase in peak current density was detected    in response to 1 µM norepinephrine (NE) was examined. As compared to untreated NRVMs, a significant hypertrophic response as depicted by the increase in cell surface area was detected after a 24-h stimulation with NE, and hypertrophy persisted after 48 h A,B. In p, N = 2) A,B. In h, N = 2) A,B. By c = 0.13) C.Vα2δ1 protein levels at 24 h and remained elevated at 48 h as compared to untreated NRVMs . NRVMs were stained with cardiac troponin-T (staining not shown), CaVα1C subunit, wheat germ-agglutinin (WGA), or 4′,6-diamidino-2-phenylindole . CaVα1C localization was determined by Pearson’s coefficient assessing the convergence of the immunofluorescent signal between WGA/CaVα1C and DAPI/CaVα1C. Vα1C (green). In untreated NRVMs, CaVα1C staining was detected predominantly on the plasma membrane and perinuclear region  as compared to untreated NRVMs   A,C.Vα1C (green). The co-staining of NRVMs with DAPI and CaVα1C confirmed the perinuclear localization  of the pore-forming subunit  and was similar to that observed in untreated cardiomyocytes  ( subunit B. In NE- > 0.05) B,D.Vα2δ1 was localized at the plasma membrane of neonatal mouse cardiomyocytes [Vα2δ1 protein levels secondary to NE-induced cardiac hypertrophy was associated with a greater translocation to the plasma membrane. Therefore, the colocalization of CaVα2δ1 with WGA or DAPI was examined. Vα2δ1 (green). In NRVMs treated with 1 µM NE for 24 or 48 h, CaVα2δ1 localization at the plasma membrane was not altered  when compared to untreated NRVMs  (Previous studies reported that Camyocytes . Immunof > 0.05) A,C. The Vα2δ1 (green). The co-staining of NRVMs with DAPI and CaVα2δ1 in untreated NRVMs revealed a perinuclear and nuclear signal  of the calcium subunit  ) with NRVMs treated with 1 µM NE for 24 or 48 h ; p > 0.05 (Vα2δ1 (green). The co-staining with DAPI and CaVα2δ1 in untreated NRVMs revealed the presence of the subunit in perinuclear and nuclear regions, depicted by a yellow signal    ( ~ 0.51) B. As seep > 0.05 A,C. Figu ~ 0.51) B. CaVα2δ; n = 3) B,D. In N; n = 3) B,D.1-adrenergic receptor antagonist metoprolol (100 nM) failed to inhibit NE-mediated hypertrophy , NE-mediated ERK1/2 phosphorylation was inhibited  were treated with norepinephrine as numerous studies have previously established a hypertrophic role of the sympathetic neurotransmitter. As demonstrated in previous studies, the exposure of NRVMs to sympathetic stimulation for 24 and 48 h led to a significant increase in the cell surface area as compared to untreated NRVMs. In parallel, L-type CaV1.2 peak current density was significantly elevated in NRVMs secondary to NE-induced cardiomyocyte hypertrophy. These data provided the impetus to assess the individual role of the subunits forming the oligomeric CaV1.2 channel in the increased Ca2+ influx in NE-induced hypertrophied NRVMs.Numerous in vitro and in vivo studies have delineated the role of calcium-dependent signaling events linking various stimuli to ventricular cardiomyocyte hypertrophy ,30,31. H2+ channels in ventricular cardiomyocytes in response to acute sympathetic stimulation occurs predominantly via β-adrenergic receptor-mediated recruitment of protein kinase A and subsequent channel phosphorylation [2+ channel activity. The increase in the L-type CaV1.2 peak current density in NE-treated NRVMs was associated with a significant upregulation of CaVα2δ1 with a more modest increase in the protein expression of CaVβ3. In contrast, the protein levels of the pore-forming CaVα1C and the accessory CaVβ2 were unchanged. Previous work from our lab revealed that co-expression of CaVα1C and CaVα2δ1 subunits upregulated by 5- to 10-fold the peak current density and facilitated the opening of the L-type CaV1.2 activity at physiological voltages [Vβ3 protein levels in NE-induced hypertrophy of NRVMs was reported to be insufficient to promote on its own a change in the activity of CaV1.2 [Vα2δ1 protein levels in NE-treated NRVMs was blunted by pre-treatment with the selective β1-blocker metoprolol, whereas CaVβ3 subunit upregulation remained unchanged. Moreover, metoprolol did not inhibit NE-mediated hypertrophy, which was in part consistent with the predominant role of the α1-adrenergic receptor in cardiomyocyte hypertrophy [1-adrenergic receptor-mediated upregulation of CaVα2δ1 protein levels in response to NE may have contributed in part to increased L-type Ca2+ channel activity independent of the concomitant hypertrophic response. By contrast, upregulation of CaVβ3 protein levels may be directly dependent on the hypertrophic response of NRVMs secondary to sympathetic stimulation. These data are supportive of β1-adrenergic receptor-mediated NE-induced upregulation of CaVα2δ1, but other regulators of adrenergic receptors need to be added to support the conclusion.Activation of voltage-gated L-type Carylation . The prevoltages . The morf CaV1.2 . The uprertrophy . CollectVα2δ1 protein expression was not accompanied by a significant change in its plasma membrane localization. Immunofluorescence confocal images of untreated and NE-treated NRVMs co-stained with WGA and CaVα2δ1 revealed that localization of the subunit at the plasma membrane was similar. Moreover, the co-staining of DAPI and CaVα2δ1 in perinuclear and nuclear regions was not significantly modified in NE-treated NRVMs. Similarly, we failed to observe any significant change in the localization of CaVα1C at the plasma membrane in NE-hypertrophied NRVMs. It remains to be seen if the perinuclear/nuclear distribution of CaVα2δ1 and CaVα1C is exclusive to NRVMs or is conserved in adult cardiomyocytes after postnatal development.The upregulation of the CaVα2δ1 protein levels via recruitment of the ras/MEK/ERK1/2 signaling pathway [1-adrenergic receptor is known to promote ERK1/2 phosphorylation [1-adrenergic receptor antagonist metoprolol suppressed NE-mediated ERK1/2 phosphorylation. Furthermore, pharmacological inhibition of the upstream activator MEK with U0126 suppressed NE-mediated ERK1/2 phosphorylation and the concomitant upregulation of CaVα2δ1 protein levels. By contrast, U0126 failed to inhibit NE-mediated NRVM hypertrophy and upregulation of CaVβ3 protein levels. The absence of an anti-hypertrophic effect of U0126 after NE treatment of NRVMs was consistent with previous data demonstrating that recruitment of the ERK1/2 signaling pathway alone was insufficient to promote cardiomyocyte hypertrophy in response to various stimuli [A previous study reported that EGF stimulation of GH3 pituitary cells increased Ca pathway . In NRVMrylation . NE trea stimuli .2+ influx via activation of the L-type CaV1.2 channel after sympathetic discharge [2+ channel activity. The latter response required β1-adrenergic receptor-mediated recruitment of the tyrosine kinase ERK1/2 translating to the increased expression of the CaV1.2 auxiliary subunit, CaVα2δ1. β1-adrenergic receptor-mediated upregulation of the CaVα2δ1 subunit was independent of the hypertrophic response. Upregulation in the protein expression of the CaVα2δ1 subunit secondary to sympathetic hyperactivity may hence contribute to intracellular Ca2+ overload with or without hypertrophy [The present study has revealed that in addition to the acute increase in Caischarge , chronicertrophy .


Stomata regulate gas and water exchange between the plant and external atmosphere, which are vital for photosynthesis and transpiration. Stomata are also the natural entrance for pathogens invading into the apoplast. Therefore, stomata play an important role in plants against pathogens. The pattern recognition receptors (PRRs) locate in guard cells to perceive pathogen/microbe-associated molecular patterns (PAMPs) and trigger a series of plant innate immune responses, including rapid closure of stomata to limit bacterial invasion, which is termed stomatal immunity. Many PRRs involved in stomatal immunity are plasma membrane-located receptor-like protein kinases (RLKs). This review focuses on the current research progress of RLK-mediated signaling pathways involved in stomatal immunity, and discusses questions that need to be addressed in future research. SLACreatment . Recentl OSCA1.3 . Taken tN-acetylglucosamine, such as chitin, peptidoglycan (PGN), and rhizobial nodulation factor (NF). Chitin is a β-l, 4-linked homopolymer of N-acetylglucosamine, which mainly exists in the cell walls of most of the higher fungi cyt during pathogen invasion [Usually, the FLS2-mediated signaling regulates stomatal immunity by ultimately modulating the activities of transporters or channel proteins localized in the plasma membrane through multi-pathways downstream of FLS2 to control stomatal aperture during the immune response ,53,66,73and H2O2 . Recent and H2O2  and moduand H2O2 . Some liand H2O2 . Cyclic invasion . This noinvasion ,73 (FiguEnvironmental factors, such as humidity, light, and temperature, also affect stomatal movement, and they often share similar downstream signaling events with PRR-triggered stomatal immunity, including phytohormone signaling, ROS production, and activation of channel proteins ,72,137.


Male fertility is often estimated by simple sperm assessment, and therefore, it is crucial to establish species-specific baselines for normal sperm parameters. In this paper, sperm physiology, function, and common abnormalities in stallions will be reviewed.As the use of assisted reproductive technologies (ART) and in vitro embryo production (IVP) expand in the equine industry, it has become necessary to further our understanding of semen physiology as it applies to overall fertility. This segment of our two-section review will focus on normal sperm parameters, beginning with development and extending through the basic morphology of mature spermatozoa, as well as common issues with male factor infertility in IVP. Ultimately, the relevance of sperm parameters to overall male factor fertility in equine IVP will be assessed. During natural breeding, a stallion will deposit millions of sperm within the intra-uterine environment of the mare . Among tThis significant diversity in sperm fertility within an ejaculate becomes more pertinent when applied to in vitro embryo production (IVP), during which a smaller number of sperm are generally selected for either in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI). Thus, it becomes necessary to understand which physiological and functional parameters are the biggest contributors to sperm fertility. This allows us to select the highest quality sperm within an ejaculate for IVP.Studies of equine sperm fertility encompass sperm biogenesis ,4, motilAdequate production of high-quality sperm by the male is critical to both natural and artificial reproductive processes. Therefore, it is critical to understand the pathways by which male gametes are derived. This process, known as spermatogenesis, occurs in the germinal epithelium of the seminiferous tubules of the testis, and is initiated during puberty  3]. Cro. Cro3]. The seminiferous tubule is divided into a basal and an adluminal layer, which is fully surrounded by a basal lamina ,19. LeydNGN3) gene . In . In 7]. 2+), calcium ionophore, bicarbonate (HCO3−), lysophosphatidylcholine, and progesterone leading to calcium oscillations [Prior to fertilization, the acrosome undergoes a calcium-dependent exocytotic reaction (acrosome reaction) as a result of sperm-oocyte binding that is essential for the subsequent penetration of the oocyte . In equillations ,123,124.llations . In humallations ,128,129.In the context of fertilization, capacitation involves calcium oscillations that trigger a complex cascade of intracellular events leading to the binding of specific zona ligands on the outer plasma membrane with the zona pellucida of the oocyte ,131. SubThe mitochondrial helix is a sensitive structure that can be easily damaged under extreme environmental conditions, including cryopreservation . DisruptApoptosis is also a common issue seen in sperm samples, especially those that undergo thermal, oxidative, or osmotic stressors from extending, cooling, or cryopreservation ,142. TheDNA integrity assessment is one of the most valuable assessments of sperm fertilization potential due to the strong correlation with sperm reproductive competence; in fertilization as well as in subsequent embryo development and offspring phenotype . Poor DNSurface composition and the resulting membrane charge are also of interest in sperm fertility studies. A greater net negative zeta potential, a parameter determined by surface composition as described previously, is acquired during epididymal maturation through extensive membrane remodeling and has been correlated with sperm quality in men ,155. TheThorough interpretation of sperm physiology, despite its complexity, is the best method for assessing male fertility. In particular, furthering our understanding of the relationships between sperm morphology, viability, biological composition, and metabolism for equine sperm will be extremely beneficial in understanding fertility in stallions, as well as shedding light on associated mechanisms. In addition, characterization of new biophysical properties, such as zeta potential, will not only aid in our understanding of what makes a fertile sperm, but will also allow for the development of new semen selection technologies. For a review of current and prospective sperm selection technologies, please refer to Section II of this review. In conclusion, sperm physiological assessment is an invaluable tool for the equine breeding industry and merits continued consideration in clinical and research settings.


This study aimed to analyze the role of the FcepsilonRI (FcεRI) signaling pathway in the pathogenesis of benign lymphoepithelial lesion of lacrimal gland (LGBLEL). Transcriptomic analysis was performed on LGBLEL and orbital cavernous hemangioma (CH) patients diagnosed via histopathology in Beijing Tongren Hospital, Capital Medical University, between July 2010 and October 2013. Four LGBLEL and three orbital CH patients, diagnosed between October 2018 and August 2019, were randomly selected as experimental and control groups, respectively. RT-PCR, immunohistochemical staining, and western blotting were used to verify genes and proteins related to the FcεRI signaling pathway. Transcriptomic analysis showed that the FcεRI signaling pathway was upregulated in the LGBLEL compared with the CH group. The mRNA expression levels of important genes including SYK, p38, JNK, PI3K, and ERK were significantly increased in the LGBLEL group . Immunohistochemical staining results showed that SYK, p38, and ERK were positively expressed in LGBLEL, while JNK and PI3K were not. The protein contents of P-SYK, P-p38, P-JNK, P-PI3K, and P-ERK were significantly higher in the LGBLEL than in the CH group . The FcεRI signaling pathway participates in the pathogenesis of LGBLEL. It is now recognized that lymphoepithelial lesions in glands can result from a variety of disorders ranging from reactive lymphoid proliferations to overt B cell lymphomas of mucosal associated lymphoid tissue3. Previous studies have suggested that BLEL is part of Sjögren's syndrome (SS) due to the similar pathologic features4; however, other systemic diseases and symptoms, such as salivary gland enlargement, dry eye, and dry mouth, are less commonly associated with LGBLEL than they are with SS. Therefore, the term LGBLEL has been used to describe benign lymphoepithelial changes in lacrimal glands associated with unilateral or bilateral gland enlargement in the absence of the clinical signs of SS (dry eyes and mouth)5.Lacrimal gland benign lymphoepithelial lesion (LGBLEL) is an inflammatory disease common in middle-aged women. Symptoms of LGBLEL include swelling of the eyelids and diffuse enlargement of the lacrimal glands. The typical pathological manifestations are diffuse infiltration of lymphocytes and plasma cells in lacrimal gland tissue, atrophy and disappearance of glands, and hyperplasia of fibrous tissue7. Increased expression of IgG4 in serum and tissue has been found in some cases of LGBLEL. Therefore, LGBLEL with positive expression of IgG4 is considered IgG4-related ocular disease (IgG4-ROD)8. IgG4-ROD is an immune-associated inflammatory disease, with etiology that may be related to SS, allergic rhinitis, dry eye, and asthma10.At present, the pathogenesis and mechanisms involved in LGBLEL are not clear, though several different hypotheses suggest that LGBLEL may be related to sex hormones, basal cell infiltration, autoimmune disease, and/or correlated with IgG4 levels12. FcεRI is a high-affinity IgE receptor, which has been shown to be involved in type I hypersensitivities, such as allergic asthma13. Activation of the classical IgE-mediated FcεRI signaling pathway can directly lead to increased secretion of cytokines, such as interleukin (IL)-3, IL-4, IL-5, IL-13, and tumor necrosis factor-α, leading to activation of helper T2 cells (Th2) and eosinophils, as well as aggravation of the inflammatory response, which is manifested by increased concentrations of serum IgG4 and IgE. IgE has a positive feedback effect on the FcεRI signaling pathway. Non-IgE-mediated allergic reactions can be caused by activation of IgG immune-related complexes and complement systems16. Activation of the FcεRI signaling pathway can lead to the release of inflammatory mediators and promote a chronic inflammatory response. Continued inflammation can lead to long-term lacrimal tissue injury, fibrosis, remodeling, and even malignant transformation. Therefore, we hypothesize that the IgE-mediated FcεRI signaling pathway may be involved in the pathogenesis of LGBLEL.Studies have found that complement mediated signaling pathways, T cell signaling pathways, and B cell signaling pathways are all involved in the pathogenesis of LGBLELIn this study, transcriptomics analysis was used to analyze the role of the FcεRI signaling pathway in the pathogenesis of LGBLEL. Western blotting, real time-PCR (RT-PCR), immunohistochemical staining were used to verify the expression of genes and proteins related to the FcεRI signaling pathway in LGBLEL tissues and to study the mechanism of action of the FcεRI signaling pathway.This study included an experimental group comprised of LGBLEL patients and a control group comprised of cavernous hemangioma (CH) patients. The diagnosis of LGBLEL and CH was mainly based on histopathological examination, as well as the typical pathological manifestations of LGBLEL, including diffuse infiltration of lymphocytes and plasma cells in the lacrimal gland tissue, atrophy and disappearance of glands, and hyperplasia of fibrous tissue Fig. A–D. CH wInformed consent was obtained for experiments involving human participants (including the use of tissue samples). This study was conducted in accordance with the principles of the Helsinki Declaration and was approved by the Ethics Committee of Beijing Tongren Hospital, Capital Medical University (TRECKY2019-093).Pathological tissue samples from patients with LGBLEL and orbital CH were collected by clinicians during surgery and transferred to a standardized laboratory for study. Portions of the tissue specimens were stored in a freezer at − 80 °C for later use; the other parts were soaked in 10% formalin for paraffin embedding and sectioning.http://hannonlab.cshl.edu/fastx_toolkit/, version0.0.13)17. The Benjamini–Hochberg procedure for multiple testing correction was used to control the false discovery rate (FDR) and significant differentially expressed genes (DEGs) were selected with |log2FC|> 1 and FDR < 0.01. Gene ontology (GO) functional annotation for biological processes and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways enrichment to determine differentially expressed genes (DEGs) between LGBLEL and CH were performed using the Database for Annotation, Visualization and Integrated Discovery (DAVID) online platform, version 6.8 (https://david.ncifcrf.gov/). Significantly enriched GO terms and KEGG pathways with an FDR < 0.05 were selected.Total RNA was extracted via a mirVana miRNA Isolation Kit , according to the manufacturer’s instructions and RNA quality was evaluated using an Agilent 2100 Bioanalyzer . The dataset was sequenced on an Illumina HiSeq 2000 (100-bp paired-end), and quality control processes were performed using the FASTQ software  software. The PCR reaction conditions were 95 °C/3 min, 95 °C/30 s, 55 °C/20 s, 72 °C/20 s, and 40 cycles; GAPDH was used as the internal reference. Primer sequence information is shown in Table The pathological tissue was cut, lysate added, and RNA extracted with TRIzol reagent (Invitrogen) according to the manufacturer's instructions. PCR primers were designed, and the cDNA of total RNA was synthesized via reverse transcription according to RT kit instructions and was used as a template for RT-PCR 19. The tissue was again washed three times with PBS, DAB stained, rinsed with water, hematoxylin stained, mounted, and imaged under a microscope . Polyclonal Rabbit IgG  was selected for isotype control antibody, which immunohistochemically showed all negative in 4 LGBLEL patients. The primary antibodies used for the immunohistochemical staining of proteins related to FcεRI signaling pathway were spleen tyrosine kinase (SYK) , p38  , c-Jun N-terminal kinase (JNK) , phosphoinositide 3-kinase (PI3K)  and extracellular signal-regulated kinases (ERK) .The diseased tissue sections were dewaxed, incubated at room temperature for 5–10 min, washed with distilled water, and soaked in PBS for 5 min. Drops of primary antibody were added, and the tissue sections incubated overnight at 4 °C. The tissue was then washed three times with PBS, biotin-labeled secondary antibody was added after 5 min, and the tissue sections were incubated at 37 °C for 30 min20. The ratio of the target to GAPDH light density was regarded as the relative concentration of protein expression. The primary antibodies were SYK , p38 , JNK , PI3K , and ERK .The diseased tissue was cut into pieces, lysate added, centrifuged, and the protein concentration detected using a BCA protein assay kit . An electrophoretic gel was prepared and the protein samples were mixed with 5 × loading buffer and treated at 100 °C for 5 min. An amount of 100 μg of cell protein was collected from each well, and electrophoresis was performed with a constant current power source of 15 mA per gel. The transfer tank was filled with electro-transfer solution to start the film transfer and the color-developing reagent system was added to the protein membranet-test function in the R package (version 3.5.1). The hypergeometric distribution model was used to determine biological pathways that were significantly enriched with DEGs. The Benjamini–Hochberg procedure was used to adjust the p-values to control the false discovery rate (FDR). Fisher exact probability method was used for counting data such as gender, and Wilcoxon-Mann–Whitney U two sample test was used for measuring data such as age. An unpaired t-test was used to analyze the differences in RNA content and protein concentration between the LGBLEL and CH groups. P < 0.05 was considered statistically significant.Statistical analysis was performed using SPSS version 18.0  and GraphPad Prism 8.0 . Log2 transformed data were used to calculate the difference of proteins using the This article does not include patients’ names, pictures, and other personal information. Informed consent was obtained from the patients for publication of this article and any accompanying images.All the authors read and approved the final manuscript for publication.DEGs between the LGBLEL and CH groups that had |log2FC|> 1 and FDR < 0.01 were selected. There were 5124 differentially expressed genes, among which 3,501 genes were downregulated and 1,623 genes were upregulated. The network interaction analysis for 686 upregulated and 892 downregulated DEGs with confidence scores ≥ 0.9 is shown in Fig. KEGG pathways enrichment for the DEGs was performed using the online functional annotation tool DAVID. The top 25 KEGG pathways that were significantly enriched for the downregulated and upregulated DEGs are shown in Fig. The previous results indicated that the FcεRI signaling pathway is involved in the pathogenesis of LGBLEL. Therefore, we then wanted to identify and validate the functions of important genes associated with the FcεRI signaling pathway in LGBLEL, including SYK, p38, JNK, PI3K, and ERK. RT-PCR showed that the mRNA expression levels of SYK, P38, JNK, PI3K, and ERK were significantly higher in the LGBLEL compared with the orbital CH group  (Fig. As shown in Fig. According to the results of western blotting and protein content detection, P-SYK, P-p38, P-JNK, P-PI3K, and P-ERK proteins were highly expressed in LGBLEL tissues and were significantly higher than in the CH group Fig. A. The P-23. Allergic diseases such as allergic rhinitis, chronic sinusitis, and asthma are common causes of chronic inflammatory lesions and are associated with the activation of mast cells and basophil granulocytes. LGBLEL is a chronic inflammatory disease, which clinical studies have indicated may be related to diseases including sinusitis and allergic rhinitis. Adzavon et al.24 found that the FcεRI signaling pathway may be involved in the mechanism that leads to malignant development in LGBLEL. The occurrence of malignant transformation is one of the pathological processes of LGBLEL; therefore, the FcεRI signaling pathway was examined in this study to further verify its role in LGBLEL pathogenesis.The FcεRI signaling pathway has been proven to be associated with allergic responses, inflammatory responses, and autoimmune diseases, such as rheumatoid arthritis and osteoarthritisThe transcriptome analysis showed that the FcεRI signaling pathway is involved in the pathogenesis of LGBLEL, and RT-PCR was used to quantitatively analyze the important genes related to the FcεRI signaling pathway. The mRNA expression levels of SYK, p38, JNK, PI3K, and ERK were found to be increased in LGBLEL tissues. Western blotting confirmed that the protein expressions of SYK, p38, JNK, PI3K, and ERK were significantly higher in LGBLEL tissues than in orbital CH tissues. In addition, immunohistochemical staining showed that SYK, p38, and ERK were positively expressed in LGBLEL, though JNK and PI3K were not expressed in the lymphocytes or epithelial cells in BLEL. This study confirms the involvement of the FcεRI signaling pathway in the pathogenesis of LGBLEL.26. PI3K can promote pro-inflammatory cytokine production and plays a regulatory role in immune responses28. Therefore, the negative expression of P-JNK and P-PI3K in immunohistochemical staining may indicate that JNK and PI3K are not the key signaling molecules of the FcεRI signaling pathway but may play an auxiliary role in the mechanism of LGBLEL. SYK is a cytoplasmic tyrosine kinase that is involved in many cellular signaling processes and drives immune inflammation29. Studies have shown that SYK, JNK, and ERK play important regulatory roles in B cell receptor signaling pathways30. SYK, PI3K, JNK, and p38 can jointly affect T cell activation and participate in the T cell receptor signaling pathway33. SYK and JNK can participate in complement system-mediated phagocytosis, T cell activation, and inflammatory responses35. Previous studies have confirmed that the B cell receptor signaling pathway, the T cell receptor signaling pathway, and the complement signaling pathway are involved in LGBLEL pathogenesis12. The B cell receptor, T cell receptor, and FC receptor are classical immune-related receptors29. Therefore, we hypothesized that these signaling pathways may interact with FcεRI-mediated signaling pathways to promote inflammatory infiltration of lacrimal tissue, leading to lacrimal gland enlargement and fibrosis.The FcεRI signaling pathway plays a functional role in various ways. JNK is highly responsive to a diverse array of cellular stimuli, such as inflammatory cytokines, and plays a significant role in innate and adaptive immune responses36. Studies of IgG4-RD have shown infiltration of many CD4 + and CD25 + Treg cells in the affected tissues and an increase in the number of CD4 + and CD25 + Treg cells in the blood, suggesting activation of Treg cells in IgG4-RD37. Treg cells and Th2 cells can produce inflammatory mediators, such as IL-10, IL-4, IL-5, IL-13, and transforming growth factor-β, leading to chronic inflammation. IgE-mediated activation of the FcεRI signaling pathway can directly lead to increased secretion of cytokines, such as IL-3, IL-4, IL-5, IL-13, and tumor necrosis factor-α, leading to Th2 cell activation, eosinophilic activation, and aggravation of the inflammatory response15. The release of inflammatory mediators can activate the B cell receptor pathway, T cell receptor pathway, and complement signaling pathway, leading to lacrimal gland injury, tumor-like hyperplasia, and dysfunction.IgG4-RD is considered to be a Th2 cell-dominated disease, and cytokines, such as IL-4 and IL-13, can promote IgG4 conversion16. Beutier et al. showed that FcγRIII receptors were the main receptors for IgG-dependent passive systemic allergic reactions induced by IgG1, IgG2a, and IgG2b antibodies, and that macrophages, mast cells, basophils, and neutrophils participated in the IgG-mediated pathway through the recruitment of FcγR38. IgG immune complex can also cause the release of complement C3a, C5a, and C5b-9, which then activates mast cells and basophils, causing cell degranulation and release of soluble mediators39. The elevated expression of IgG and IgG-subtypes in the serum of some LGBLEL patients suggests that the IgG-mediated replacement pathway and complement system may also play important roles in the pathogenesis of LGBLEL.In this study, we demonstrated the role of IgE-mediated FcεRI signaling in the pathogenesis of LGBLEL. Studies have shown that, in the IgG-mediated replacement pathway, IgG acts on a variety of immune cells by cross-linking with FcγR12. The role of the IgG-mediated substitution pathway and complement system in the pathogenesis of LGBLEL and the relationship between the FcεRI signaling pathway and other signaling pathways need to be further studied and verified. The immunohistochemical staining results of JNK and PI3K may have included false negatives due to the influence of antibodies, tissue locations, and experimental operations. In addition, the expression levels of inflammatory mediators still need to be determined in serum from LGBLEL patients. The pathogenesis of LGBLEL is complex and involves multiple signaling pathways. Based on previous studies, the results of this study indicate that the FcεRI signaling pathway participates in LGBLEL pathogenesis and provides new evidence for understanding the pathogenesis of LGBLEL.Due to the difficulty in obtaining normal lacrimal gland tissue, this study used orbital cavernous hemangioma as the control group, which is consistent with previous studiesSupplementary Information.


Porphyromonas and Aggregatibacter and elevated proinflammatory cytokines in patients with OSAHS of varying severity compared with individuals without OSAHS; however, the two organisms were not measured in the blood samples from all participants. High levels of specific Porphyromonas bacteria were detected in patients with OSAHS with and without HTN, whereas the relative abundance of Aggregatibacter was negatively correlated with the homocysteine level. The receiver operating characteristic curve analysis of controls and patients with OSAHS resulted in area under the curve values of 0.759 and 0.641 for patients with OSAHS with or without HTN, respectively. We found that the predictive function of oral microbiota was different in patients with OSAHS with and without HTN. However, there was no direct invasion by the two organisms causing endothelial cell injury, leading to speculation regarding the other mechanisms that may lead to HTN. Elucidating the differences in the oral microbiome will help us understand the pathogenesis of OSAHS-related HTN.Obstructive sleep apnea–hypopnea syndrome (OSAHS) is an independent risk factor for hypertension (HTN). The oral microbiota plays a pathophysiological role in cardiovascular diseases; however, there are few reports directly investigating and identifying the organisms involved in OSAHS-related HTN. Therefore, this study aimed to identify those organisms. We obtained 139 oral samples and determined the microbiome composition using pyrosequencing and bioinformatic analyses of the 16S rRNA. We examined the fasting levels of cytokines and homocysteine in all participants and analyzed the correlations between the oral microbiota and homocysteine levels. We determined the molecular mechanism underlying HTN by investigating the genetic composition of the strains in the blood. We detected higher relative abundances of  Recent evidence has shown an association between obstructive sleep apnea–hypopnea syndrome (OSAHS), a common sleep breathing disorder, and increased cerebral/cardiovascular morbidity and mortality . HyperteNumerous surveys have implicated microbiota in the pathogenesis of HTN –10. A deAggregatibacter spp. and Porphyromonas spp  were significantly higher among patients with OSAHS than among the controls; post hoc testing indicated higher levels among those with AHI ≥15 than among those with AHI <15  = 3.211, <15 Fig. .The relative abundances of energy and biotin metabolism were significantly higher in Group 1 than in the controls, whereas methane and thiamin metabolism were significantly higher in Group 1 than in the controls and Group 2. Protein folding and associated processing were significantly higher in Group 3 than in Group 2. The relative abundances of cell motility and secretion, protein kinases, cyanoamino acid metabolism, and ion channels were significantly higher in Group 4 than in Group 1. Conversely, mitogen-activated protein kinase signaling pathway-yeast was significantly higher in Group 1 than in Group 4, and porphyrin and chlorophyll metabolism was significantly higher in Group 1 than in both Group 4 and Group 2. Bacterial invasion of epithelial cells was significantly higher in Group 2 than in the controls  of 0.759 and 0.750, respectively, and discriminated patients with OSAHS from those with OSAHS without HTN with an ROC–AUC of 0.641 Fig. .P. gingivalis nor A. actinomycetemcomitans 18S recombinant DNA genes were detected in the 139 samples   might enter atherosclerotic lesions directly and play a role in plaque formation [The entry of oral microbiota and their metabolic products into systemic circulation is mechanism initiating cardiovascular events , 18. Aoyormation .Aggregatibacter and Porphyromonas particularly had substantial effects.In fact, periodontitis, an infectious disease of the tooth-supporting structures caused by oral pathogens, has been found to be a risk factor for CVD . RecentlP. gingivalis and A. actinomycetemcomitans existed in the blood to confirm the possible pathophysiological mechanisms involving the two organisms in OSAHS-related HTN.However, the mechanism by which the oral microbiome contributes to CVD and HTN remains unclear. Direct invasion is an easy and fairly simple pathway. Evidence that identical oral bacterial pathogens are present in the subgingival and atherosclerotic plaques , 33 suggP. gingivalis nor A. actinomycetemcomitans genes in any of the patients. Pathogenic oral bacteria can induce proinflammatory factors [Nevertheless, we found neither  factors  that med factors . We suggDNA and lipopolysaccharides in oral bacterial pathogens activate nuclear factor-kappa B (NF-κB) and activator protein 1 (AP1), resulting in the increased production of inflammatory factors, which synergize with bacterial cell wall components, activate neutrophils, and produce large amounts of reactive oxygen species (ROS) . AdditioAggregatibacter and HCY levels. Theoretically, a positive correlation exists because A. actinomycetemcomitans contains the gene required for HCY synthesis and can thus stimulate HCY production; [Porphyromonas [On the other hand, it has been noted that HCY levels are associated with an increased risk of metabolic abnormalities and HTN in patients with OSAHS . Howeverduction;  this is yromonas . HoweverPorphyromonas and Aggregatibacter genera and OSAHS-linked HTN. Our findings regarding the close association of some oral microbiota with HTN and the clinical characteristics of CVD may also enhance our understanding of the pathogenesis of OSAHS-linked HTN by elucidating the basis for the differences in the oral microbiome. Moreover, this could support effective intervention for, management of, and prognostic evaluations of modifying oral microbiota in patients with OSAHS.Overall, we found evidence of changes in the oral microbiota in patients with OSAHS and an association between oral bacteria of the Supplementary Table 1Supplementary Table 2Supplementary Table 3Supplementary Table 4Supplementary Table 5Supplementary Figure 1Supplementary Figure 2


Liver cancer has become one of the most common cancers and has a high mortality rate. Hepatocellular carcinoma is one of the most common liver cancers, and its occurrence and development process are associated with chronic hepatitis B virus (HBV) and hepatitis C virus (HCV) infections.Main bodyThe serious consequences of chronic hepatitis virus infections are related to the viral invasion strategy. Furthermore, the viral escape mechanism has evolved during long-term struggles with the host. Studies have increasingly shown that suppressor of cytokine signaling (SOCS) proteins participate in the viral escape process. SOCS proteins play an important role in regulating cytokine signaling, particularly the Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling pathway. Cytokines stimulate the expression of SOCS proteins, in turn, SOCS proteins inhibit cytokine signaling by blocking the JAK-STAT signaling pathway, thereby achieving homeostasis. By utilizing SOCS proteins, chronic hepatitis virus infection may destroy the host’s antiviral responses to achieve persistent infection.This review provides recent knowledge regarding the role of SOCS proteins during chronic hepatitis virus infection and provides some new ideas for the future treatment of chronic hepatitis. Although substantial progress has been achieved in modern medical standards, unknown and existing viruses still pose serious risks to human and animal lives. For example, the epidemics of hepatitis B virus (HBV) present an escalating trend of global infection risk which causes a large number of people to suffer from chronic HBV. More than 257 million people worldwide are chronically infected with HBV, even until now, the virus has not been completely eradicated . UnfortuSome cytokines, such as Type I interferon, have direct or indirect antiviral effects on fighting virus infection. Cytokines constitute a class of signaling molecules secreted by cells that play critical regulatory roles in communication with surrounding cells . CytokinCytokine-inducible SH2 (CIS) and suppressor of cytokine signaling (SOCS) proteins play important roles in regulating cytokine signaling, particularly the JAK-STAT signaling pathway. Numerous studies have found that cytokines stimulate the expression of SOCS proteins; however, SOCS proteins inhibit cytokine signaling by blocking the JAK-STAT signaling pathway, thereby achieving homeostasis . In addi. found that CIS is a physiological inhibitor of GM-CSF signaling by restraining the inflammatory properties of myeloid cells [The CIS/SOCS family includes CIS and SOCS proteins, can also further be subdivided into SOCS1, SOCS2, SOCS3, SOCS4, SOCS5, SOCS6, and SOCS7 , 20. Theid cells .Fig. 1a SOCS proteins were originally shown to inhibit cytokine signaling through the JAK-STAT signaling pathway . With moInnate immunity is the first-line defense against invading pathogens and plays an important role in maintaining the host’s safety. The innate immune system includes the following three classical pattern-recognition responses (PRRs): Toll-like receptors (TLRs), retinoic acid-inducible gene I-like receptors (RLRs), and nucleotide oligomerization domain-like receptors (NLRs). Among these receptors, TLRs and RLRs play a pivotal role in the production of type I interferon and various cytokines, and NLRP3, which is the most widely studied NLRs, regulates the maturation of IL-1β and IL-18 via the activation of caspase-1.After recognition, TLRs immediately recruit Toll/interleukin-1 receptor (TIR)-containing adaptors, such as MyD88 and TIR domain-containing adapters inducing IFN-β (TRIF), thus transmitting related signals to downstream molecules. Following the recognition of viral double-stranded RNA, the TLR3 signaling pathway is activated by the TRIF adaptor, and TNF receptor-associated factor 3 (TRAF3) is recruited in response. TRAF3 activates two related kinases, inducible I κB kinase (IKKi) and TANK-binding kinase 1 (TBK1), which, in turn, mediate the phosphorylation of interferon regulatory factor 3/7 (IRF-3/7). Finally, IRF-3/7 enters the nucleus and stimulates the production of type I interferon and cytokines. Type I interferon binds a heterodimeric transmembrane receptor, i.e., the IFNα receptor (IFNAR), which is composed of IFNAR1 and IFNAR2 subunits. Once IFNAR is bound by Type I interferon, the receptor-associated protein tyrosine kinases JAK1 and tyrosine kinase 2 (TYK2) are activated; the transcription factors STAT1 and STAT2 are then phosphorylated and form a dimer. Dimerized STAT1 and STAT2 assembles with IRF9 to form a trimolecular complex called IFN-stimulated gene factor 3 (ISGF3). Finally, ISGF3 translocates to the nucleus and binds its cognate DNA sequences called IFN-stimulated response elements (ISRE), thereby initiating the transcription of ISGs. –30Next, the SOCS proteins begin to express and play their roles in type I interferon signaling. CIS, SOCS1, SOCS2, and SOCS3 aim to execute their negative feedback regulation in JAK-STAT-mediated cytokine signaling, while SOCS4, SOCS5, SOCS6 and SOCS7 primarily focus on regulating the growth factor receptor signal. Among these proteins, SOCS1 and SOCS3 are the most widely studied . SOCS1 aIn addition to regulating the JAK-STAT signaling pathway, SOCS proteins can also modulate the TLR signaling pathway. Similar to the negative feedback regulation of the JAK-STAT signaling pathway, SOCS proteins mainly bind key molecules in the Toll receptor signaling pathway and then use the SOCS box region to ubiquitinate and degrade the target molecule, thereby suppressing the signaling pathway. As SOCS1 and SOCS3 are amongst the most widely reported in the past studies. The findings have shown that SOCS1 and SOCS3 use the SH2 region to recognize and bind the myeloid differentiation factor 88 (MYD88)-adaptor-like  protein , TRAF3/6A complex balance system exists in the host that ensures that innate and adaptive immune responses are properly activated and terminated via the regulation of positive and negative signaling to clear invading viruses and regulate uncontrolled inflammation. However, persistent pathogen infection may disrupt this balanced mechanism to achieve long-term stable replication and proliferation in the host. Host cells recognize the components of a virus through PRRs and activate innate immune responses, while adaptive immunity plays an important role in preventing the reinvasion of pathogens. Once a virus invades the host, PRRs quickly recognize the virus, activate complex intracellular signaling pathways and target inflammatory and antiviral responses, ultimately removing the invading virus rapidly. TLRs not only activate the host's innate immunity but also promote specific adaptive immunity by acting on antigen presenting cells. Among these processes, cytokine production is inseparable from the key signaling pathway, i.e., the JAK-STAT signaling pathway. The JAK-STAT signaling pathway regulates more than 50 downstream cytokines and growth factors as a central communication node in the immune system. This signaling pathway mainly comprises JAK and STAT proteins; JAKs can be subdivided into JAK1, JAK2, JAK3 and TYK2, while STATs can be subdivided into STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B and STAT6 . As a neHCV was the first virus used to explore the function of SOCS proteins, which were revealed to inhibit type I interferon signaling. Once HCV stably proliferates in the liver, the virus can compete with the antiviral response produced by the host and interact with various immune evasion mechanisms to damage the host's antiviral response, such as the type I interferon response. A recent study revealed that HCV increased SOCS1 expression as the viral load increased . HCV infDue to the fact that HCV infection mainly evades host innate immunity by regulating SOCS proteins. Overexpressed SOCS1 protein also inhibits the antiviral effect of type I interferon  and typePatients with chronic HCV infection often have multiple associated diseases, several of which have found to be linked to SOCS protein expression. Empirical epidemiological studies show strong evidences in which HCV infection is linked to type 2 diabetes, which is thought to originate from the downregulation of IRS1 and IRS2 by upregulating SOCS3 during HCV infection , 56. FurRecently, numerous reports have shown that miRNAs play an important role in hepatitis virus infections. In these reports, miR146a is upregulated in monocytes from HCV-infected patients , and miRIn another hepatitis virus study, HBV infection increased SOCS3 expression in Huh7 cells, resulting in sustained STAT3 activation . Additio. also found that the gene loss and epigenetic silencing of SOCS1 are strongly associated with HBV-related HCC [HBV inhibits the level of endogenous interferon and induces SOCS1 and SOCS3 expression; however, following interferon treatment, HBV replication is inhibited, and the expression levels of SOCS1 and SOCS3 begin to decrease, indicating that HBV may achieve sustained infection in vivo by upregulating the expression of the SOCS proteins . SOCS1 eated HCC . Howeverated HCC . Furtherated HCC . The relOnly a few studies have reported the interactions between SOCS proteins and hepatitis A virus (HAV). However, in various animal models of HAV, SOCS proteins are involved in HAV infection. For example, previous reports have shown that ducks can be used as a small animal model for studying HAV, and duck hepatitis A virus (DHAV) can induce acute and chronic hepatitis in ducks . During . found that the KIR region of SOCS-1 binds directly to the phosphorylation site of JAK2 and they synthesized a peptide of pJAK2  as an antagonist of SOCS1 [Considering the relationships between chronic hepatitis and SOCS proteins, small-molecule antagonists of SOCS signaling and SOCS protein silencing could be viable strategies for improving the treatment of chronic hepatitis as well as effectively mitigating hepatitis virus infection. Waiboci et alof SOCS1 . This anof SOCS1 . HBV andof SOCS1 . HBV infIn recent years, we have enhanced our understanding of the structure and function of SOCS proteins by conducting numerous in-depth researches. By functioning as negative feedback regulators, SOCS proteins are involved in the participation of the life cycle of the host, especially during the process of hepatitis virus infection. Studying the role of SOCS proteins in hepatitis virus-induced hepatocellular carcinoma provides some new ideas for improving the future treatment of hepatitis.


Although HTLV-1 was transmitted to salivary gland epithelial cells (SGECs) by a biofilm-like structure, no viral synapse formation was observed. After infection to SGECs derived from SS patients, adhesion molecules and migration factors were time-dependently released from infected SGECs. The frequency of the appearance of autoantibodies including anti-Ro/SS-A, La/SS-B antibodies in SS patients complicated with HAM is unknown; the observation of less frequent ectopic germinal center formation in HTLV-1-seropositive SS patients was a breakthrough. In addition, HTLV-1 infected cells inhibited B-lymphocyte activating factor or C-X-C motif chemokine 13 through direct contact with established follicular dendritic cell-like cells. These findings show that HTLV-1 is directly involved in the pathogenesis of SS.Viruses are a possible cause for Sjögren’s syndrome (SS) as an environmental factor related to SS onset, which exhibits exocrine gland dysfunction and the emergence of autoantibodies. Although retroviruses may exhibit lymphocytic infiltration into exocrine glands, human T-cell leukemia virus type 1 (HTLV-1) has been postulated to be a causative agent for SS. Transgenic mice with HTLV-1 genes showed sialadenitis resembling SS, but their phenotypic symptoms differed based on the adopted region of HTLV-1 genes. The dominance of  A controversial question is whether retroviruses  and SS compared to the above-mentioned viruses, some important descriptions of an association between CMV and SS have been obtained. Shillitoe et al. reported in 1982 that patients with SS had significantly high IgG and IgM class antibody levels against CMV , but an gag, pol, and env [pX region is a major feature of HTLV-1. It was long thought that the tax gene derived from the pX region that is present in the plus strand of HTLV-1 plays a central role among the genes that drive HTLV-1, because Tax protein was shown to have the ability to activate nuclear factor kappa B (NF-κB) [HBZ) [HTLV-1, the central microorganism in this review, is a type of ribonucleic acid (RNA) virus. In addition to the three regions of  and env ,26, whic (NF-κB) , which iB) [HBZ) ,29 derivHTLV-1 was isolated by Gallo et al. in 1980 , but in tax gene which is included in pX region that regulates HTLV-1 gene expression was introduced [Transgenic (Tg) mice have been used to reproduce the pathophysiology of SS due to HTLV-1 infection. Green et al. created mice in which the troduced   without lymphocyte infiltration in the submandibular, parotid, and labial salivary glands at 4–5 weeks of age. After the proliferation of SGECs, lymphocytic infiltration started around the SGECs, and at 6–8 weeks, lymphocytic infiltration was enhanced, and epithelial islands were formed. The amount of Tax protein in the salivary glands shown by Western blotting increased after 5 weeks of age in accord with the severity of salivary gland inflammation. These results not only showed an association between SS and HTLV-1 but also provided important evidence of the process by which lymphocyte infiltration causes the sialadenitis that occurs in SS.tax Tg mice expressed nerve growth factor [tax Tg mouse had neurofibromas that had the potential to activate granulocyte-macrophage colony-stimulating factor [tax transgenic mice have a phenotype that may be different from the usual SS phenotype. Those HTLV-1 tax Tg mice exhibited immunoglobulin abnormalities, and cell lines derived from these mice were used to confirm B-cell proliferation and increased IgM secretion [J.E. Green also reported that transgenic tumor cells in HTLV-1 h factor , and othg factor ,41, indiecretion .env-pX region transgenic mice that showed rheumatoid arthritis (RA)-like chronic arthritis [tax-only transgenic mice and reported that the causative gene for arthritis in env-pX Tg mice was tax, but they did not mention salivary gland inflammation in their report [tax gene is able to induce autoimmunity. Since the publication of these studies in the 1980s/1990s, there have been no reports describing the association between tax Tg mice and SS.Iwakura et al. developed HTLV-1 rthritis . In thesr report . These rAn intraperitoneal or intravenous administration of HTLV-1-infected cells to rats results in persistent HTLV-1 infection . Yoshikienv-pX region was introduced [pX protein expression would occur due to the action of this promoter. Unlike the WKAH rat mentioned above, this env-pX Tg rat model exhibited various organ disorders that occur in autoimmune diseases. The env-pX Tg rats were characterized by presenting SS-like sialadenitis, but they also presented RA-like synovitis and necrotizing vasculitis. In addition, since env-pX Tg rats show expressions of CD80/86 [Yamazaki et al. also created rats in which the troduced . Since t CD80/86  and inte CD80/86 , which atax activates nuclear transcription factors such as NF-kB, activator protein 1 (AP-1), and cyclic AMP response element binding protein (CREB) [fos [egr-1/2 [HBZ suppresses the action of tax. With respect to the action of transforming growth factor-beta (TGF-b) via Smad or co-activator p300 [HBZ acts to activate TGF-b via Smad [tax. These effects were clarified by the phenotype analysis of HBZ Tg mice performed by Satou et al. [HBZ has two conflicting functions: a transformation of T cells and the induction of chronic inflammation [HBZ Tg mice have been confirmed to have chronic inflammation of the lungs and skin [Although n (CREB) ,54 and aEB) [fos  or egr-1[egr-1/2 ,57, HBZ tor p300 , HBZ actvia Smad , contraru et al. , who alsammation . HBZ Tg and skin , but thaHBZ Tg mice, HBZ-induced Foxp3 was observed to exert a reduced action of the commonly known inhibitory Foxp3. Foxp3 is the center of the master regulator of regulatory T cells (Tregs) [However, in  (Tregs) ,63. It i (Tregs) . This seIt is estimated that more than 10 million people are infected with HTLV-1 worldwide, but the distribution is highly biased. South Africa, central Australia, the Caribbean islands, South America, and Japan are known as endemic areas ,66,67. IThere are reports from the endemic areas regarding the association between HTLV-1 infection and SS. Lima et al. investigated the cases of 272 anti-HTLV-1 antibody-positive patients at an HTLV-1 clinic in Brazil, and they reported that 21.7% of the patients had sicca syndrome . InteresThere are reports suggesting an association between HTLV-1 infection and SS even in non-endemic areas. There is a report from France of 15 cases of anti-HTLV-1 antibody-positive SS patients, and although hypergamma globulinemia was observed, no autoantibodies were detected in these cases . The finThe question of whether HTLV-1 is expressed in the exocrine glands of SS patients is addressed next. The expression of HTLV-1-related genes in LSGs of individuals with SS was reported mainly in the 1990s .tax gene in anti-HTLV-1 antibody-seronegative SS salivary glands in 1993 [tax [pX region, Ohyama et al. confirmed the expression of the HTLV-1 gene only in the infiltrated mononuclear cells (MNCs) of the salivary glands of anti-HTLV-1 antibody-positive SS patients [gag, pol, env, and pX only in the salivary glands of anti-HTLV-1 antibody-positive SS patients. Interestingly, pX gene expression was observed in T-cell lines established from anti-HTLV-1 antibody-positive patients, but not in SGEC lines [Mariette et al. first reported the expression of the HTLV-1  in 1993 . The fol993 [tax . With thpatients . They alEC lines .gag, pol, and env in the salivary glands of HAM patients, but they also observed that tax was expressed in some healthy subjects and patients with dry eye and dry mouth [tax expression not only in infiltrating MNCs but also in SGECs. We performed a nested PCR on the salivary gland tissue of anti-HTLV-1 antibody-negative patients from an endemic area, and the results revealed that the tax detection rate was low at 18% [tax was identified by PCR in 30% of salivary glands of SS patients, but also in 28% of individuals with other inflammatory diseases [tax gene is detected infrequently regardless of whether the subjects are in an endemic area. Lee et al. later showed that tax was present in only 3.8% and pX, p19, and env were not detected in the peripheral blood mononuclear cells (PBMC) of SS patients [Tangy et al. identified ry mouth . They alw at 18% . Mariettdiseases . These fpatients .tax has thus been a focus of research, but there has been no study on HBZ read from the HTLV-1 gene minus strand. Focusing on both tax and HBZ, we first examined their expression in HTLV-1-infected cell lines. The infected cell line HCT derived from HAM showed a predominant expression of tax [tax and HBZ were also observed, and the expression of tax was lower in an ATL-derived cell line than in HCT-5 cells. The results of ISH using the same probe revealed the HBZ was dominant in SS with ATL, but tax was dominant in SS with HAM, clarify a difference in HTLV-1 gene expression between ATL and HAM. Interestingly, HBZ and tax gene expressions were clearly observed not only in MNCs but also in ductal epithelium. To the best of our knowledge, there is no other published study of HBZ expression in SS, and we plan to investigate this in the future.The expression of n of tax , but exptax Tg mouse established by Green et al., who observed the expression of Tax protein by conducting Western blotting of salivary glands and muscle tissue [The detection of HTLV-1-related protein expression at the protein level in vivo is technically difficult, and there are few reports of such a detection. The first report was a e tissue . Lee et e tissue .Shattles et al. stained salivary gland tissues with a monoclonal antibody against p19 belonging to HTLV-1 gag and reported that 31% were positive . HoweverHBZ and SS by using ISH, we also examined HBZ-induced Foxp3 expression due to the limited expression of HBZ in the salivary gland MNCs and duct epithelium of HAM-SS patients [In addition to investigating the association between patients ; we obsepatients . Howeverpatients . In factpatients .Cell-to-cell contact is important for the transfer of viral particles from HTLV-1-infected lymphocytes to non-infected lymphocytes, and there are also reports of the possibility of cell-free infection. The three known types of virus particle migration via cell-to-cell contact are: (1) transmission through viral synapses with microtubule organizing center formation ,87; (2) The role of dendritic cells (DCs) in cell-free transmission is gaining attention . Jain etOnly a few studies have investigated the mechanism of infection of HTLV-1 particles to epithelial cells. Martin-Latil et al. evaluated HTLV-1 transmission efficiency by observing passage through the epithelial barrier, and they reported that the epithelial barrier was maintained unbroken during the co-culture of HTLV-1-infected cells and epithelial cells . Other tWe reported the involvement of viral synapses, biofilm, and cellular conduits in HCT-5 infected cells from HAM patients ,101: theThe infection of epithelial cells with HTLV-1 and the question of whether or not immune abnormalities are subsequently induced are important when considering the immune pathology of HTLV-1 infection. Infection of HTLV-1 epithelial cells has been confirmed in thymic epithelial cells , retinalWe have clarified the time-dependent increases in the levels of intercellular adhesion molecule-1 (ICAM-1) , IFN-γ-iSecondary lymphoid follicles and the germinal center (GC) play major roles in the production of autoantibodies in autoimmune diseases . FollicuTo investigate the effect of HTLV-1 on FDCs, we established an FDC-like cell line from human tonsil tissue and observed the effects of HCT-5 and MT-2 cell infections . The co-We also observed that the BAFF and CXCL13 concentrations in the HTLV-1 carrier serum were lower than those in the anti-HTLV-1 negative SS serum . This watax, env-pX, and HBZ showed salivary gland changes similar to those seen in human SS. However, it has yet to be established whether HTLV-1 genes truly cause SS in humans.The main findings regarding the relationship between HTLV-1 and SS are summarized . Tg miceHBZ Tg has been confirmed to cause inflammation of the lungs and skin, but it is unclear whether it causes exocrine gland damage similar to SS. In HTLV-1 tax and env-pX Tg animals, in addition to the lymphocyte infiltration of salivary glands, researchers have also observed plasma cell infiltration and the appearance of rheumatoid factor and anti-DNA antibody in serum. This point contradicts the low frequency of the appearance of autoantibodies such as anti-Ro/SS-A antibody in anti-HTLV-1 antibody-positive SS patients reported in endemic areas such as Brazil and the frequency of autoantibodies in SS with HAM. Whether the appearance of autoantibodies is the main serological abnormality in Tg mice is an issue to be elucidated in light of the low frequency of autoantibodies in SS associated with HAM. We have discovered that the direct effects of HTLV-1 may result in dysfunction of FDCs and a decrease in autoantibody production. However, the mechanisms by which HTLV-1 acts on the autoantibody production system remain a topic for further investigation.In particular, tax is expressed at a low level in non-SS LSGs, but both infiltrating mononuclear cells and SGECs in the LSGs of anti-HTLV-1 antibody-positive SS patients were infected with HTLV-1. In addition, HTLV-1 was integrated after transmission to SGECs via at least biofilm. These observations indicate that HTLV-1 has the ability to infect SGECs and induce inflammation. However, the behavior and effects of HTLV-1 in SS patients remain unclear, and it is necessary to conduct studies using immunological and molecular biological techniques.Regarding the expression of the HTLV-1 gene in LSGs, only In summary, both epidemiological and immunological basic research investigations have begun to reveal the effects of HTLV-1 on SS. However, it cannot yet be ruled out that infiltration of HTLV-1 infected cells and direct infection of SGECs themselves may result in inflammation of the exocrine glands, similar to SS. Further research is essential to confirm whether SS can be recognized as an HTLV-1-related disease.