Macrophage-stimulating Protein Research Articles

Serum biomarker signature is predictive of the risk of hepatocellular cancer in patients with cirrhosis

BackgroundInflammatory and metabolic biomarkers have been associated with hepatocellular cancer (HCC) risk in phases I and II biomarker studies. We developed and internally validated a robust metabolic biomarker panel predictive...

The Modulation of Phospho-Extracellular Signal-Regulated Kinase and Phospho-Protein Kinase B Signaling Pathways plus Activity of Macrophage-Stimulating Protein Contribute to the Protective Effect of Stachydrine on Acetaminophen-Induced Liver Injury.

Stachydrine, a prominent bioactive alkaloid derived from Leonurus heterophyllus, is a significant herb in traditional medicine. It has been noted for its anti-inflammatory and antioxidant characteristics. Consequently, we conducted a study of its hepatoprotective effect and the fundamental mechanisms involved in acetaminophen (APAP)-induced liver injury, utilizing a mouse model. Mice were intraperitoneally administered a hepatotoxic dose of APAP (300 mg/kg). Thirty minutes after APAP administration, mice were treated with different concentrations of stachydrine (0, 2.5, 5, and 10 mg/kg). Animals were sacrificed 16 h after APAP injection for serum and liver tissue assays. APAP overdose significantly elevated the serum alanine transferase levels, hepatic pro-inflammatory cytokines, malondialdehyde activity, phospho-extracellular signal-regulated kinase (ERK), phospho-protein kinase B (AKT), and macrophage-stimulating protein expression. Stachydrine treatment significantly decreased these parameters in mice with APAP-induced liver damage. Our results suggest that stachydrine may be a promising beneficial target in the prevention of APAP-induced liver damage through attenuation of the inflammatory response, inhibition of the ERK and AKT pathways, and expression of macrophage-stimulating proteins.

Glucose-oxygen coupling can serve as a biomarker for neuroinflammation-related genetic variants.

Abstract The single-nucleotide polymorphism rs3197999 in the macrophage-stimulating protein 1 gene is a missense variant. Studies have indicated that macrophage-stimulating protein 1 mediates neuronal loss and synaptic plasticity damage, and overexpression of the macrophage-stimulating protein 1 gene leads to the excessive activation of microglial cells, thereby resulting in an elevation of cerebral glucose metabolism. Traditional diagnostic models may be disrupted by neuroinflammation, making it difficult to predict the pathological status of patients solely based on single-modal images. We hypothesize that the macrophage-stimulating protein 1 rs3197999 single-nucleotide polymorphism may lead to imbalances in glucose and oxygen metabolism, thereby influencing cognitive resilience and the progression of Alzheimer’s disease. In this study, we found that among 121 patients with mild cognitive impairment, carriers of the macrophage-stimulating protein 1 rs3197999 risk allele showed a significant reduction in the coupling of glucose and oxygen metabolism in the dorsolateral prefrontal cortex region. However, the rs3197999 variant did not induce significant differences in glucose metabolism and neuronal activity signals. Furthermore, the rs3197999 risk allele correlated with a higher rate of increase in clinical dementia score, mediated by the coupling of glucose and oxygen metabolism. Highlight

Genetic vulnerability to Crohn's disease reveals a spatially resolved epithelial restitution program.

Effective tissue repair requires coordinated intercellular communication to sense damage, remodel the tissue, and restore function. Here, we dissected the healing response in the intestinal mucosa by mapping intercellular communication at single-cell resolution and integrating with spatial transcriptomics. We demonstrated that a risk variant for Crohn's disease, hepatocyte growth factor activator (HGFAC) Arg509His (R509H), disrupted a damage-sensing pathway connecting the coagulation cascade to growth factors that drive the differentiation of wound-associated epithelial (WAE) cells and production of a localized retinoic acid (RA) gradient to promote fibroblast-mediated tissue remodeling. Specifically, we showed that HGFAC R509H was activated by thrombin protease activity but exhibited impaired proteolytic activation of the growth factor macrophage-stimulating protein (MSP). In Hgfac R509H mice, reduced MSP activation in response to wounding of the colon resulted in impaired WAE cell induction and delayed healing. Through integration of single-cell transcriptomics and spatial transcriptomics, we demonstrated that WAE cells generated RA in a spatially restricted region of the wound site and that mucosal fibroblasts responded to this signal by producing extracellular matrix and growth factors. We further dissected this WAE cell-fibroblast signaling circuit in vitro using a genetically tractable organoid coculture model. Collectively, these studies exploited a genetic perturbation associated with human disease to disrupt a fundamental biological process and then reconstructed a spatially resolved mechanistic model of tissue healing.

Importance of microRNAs by mRNA-microRNA integration analysis in acute ischemic stroke patients

ObjectsThe roles of mRNA and microRNA (miRNA) are well known in many diseases, including ischemic stroke; thus, integration analysis using mRNA and miRNA is important to elucidate pathogenesis. However, their contribution, especially that of miRNA-targeted mRNA, to the severity of acute ischemic stroke remains unclear. Therefore, we examined mRNA and miRNA integration analysis targeted for acute ischemic stroke to clarify the pathway related to acute stroke severity. Material and methodsWe performed Ingenuity Pathway Analysis (IPA) using RNA extracted from the whole blood of four healthy controls, six minor acute ischemic stroke patients (MS; National Institutes of Health Stroke Scale [NIHSS] < 8), and six severe acute ischemic stroke patients (SS; NIHSS ≥ 8) on admission. mRNA and miRNA were measured using RNA sequencing and RNA expression variation; canonical pathway analysis (CPA) and upstream regulator analyses were performed. ResultsAcute ischemic stroke patients demonstrated different RNA expressions to healthy controls. Compared to MS patients, in the SS patients, 1222 mRNA, 96 miRNA, and 935 miRNA-targeted mRNA expressions were identified among differentially expressed RNA expressions (p<0.05, |log2 fold change| >1.1). CPA by IPA using mRNAs or miRNA-targeted mRNAs showed that macrophage-stimulating protein (MSP)-recepteur d'origine nantais (RON) signaling was mostly activated in SS patients compared to in MS patients. In addition, upstream regulator analysis in IPA showed that most mRNAs located upstream are miRNAs. ConclusionsIn severe acute stroke, integration of mRNA and microRNA analysis showed activated MSP-RON signaling in macrophages, and multiple miRNAs comprehensively controlled the overall pathophysiology of stroke.

Causal associations between cardiorespiratory fitness and type 2 diabetes

Higher cardiorespiratory fitness is associated with lower risk of type 2 diabetes. However, the causality of this relationship and the biological mechanisms that underlie it are unclear. Here, we examine genetic determinants of cardiorespiratory fitness in 450k European-ancestry individuals in UK Biobank, by leveraging the genetic overlap between fitness measured by an exercise test and resting heart rate. We identified 160 fitness-associated loci which we validated in an independent cohort, the Fenland study. Gene-based analyses prioritised candidate genes, such as CACNA1C, SCN10A, MYH11 and MYH6, that are enriched in biological processes related to cardiac muscle development and muscle contractility. In a Mendelian Randomisation framework, we demonstrate that higher genetically predicted fitness is causally associated with lower risk of type 2 diabetes independent of adiposity. Integration with proteomic data identified N-terminal pro B-type natriuretic peptide, hepatocyte growth factor-like protein and sex hormone-binding globulin as potential mediators of this relationship. Collectively, our findings provide insights into the biological mechanisms underpinning cardiorespiratory fitness and highlight the importance of improving fitness for diabetes prevention.

Brain and immune system-derived extracellular vesicles mediate regulation of complement system, extracellular matrix remodeling, brain repair and antigen tolerance in Multiple sclerosis

BackgroundMultiple sclerosis (MS) is an immune-mediated central nervous system disease whose course is unpredictable. Finding biomarkers that help to better comprehend the disease’s pathogenesis is crucial for supporting clinical decision-making. Blood extracellular vesicles (EVs) are membrane-bound particles secreted by all cell types that contain information on the disease’s pathological processes. PurposeTo identify the immune and nervous system-derived EV profile from blood that could have a specific role as biomarker in MS and assess its possible correlation with disease state. ResultsHigher levels of T cell-derived EVs and smaller size of neuron-derived EVs were associated with clinical relapse. The smaller size of the oligodendrocyte-derived EVs was related with motor and cognitive impairment. The proteomic analysis identified mannose-binding lectin serine protease 1 and complement factor H from immune system cell-derived EVs as autoimmune disease-associated proteins. We observed hepatocyte growth factor-like protein in EVs from T cells and inter-alpha-trypsin inhibitor heavy chain 2 from neurons as white matter injury-related proteins. In patients with MS, a specific protein profile was found in the EVs, higher levels of alpha-1-microglobulin and fibrinogen β chain, lower levels of C1S and gelsolin in the immune system-released vesicles, and Talin-1 overexpression in oligodendrocyte EVs. These specific MS-associated proteins, as well as myelin basic protein in oligodendrocyte EVs, correlated with disease activity in the patients with MS. ConclusionNeural-derived and immune-derived EVs found in blood appear to be good specific biomarkers in MS for reflecting the disease state.

The SHDRA syndrome-associated gene TMEM260 encodes a protein-specific O-mannosyltransferase

Mutations in the TMEM260 gene cause structural heart defects and renal anomalies syndrome, but the function of the encoded protein remains unknown. We previously reported wide occurrence of O-mannose glycans on extracellular immunoglobulin, plexin, transcription factor (IPT) domains found in the hepatocyte growth factor receptor (cMET), macrophage-stimulating protein receptor (RON), and plexin receptors, and further demonstrated that two known protein O-mannosylation systems orchestrated by the POMT1/2 and transmembrane and tetratricopeptide repeat-containing proteins 1-4gene families were not required for glycosylation of these IPT domains. Here, we report that the TMEM260 gene encodes an ER-located protein O-mannosyltransferase that selectively glycosylates IPT domains. We demonstrate that disease-causing TMEM260 mutations impair O-mannosylation of IPT domains and that TMEM260 knockout in cells results in receptor maturation defects and abnormal growth of 3D cell models. Thus, our study identifies the third protein-specific O-mannosylation pathway in mammals and demonstrates that O-mannosylation of IPT domains serves critical functions during epithelial morphogenesis. Our findings add a new glycosylation pathway and gene to a growing group of congenital disorders of glycosylation.

A network map of macrophage-stimulating protein (MSP) signaling.

Macrophage-stimulating protein (MSP), a serum-derived growth factor belonging to the plasminogen-related kringle domain family, is mainly produced by the liver and released into the blood. MSP is the only known ligand for RON ("Recepteur d'Origine Nantais", also known as MST1R), which is a member of the receptor tyrosine kinase (RTK) family. MSP is associated with many pathological conditions, including cancer, inflammation, and fibrosis. Activation of the MSP/RON system regulates main downstream signaling pathways, including phosphatidylinositol 3-kinase/ AKT serine/threonine kinase/ (PI3-K/AKT), mitogen-activated protein kinases (MAPK), c-Jun N-terminal kinase (JNK) & Focal adhesion kinase (FAK). These pathways are mainly involved in cell proliferation, survival, migration, invasion, angiogenesis & chemoresistance. In this work, we created a pathway resource of signaling events mediated by MSP/RON considering its contribution to diseases. We provide an integrated pathway reaction map of MSP/RON that is composed of 113 proteins and 26 reactions based on the curation of data from the published literature. The consolidated pathway map of MSP/RON mediated signaling events contains seven molecular associations, 44 enzyme catalysis, 24 activation/inhibition, six translocation events, 38 gene regulation events, and forty-two protein expression events. The MSP/RON signaling pathway map can be freely accessible through the WikiPathways Database URL: https://classic.wikipathways.org/index.php/Pathway:WP5353 .

Induction of macrophages matriptase and activation of macrophage stimulating protein 1 in sickle cell anemia - related chronic kidney disease

BACKGROUND: More than 50% of sickle cell anemia (SCA) patients develop chronic kidney disease (CKD). The mechanism of CKD is multifactorial and is not yet fully understood. SCA is a vasculopathy associated with multiple mechanisms driven by cell-free mutated hemoglobin (HbS). We have demonstrated that activation of recepteur d'origine nantais (RON) kinase contributes to the renal endothelial injury in SCA mouse model. Inhibition of RON reduces endothelial injury and ameliorates renal disease. Macrophage-stimulating protein 1 (MSP1) is the only known ligand for RON. MSP1 is synthesized by the liver and released in the circulation as biologically inactive pro-MSP1 protein that is activated by proteolytic cleavage. Macrophage membrane matriptase (MT-SP1) cleaves MSP1, making it able to bind RON receptor. The levels of plasma MSP1 and macrophage MT-SP1 in SCA have not been investigated. OBJECTIVES: To evaluate MT-SP1 expression and serum MSP1 levels in SCA patients and mouse model. HYPOTHESIS: The level of circulating MSP1 is reduced in SCA patients and mice due to its accumulation in organs. METHODS: The study was approved by Howard University IRB.All subjects provided written informed consent. Whole blood samples were obtained from 44 SCA patients enrolled at HU CSCD Registry study and 6 healthy control individuals. SCA mice (Townes) were housed in Howard University; the animal protocol was approved by the IACUC. ELISA kits were used to test human and mouse MSP1. Human THP-1 promonocytic cells were differentiated into macrophages with 25 nM PMA for 72 hrs and treated with either purified HbS or HbA (5 μM). Total RNA was used for RNA Seq. Comparisons were conducted using Dragen differential expression software v. 3.6.3 (Illumina), and gene counts were normalized using DESeq2 package from Bioconductor. RESULTS: RNA Seq results demonstrated increased levels of MT-SP1 in THP-1-derived macrophages after treatment with HbS compared to treatment with normal hemoglobin (HbA) (p=0.036). The results were further confirmed by real-time RT-PCR. The levels of plasma MSP1 were about 7-fold lower in SCD patients compared to non-SCD controls (146.8±111.7 ng/ml, N=6 in control vs. 20.6 ng/ml, N=44 in SCD, p=3.97x10-10). Log2 (MSP1) slightly correlated with albuminuria log2 (ALB/CRE) (r=0.1243, R2=0.0154, p=0.4216, N=44). This correlation was more profound in females (r=0.3158, R2=0.0997, p=0.1632, N=21). No correlation was found in males. We found moderate inverted correlation between eGFR and log2 (MSP1) (r=-0.3313, R2=0.1094, p=0.0280, N=44). In SCA patients with renal disease, MSP1 levels positively correlated with stages of CKD (r=0.3990, R2=0.1592, p=0.0320, N=29). In mouse, serum MSP1 levels were about 2.3-fold reduced in SCA compared to control mice (441.0±97.98 ng/ml, N=5 in control vs. 192.2 ±27.02, N=7 in SCD, p=0.0173). Immunostaining demonstrated the high levels of MSP1 accumulation on the surface of endothelial cells in the ki This work was supported by NIH Research Grants SC1HL150685, P50HL118006, R01HL125005, and G12MD007597. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract 2242: KRAS mutations suppress the tumor immune microenvironment in colorectal cancer

Abstract Background: KRAS mutations (KRASmut) drive tumorigenesis through various signaling pathways that are associated with worse survival in colorectal cancer patients (CRC). Immune evasion is a hallmark of KRAS-driven cancer, but the role of KRASmut in the regulation of the immune response is unclear, particularly in the tumor microenvironment (TME), which often assists cancer cells to thrive and successfully escape immune surveillance. Here, we explore the immunobiological impacts of KRASmut in CRC patients. Methods: A total of 97 fresh frozen tumor samples were collected from each CRC patient with stage I-IV who underwent surgical treatment at the Department of Surgery, Faculty of Medicine Siriraj Hospital between October 2010 and March 2011. First, all patients were identified KRAS mutational status and compared overall survival (OS) with wild-type KRAS (KRASwt). Next, we analyzed the gene expression profiling of KRASmut which was performed using the Nanostring platform. Finally, we performed the biological network analysis focusing on immunological pathways associated with KRASmut using the Ingenuity Pathway Analysis (IPA). Results: We observed KRASmut in 41.24% (n=40) of the total cases. Mutations occurred primarily in exon 2 (total n=36; codon 12, n=28; codon 13, n=8) and rarely in exon 3 (n=4). Patients with KRASmut CRC in exon 2 and exon 3, showed significantly worse OS compared to KRASwt (HR 1.77, 95% CI 1.07-2.93; P&amp;lt;0.05). Comparing gene expression profiles between KRASmut and KRASwt, found that the survival outcome of patients with KRASmut was correlated with immunosuppressive TME by activation of genes encoded in TGFβ signaling pathway. In addition to activating TGFβ pathway, the gene expression profile of KRASmut also interrupted gene encoded pro-inflammatory cytokines, including TNFRSF12, IRF6, CCR2, CCL8, CCL11, CXCR3 and CXCL10. IPA analysis revealed that differentially expressed genes of KRASmut were significantly enriched in regulation of epithelial mesenchymal transition (EMT) and macrophage-stimulating protein receptor d'origine nantais (MSP-RON) signaling pathways (P value&amp;lt;0.001), which are strongly associated with maintaining the stability of the TME and contributing to immune escape in the immune TME. Conclusions: Our results suggested that KRASmut activated TGFβ and interrupted pro-inflammatory cytokine to suppress tumor immune microenvironment in CRC. Future studies and clinical trials in patients with CRC with KRASmut should take these transcriptional profiles into account. Citation Format: Pariyada Tanjak, Amphun Chaiboonchoe, Tharathorn Suwatthanarak, Onchira Acharayothin, Kullanist Thanormjit, Jantappapa Chanthercrob, Thanawat Suwatthanarak, Bundit Wannasuphaphol, Kemmapon Chumchuen, Bhoom Suktitipat, Somponnat Sampattavanich, Krittiya Korphaisarn, Ananya Pongpaibul, Naravat Poungvarin, Harald Grove, Woramin Riansuwan, Atthaphorn Trakarnsanga, Asada Methasate, Manop Pithukpakorn, Vitoon Chinswangwatanakul. KRAS mutations suppress the tumor immune microenvironment in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2242.

Abstract LB524: Discovery of highly selective RON kinase inhibitors

Abstract RON is a receptor tyrosine kinase of the MET proto-oncogene family, known as macrophage stimulating 1 receptor (MST1R) that recognizes macrophage-stimulating protein (MSP). RON has been known to be highly expressed in several epithelial tumors, promoting tumor progression and metastasis, and on macrophages surface, facilitating M2-like phenotype polarization of tumor-associated macrophages (TAM) to impair anti-tumor functions of CD8+ T cells. Although pharmacological inhibition of RON kinase has received attention from many research groups, monoclonal antibody drug showed a limited clinical efficacy due to RON variant of receptor region. Because the structure homology of kinase domain between RON and MET is 80%, discovery of RON-specific small molecule inhibitor is considered to be highly challenging to achieve. At first, we discovered a lead compound with about 50-fold of RON selectivity against MET kinase. Our medicinal chemistry efforts elevated RON/MET selectivity up to 500~10,000-fold (IC50 = 1~50 nM in RON enzyme assay), and the target specificity was proved through a panel assay of 372 kinases (Reaction Biology Corp.). A series of compounds effectively modulated p-RON and downstream signals (e.g. p-ERK and p-AKT) in MDA-MB-453, and inhibited cell proliferation of Ba/F3 cell-line engineered with human RON kinase. Significant anti-tumor effects were shown in syngeneic mouse models compared to a control group after oral administration, and studies related to immuno-oncology are ongoing in in-vitro or ex-/in-vivo assay systems. In conclusion, we have identified RON selective small molecule inhibitors that will be useful to figure out the biology following RON selective inhibition against MET kinase, and to validate the translational potential of RON kinase as a therapeutic target for human diseases including cancer. Citation Format: Young Jin Ham, GyeongHwan Kim, Juhyun Lee, Jaeyoung Lee, Min Kyung Kim, Sunhong Kim, Woosook Kim, Han Byoul Kim, Sung Woong Jang, Hee Dong Park, Seonguk Jeon, Seok-Joo Kim, Joongheui Cho, Jungjoon Kim, Hong bin Yoon, Youngshin Kwak. Discovery of highly selective RON kinase inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB524.

Содержание некоторых сосудистых факторов роста в сыворотке крови больных с гнойными осложнениями высокоэнергетической травмы

Introduction There is a lack of studies analyzing the molecular aspects of body functioning in purulent complications of bone tissue. The purpose of the review was to explore the serum vascular endothelial growth factor (VEGF) levels in patients with high-energy trauma complicated with infection. Material and methods The study included 36 individuals who sustained a high-energy injury. The participants were identified as no-infection patients (Group I) and those who developed infection during treatment (Group II). Enzyme immunoassay was employed to quantify VEGF with Thermofisher immunoassay analyzer (USA). The study was performed according to the methodology from the R &amp; D Systems kit manual (Angiopoietin-1 and -2 (Ang-1, Ang-2)), platelet-derived growth factors (PDGF-AB, PDGF-BB), Invitrogen (epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), RayBiotech (macrophage stimulating protein (MSP), Biomerika Inc. (Calcitonin-calciotonin) USA) and Immunodiagnostic (bone sialoprotein (BSP) (Germany). HITACHI HI Vision Avius ultrasound scanner was used to explore vessels of the soft tissues and the periosteum at the site of regeneration (defect). Results There were no differences in the serum growth factor levels among the participants by gender. Patients of Group II showed 60% reduction in the Ang-2 and VEGF concentration as compared with that in the no-infection group. The serum EGF concentration was 1.6 times higher in individuals suffering from complications of high-energy trauma than in no-infection patients. There were no significant differences in platelet-derived growth factor PDGF-AB, PDGF-BB between the groups. Сhanges in the VEGF concentration were confirmed by the results of Doppler ultrasonography. Conclusion The findings suggest that the VEGFs examined can be additional diagnostic and prognostic markers for the cohort of patients.

The MSP-RON pathway regulates liver fibrosis through transforming growth factor beta-dependent epithelial-mesenchymal transition.

Liver fibrosis is pathologically important in the liver cirrhosis progression. The epithelial-mesenchymal transition (EMT) is crucial for organ fibrosis. Macrophage-stimulating protein (MSP) and its receptor tyrosine kinase, RON, promote cellular EMT. However, their role in liver fibrosis is unclear. Here, we clarify the biological profile, potential mechanisms and therapeutic targets of the MSP-RON pathway in liver fibrosis. Macrophage-stimulating protein expression and its correlation with clinicopathological characteristics of cirrhosis were evaluated in 57 clinical cases and a control group. The effect of MSP-RON pathway in liver fibrosis was determined in vitro and in vivo. The therapeutic effects of MSP or RON inhibition on liver fibrosis were evaluated in a mouse liver fibrosis model. Macrophage-stimulating protein is upregulated in liver cirrhosis, which was associated with poor patient prognosis. The MSP-RON pathway promoted hepatocytes EMT. MSP-RON-induced EMT depends on the transforming growth factor beta (TGF-β) pathway and is regulated by TGF-β inhibitors. In animal models, an MSP blocking antibody and a small molecule inhibitor of RON, BMS-777607, both inhibited liver fibrosis progression. Our study revealed that MSP is an important biomarker in liver cirrhosis progression and can be used to prognose patients. The MSP-RON pathway promotes the EMT of hepatocytes and the progress of fibrosis via a TGF-β related pathway. Consequently, we identified a new treatment strategy for liver cirrhosis through targeted inhibition of MSP/RON. This research increases the understanding of EMT-modulated liver fibrosis and provides new insights into biomarkers and therapeutic targets of liver fibrosis.

Differential proteomic screening of plasma exosomes before and after magnesium isoglycyrrhizinate treatment in chronic hepatitis B

Objective: To screen the differential proteomic of plasma exosomes before and after magnesium isoglycyrrhizinate (MgIG) treatment in chronic hepatitis B patients. Methods: Plasma samples were collected from 36 cases with chronic hepatitis B before and after MgIG treatment (2 ml/case). Plasma exosomes were extracted by ultracentrifugation. Exosomal particles concentration and inner diameter were detected by Nanosight NS300 particle size analyzer. Three cases of plasma exosomes were randomly selected before and after MgIG treatment. Proteins were extracted after lysis and digested with trypsin. Label-free differential proteomics analysis was performed by liquid chromatography-tandem mass spectrometry to screen out differential proteins that changed more than 1.5 times. Enzyme linked immunosorbent assay (ELISA) was used to verify the quantitative differential protein expression (n = 30). Measurement data were compared by paired sample t-test. Results: The average particle concentration of the extracted exosomes was 2.2×10(9)/ml, and the average size was (107 ± 52) nm, which was consistent with the theoretical value of plasma exosome size, proving that the plasma exosomes were successfully extracted. Proteomics results showed that before and after MgIG treatment in chronic hepatitis B patients, a total of 153 differentially expressed proteins were screened, including 85 up-regulated and 68 down-regulated proteins. Enzyme-linked immunosorbent assay results showed that compared with the MgIG before and after treatment group of chronic hepatitis B patients, the differences in the concentrations of hepatocyte growth factor activator and hepatocyte growth factor like protein in plasma exosomes were statistically significant (P < 0.05). Hepatocyte growth factor activator concentration in the plasma exosomes before and after MgIG treatment group was (45.9 ± 9.4) μg/ml and (13.9 ± 2.0) μg/ml, respectively, and it was down-regulated by about 3 times. Hepatocyte growth factor-like protein concentration in the plasma exosomes before and after MgIG treatment group was (23.4 ± 4.9) μg/ml and (13.8 ± 2.2) μg/ml, respectively, and it was down-regulated by about 2 times. Enzyme-linked immunosorbent assay results had consistency with the proteomics results. Conclusion: This study successfully screened the differential proteomic of plasma exosomes before and after MgIG treatment in chronic hepatitis B, and provided experimental basis for studying the molecular mechanism of MgIG treatment for chronic hepatitis B.

RON Expression Mediates Lipopolysaccharide-Mediated Dendritic Cell Maturation via March-I.

The macrophage stimulating protein (MSP)–Recepteur d’origine nantais (RON) signaling pathway regulates macrophage function. Here, we verified RON receptor expression in bone marrow-derived dendritic cells (BMDCs) by real time-PCR, Western blot, and flow cytometry. Flow cytometry was used to detect the changes in MHC II and CD86 expression following the inhibition of RON in BMDCs and splenic dendritic cells (DCs). Immunoprecipitation and Western blot were used to detect the level of MHC II and CD86 ubiquitination. An enzyme-linked immunosorbent assay was used to detect cytokine release, and a mixed lymphocyte reaction was performed to evaluate DC maturity. The results show that the inhibition of RON leads to an increase in March-1 transcription, which intensifies the ubiquitination of MHC II and CD86 and ultimately leads to a decreased level of these two molecules. The mixed lymphocyte reaction provided evidence that RON inhibition decreased the ability of DCs to promote the proliferation of T cells. The MSP-RON signaling pathway may play an important role in lipopolysaccharide (LPS)-stimulated DC maturation through March-I and may protect DC differentiation following LPS stimulation.

Identification of new potential antigen recognized by γδT cells in hepatocellular carcinoma.

In recent years, the application of chimeric antigen receptor T-cell (CAR-T) therapy based on gamma delta T (γδT) cells in hepatocellular carcinoma (HCC) immunotherapy has attracted more and more attention. However, specific antigens recognized by γδT cells are rarely identified, which has become the main restriction on such therapeutic application of γδT cells. In this report, we identified a new peptide and protein antigen recognized by γδT cells in HCC using our previous established strategy. First, we investigated the diversity of the γ9/δ2 T-cell immunorepertoire by sequence analyses of the expressed complementarity-determining region 3 (CDR3) in HCC patients. Then, we constructed γ9/δ2 T-cell receptor (TCR)-transfected cell lines expressing significant HCC CDR3 sequence and identified a series of peptides capable of binding to γδT cells specifically. Next, we identified, further tested and verified the biological functions of these peptides and their matched protein by bioinformatics analysis. We identified that the new protein hepatocyte growth factor-like protein, also called as macrophage-stimulating protein (MSP), and peptide HP1, not only bound to HCC-predominant γδTCR but also effectively activated γδT cells isolated from HCC patients. Moreover, they could stimulate γδT cells in peripheral blood from HCC patients to produce cytokines, which contributed to inhibiting HCC and played an important role in mediating cytotoxicity to HCC cell lines. In conclusion, we identified MSP and HP1, which showed potential as candidates for antigens recognized by γδT cells in HCC.

Parameters of macrophage reaction and humoral immunity in patients with implant-associated in-flammation after primary endoprosthetics of knee joint

Aim. To study peculiarities of macrophage reaction and humoral immunity in patients with signs of implant-associated inflammation after the primary endoprosthetics of the knee joint.&#x0D; Materials and Methods. Examination of 48 patients with signs of implant-associated inflammation after the primary endoprosthetics of the knee joints (the main group) and of 44 patients without signs of inflammatory reactions (comparison group) was carried out. Control group involved 30 healthy donors without diseases of the musculoskeletal system.&#x0D; Results. In 1 and 12 months after the surgery, patients of the main group showed elevated levels of the factor inhibiting migration of macrophages (MIF), of macrophage stimulating protein (MSP), class A immunoglobulins (IgA), С3 and С4 complement components as compared to control, and also to each previous period. In 1 month after the surgery, the content of class M and G immunoglobulins (IgM and IgG) decreased relative to control; in 12 months after the surgery a more pronounced reduction was noted. Increase in serum concentrations of MIF, MSP, IgA, С3 and С4-complement components was noted in the comparison group in 1 and 12 months and also in comparison with the previous period. Reduction of the levels of IgM, IgG in 1 and 12 months after the operation was noted in comparison with control and with the previous period.&#x0D; Conclusion. A study of parameters of macrophage reaction, proteins of complement system and the main classes of immunoglobulins permits to identify signs of implant-associated inflammation in 1 and 12 months after surgery, and, consequently, to determine patients of risk groups that require additional diagnostics measures.

Macrophage-Stimulating Protein Enhances Osteoblastic Differentiation via the Recepteur d&apos;Origine Nantais Receptor and Extracellular Signal-Regulated Kinase Signaling Pathway.

BackgroundMacrophage-stimulating protein (MSP; also known as macrophage stimulating 1 and hepatocyte growth factor-like protein) has been shown to play a crucial role in calcium homeostasis and skeletal mineralization in zebrafish. However, the precise role of MSP in osteoblasts has not been elucidated. In this study, we investigated the effect of MSP on osteoblast differentiation of pre-osteoblast cells.MethodsOsteoblast differentiation upon MSP treatment was evaluated by analyzing the osteogenic gene expression, alkaline phosphatase (ALP) activity, and mineralized nodule formation. To assess changes in the MSP-RON signaling pathway, knockdown of Ron gene was performed using siRNA and pharmacological inhibitor treatment.ResultsExpression of the tyrosine kinase receptor RON, a receptor of MSP, was found to be significantly increased during osteoblast differentiation. MSP treatment significantly upregulated the expression of osteogenic marker genes and remarkably increased ALP activity and mineralized nodule formation. Conversely, knockdown of Ron significantly attenuated the expression of osteogenic marker genes and ALP activity that were induced upon MSP treatment. Mechanistically, MSP treatment significantly enhanced the phosphorylation of extracellular signal-regulated kinase (ERK); however, additional treatment with the selective ERK inhibitor PD98059 attenuated the effect of MSP on osteoblast differentiation.ConclusionsAltogether, these results indicate that the MSP-RON axis is involved in promoting osteoblast differentiation via activation of the ERK signaling pathway.

CircCRAMP1L is a novel biomarker of preeclampsia risk and may play a role in preeclampsia pathogenesis via regulation of the MSP/RON axis in trophoblasts

BackgroundPreeclampsia is a severe disease in pregnant women, which is primarily managed by early screening and prevention. Circular RNAs (circRNAs) have increasingly been shown to be important biological regulators involved in numerous diseases. Further, increasing evidence has demonstrated that circRNAs can be used as diagnostic biomarkers. This study was conducted to evaluate the potential of circCRAMP1L, previously identified to be downregulated in preeclampsia, as a novel biomarker for predicting the development of preeclampsia.MethodsWe measured the expression of circCRAMP1L, which is reportedly relevant to trophoblast physiology, in plasma samples from 64 patients with preeclampsia and 64 age-, gestational age-, and body mass index-matched healthy pregnant women by qRT-PCR. MTT proliferation and transwell invasion assays revealed the biological role of circCRAMP1L in preeclampsia pathogenesis. RNA immunoprecipitation and dual-luciferase reporter assays clarified the mechanism underlying the biological function of circCRAMP1L in TEV-1 cells.ResultscircCRAMP1L circulating levels were significantly lower in patients with preeclampsia (2.66 ± 0.82, △Ct value) than in healthy pregnant women (3.95 ± 0.67, △Ct value, p < 0.001). The area under the receiver operating characteristic curve for circCRAMP1L was 0.813. Univariate and multivariate analyses identified circCRAMP1L as an independent predictor of preeclampsia. Furthermore, when circCRAMP1L was utilised in combination with its target protein macrophage stimulating protein (MSP), the predictive performance increased, with an area under the receiver operating characteristic curve of 0.928 (95% CI 0.882–0.974), 80.0% sensitivity, and 80.0% specificity. The in vitro results indicated that circCRAMP1L regulates cell proliferation, and invasion via MSP and RON proteins. We investigated the molecular mechanisms of these effects. In vitro, relative to the control group, circCRAMP1L overexpression significantly enhanced cell proliferation; furthermore, trophoblast cell invasion increased proportionally with circCRAMP1L expression. RNA immunoprecipitation and luciferase reporter gene illustrated that circCRAMP1L participated in regulation of trophoblast cell by regulating MSP.ConclusionReduced plasma levels of circCRAMP1L may be associated with an increased risk of preeclampsia, and circCRAMP1L may be a novel biomarker of preeclampsia risk.