The extracellular matrix protein EFEMP2 is involved in the response to VHSV infection in the olive flounder Paralichthys olivaceus.

The EGF-containing fibulin-like extracellular matrix protein 2 (EFEMP2) is involved in connective tissue development, elastic fiber formation, and tumor growth. In this study, we characterized the cDNA of EFEMP2 (PoEFEMP2), a member of the fibulin family of ECM proteins, in the olive flounder Paralichthys olivaceus. The coding region of PoEFEMP2 encodes a protein that contains six calcium-binding EGF-like (EGF-CA) domains and four complement Clr-like EGF-like (cEGF) domains. PoEFEMP2 shows 67.51-96.77 % similarities to orthologs in a variety of fish species. PoEFEMP2 mRNA was detected in all tissues examined; the highest levels of PoEFEMP2 mRNA expression were observed in the heart, testis, ovary and muscle. The PoEFEMP2 mRNA level increases during early development. In addition, the PoEFEMP2 mRNA level increased at 3 h post-infection (hpi) and decreased from 6 to 48 hpi in flounder Hirame natural embryo (HINAE) cells infected with viral hemorrhagic septicemia virus (VHSV). Disruption of PoEFEMP2 using the clustered regularly interspaced short palindromic repeats/CRISPR-associated-9 (CRISPR/Cas9) system resulted in a significant upregulation of VHSV G mRNA levels and immune-related genes expression in knockout cells. These findings implicate PoEFEMP2 in antiviral responses in P. olivaceus.

N-extended photoprotein obelin to competitively detect small protein tumor markers.

Two variants of Ca2+-regulated photoprotein obelin, extended from the N-terminus with small tumor markers - melanoma inhibitory activity protein (MIA) and survivin, one of the protein inhibitors of apoptosis, were designed, obtained and studied. Both domains in the obtained hybrid proteins exhibit the properties of the initial molecules: the main features of Ca2+-triggered bioluminescence are close to those of obelin, and the tumor markers' domains are recognized and bound by the corresponding antibodies. The obtained hybrids compete with the corresponding tumor markers for binding with antibodies, immobilized on the surface and their use has been shown to be promising as bioluminescent labels in a one-stage solid-phase competitive immunoassay.

HLA-A2.1-restricted ECM1-derived epitope LA through DC cross-activation priming CD8+ T and NK cells: a novel therapeutic tumour vaccine.

BACKGROUND: CD8+ T cell-mediated adaptive cellular immunity and natural killer (NK) cell-mediated innate immunity both play important roles in tumour immunity. This study aimed to develop therapeutic tumour vaccines based on double-activation of CD8+ T and NK cells. METHODS: The immune Epitope database, Molecular Operating Environment software, and enzyme-linked immunosorbent assay were used for epitope identification. Flow cytometry, confocal microscopy, UPLC-QTOF-MS, and RNA-seq were utilized for evaluating immunity of PBMC-derived DCs, CD8+ T or NK cells and related pathways. HLA-A2.1 transgenic mice combined with immunologically reconstituted tumour-bearing mice were used to examine the antitumour effect and safety of epitope vaccines. RESULTS: We identified novel HLA-A2.1-restricted extracellular matrix protein 1(ECM1)-derived immunodominant epitopes in which LA induced a potent immune response. We also found that LA-loaded DCs upregulated the frequency of CD3+/CD8+ T cells, CD45RO+/CD69+ activated memory T cells, and CD3-/CD16+/CD56+ NK cells. We demonstrated cytotoxic granule release of LA/DC-CTLs or LA/DC-NK cells and cytotoxicity against tumour cells and microtissue blocks via the predominant IFN-γ/perforin/granzyme B cell death pathway. Further investigating the mechanism of LA-mediated CD8+ T activation, we found that LA could be internalized into DCs through phagocytosis and then formed a LA-MHC-I complex presented onto the DC surface for recognition of the T cell receptor to upregulate Zap70 phosphorylation levels to further activate CD8+ T cells by DC-CTL interactions. In addition, LA-mediated DC-NK crosstalk through stimulation of the TLR4-p38 MAPK pathway increased MICA/B expression on DCs to interact with NKG2D for NK activation. Promisingly, LA could activate CD8+ T cells and NK cells simultaneously via interacting with DCs to suppress tumours in vivo. Moreover, the safety of LA was confirmed. CONCLUSIONS: LA-induced immune antitumour activity through DC cross-activation with CD8+ T and NK cells, which demonstrated proof-of-concept evidence for the capability and safety of a novel therapeutic tumour vaccine.

Immunomodulatory Role of the Extracellular Matrix Within the Liver Disease Microenvironment.

Chronic liver disease when accompanied by underlying fibrosis, is characterized by an accumulation of extracellular matrix (ECM) proteins and chronic inflammation. Although traditionally considered as a passive and largely architectural structure, the ECM is now being recognized as a source of potent damage-associated molecular pattern (DAMP)s with immune-active peptides and domains. In parallel, the ECM anchors a range of cytokines, chemokines and growth factors, all of which are capable of modulating immune responses. A growing body of evidence shows that ECM proteins themselves are capable of modulating immunity either directly via ligation with immune cell receptors including integrins and TLRs, or indirectly through release of immunoactive molecules such as cytokines which are stored within the ECM structure. Notably, ECM deposition and remodeling during injury and fibrosis can result in release or formation of ECM-DAMPs within the tissue, which can promote local inflammatory immune response and chemotactic immune cell recruitment and inflammation. It is well described that the ECM and immune response are interlinked and mutually participate in driving fibrosis, although their precise interactions in the context of chronic liver disease are poorly understood. This review aims to describe the known pro-/anti-inflammatory and fibrogenic properties of ECM proteins and DAMPs, with particular reference to the immunomodulatory properties of the ECM in the context of chronic liver disease. Finally, we discuss the importance of developing novel biotechnological platforms based on decellularized ECM-scaffolds, which provide opportunities to directly explore liver ECM-immune cell interactions in greater detail.

Elevated Circulatory Levels of Microparticles Are Associated to Lung Fibrosis and Vasculopathy During Systemic Sclerosis.

Background: Microparticles (MPs) are vesicular structures that derive from multiple cellular sources. MPs play important roles in intercellular communication, regulation of cell signaling or initiation of enzymatic processes. While MPs were characterized in Systemic Sclerosis (SSc) patients, their contribution to SSc pathogenesis remains unknown. Our aim was to investigate the potential role of MPs in SSc pathophysiology and their impact on tissue fibrosis. Methods: Ninety-six SSc patients and 37 sex-matched healthy donors (HD) were enrolled in this study in order to quantify and phenotype their plasmatic MPs by flow cytometry. The ability of MPs purified from SSc patients and HD controls to modulate fibroblast's extra-cellular matrix genes expression was evaluated in vitro by reverse transcriptase quantitative polymerase chain reaction. Results: SSc patients exhibited a higher concentration of circulatory MPs compared to HD. This difference was exacerbated when we only considered patients that were not treated with methotrexate or targeted disease-modifying antirheumatic drugs. Total circulatory MPs were associated to interstitial lung disease, lung fibrosis and diminished lung functional capacity, but also to vascular involvement such as active digital ulcers. Finally, contrary to HD MPs, MPs from SSc patients stimulated the production of extracellular matrix by fibroblast, demonstrating their profibrotic potential. Conclusions: In this study, we provide evidence for a direct profibrotic role of MPs from SSc patients, underpinned by strong clinical associations in a large cohort of patients.

Acetylated K676 TGFBIp as a severity diagnostic blood biomarker for SARS-CoV-2 pneumonia.

The outbreak of the highly contagious and deadly severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), also known as coronavirus disease 2019 (COVID-19), has posed a serious threat to public health across the globe, calling for the development of effective diagnostic markers and therapeutics. Here, we report a highly reliable severity diagnostic biomarker, acetylated 676th lysine transforming growth factor-beta-induced protein (TGFBIp K676Ac). TGFBIp K676Ac was consistently elevated in the blood of patients with SARS-CoV-2 pneumonia (n = 113), especially in patients in the intensive care unit (ICU) compared to non-ICU patients. Patients' blood samples showed increased cytokines and lymphopenia, which are exemplary indicators of SARS-CoV-2 pneumonia. Treatment with TGFBIp neutralizing antibodies suppressed the cytokine storm. The increased level of TGFBIp K676Ac in ICU patients suggests the promise of this protein as a reliable severity diagnostic biomarker for severe SARS-CoV-2 disease.

An IL6-Adenosine Positive Feedback Loop between CD73+ γδTregs and CAFs Promotes Tumor Progression in Human Breast Cancer.

The tumor microenvironment induces immunosuppression via recruiting and expanding suppressive immune cells such as regulatory T cells (Treg) to promote cancer progression. In this study, we documented that tumor-infiltrating CD73+ γδTregs were the predominant Tregs in human breast cancer and exerted more potent immunosuppressive activity than CD4+ or CD8+ Tregs. We further demonstrated that cancer-associated fibroblast (CAF)-derived IL6, rather than TGFß1, induced CD73+ γδTreg differentiation from paired normal breast tissues via the IL6/STAT3 pathway to produce more adenosine and become potent immunosuppressive T cells. CD73+ γδTregs could in turn promote IL6 secretion by CAFs through adenosine/A2BR/p38MAPK signaling, thereby forming an IL6-adenosine positive feedback loop. CD73+ γδTreg infiltration also impaired the tumoricidal functions of CD8+ T cells and significantly correlated with worse prognosis of patients. The data indicate that the IL6-adenosine loop between CD73+ γδTregs and CAFs is important to promote immunosuppression and tumor progression in human breast cancer, which may be critical for tumor immunotherapy.

A novel biomarker of MMP-cleaved prolargin is elevated in patients with psoriatic arthritis.

Psoriatic arthritis (PsA) is a chronic musculoskeletal inflammatory disease found in up to 30% of psoriasis patients. Prolargin-an extracellular matrix (ECM) protein present in cartilage and tendon-has been previously shown elevated in serum of patients with psoriasis. ECM protein fragments can reflect tissue turnover and pathological changes; thus, this study aimed to develop, validate and characterize a novel biomarker PROM targeting a matrix metalloproteinase (MMP)-cleaved prolargin neo-epitope, and to evaluate it as a biomarker for PsA. A competitive ELISA was developed with a monoclonal mouse antibody; dilution- and spiking-recovery, inter- and intra-variation, and accuracy were evaluated. Serum levels were evaluated in 55 healthy individuals and 111 patients diagnosed with PsA by the CASPAR criteria. Results indicated that the PROM assay was specific for the neo-epitope. Inter- and intra- assay variations were 11% and 4%, respectively. PROM was elevated (p = 0.0003) in patients with PsA (median: 0.24, IQR: 0.19-0.31) compared to healthy controls (0.18; 0.14-0.23) at baseline. AUROC for separation of healthy controls from PsA patients was 0.674 (95% CI 0.597-0.744, P < 0.001). In conclusion, MMP-cleaved prolargin can be quantified in serum by the PROM assay and has the potential to separate patients with PsA from healthy controls.

Versican and Versican-matrikines in Cancer Progression, Inflammation, and Immunity.

Versican is an extracellular matrix proteoglycan with key roles in multiple facets of cancer development, ranging from proliferative signaling, evasion of growth-suppressor pathways, regulation of cell death, promotion of neoangiogenesis, and tissue invasion and metastasis. Multiple lines of evidence implicate versican and its bioactive proteolytic fragments (matrikines) in the regulation of cancer inflammation and antitumor immune responses. The understanding of the dynamics of versican deposition/accumulation and its proteolytic turnover holds potential for the development of novel immune biomarkers as well as approaches to reset the immune thermostat of tumors, thus promoting efficacy of modern immunotherapies. This article summarizes work from several laboratories, including ours, on the role of this central matrix proteoglycan in tumor progression as well as tumor-immune cell cross-talk.

Cochlin-cleaved LCCL is a dual-armed regulator of the innate immune response in the cochlea during inflammation.

The inner ear is a complex and delicate structure composed of the cochlea and the vestibular system. To maintain normal auditory function, strict homeostasis of the inner ear is needed. A proper immune response against infection, thus, is crucial. Also, since excessive immune reaction can easily damage the normal architecture within the inner ear, the immune response should be fine regulated. The exact mechanism how the inner ear's immune response, specifically the innate immunity, is regulated was unknown. Recently, we reported a protein selectively localized in the inner ear during bacterial infection, named cochlin, as a possible mediator of such regulation. In this review, the immunological function of cochlin and the mechanism behind its role within inner ear immunity is summarized. Cochlin regulates innate immunity by physically entrapping pathogens within scala tympani and recruiting innate immune cells. Such mechanism enables efficient removal of pathogen while preserving the normal inner ear structure from inflammatory damage. [BMB Reports 2020; 53(9): 449-452].

Active sites of human MEPE-ASARM regulating bone matrix mineralization.

The proteolytic fragment ASARM (acidic serine- and aspartate-rich motif) of MEPE (matrix extracellular phosphoglycoprotein) (MEPE-ASARM) may act as an endogenous anti-mineralization factor involved in X-linked hypophosphatemic rickets/osteomalacia (XLH). We synthesized MEPE-ASARM peptides and relevant peptide fragments with or without phosphorylated Ser residues (pSer) to determine the active site(s) of MEPE-ASARM in a rat calvaria cell culture model. None of the synthetic peptides elicited changes in cell death, proliferation or differentiation, but the peptide (pASARM) with three pSer residues inhibited mineralization without causing changes in gene expression of osteoblast markers tested. The anti-mineralization effect was maintained in peptides in which any one of three pSer residues was deleted. Polyclonal antibodies recognizing pASARM but not ASARM abolished the pASARM effect. Deletion of six N-terminal residues but leaving the recognition sites for PHEX (phosphate regulating endopeptidase homolog, X-linked), a membrane endopeptidase responsible for XLH, intact and two C-terminal amino acid residues did not alter the anti-mineralization activity of pASARM. Our results strengthen understanding of the active sites of MEPE-pASARM and allowed us to identify a shorter more stable sequence with fewer pSer residues still exhibiting hypomineralization activity, reducing peptide synthesis cost and increasing reliability for exploring biological and potential therapeutic effects.

Fibroblast heterogeneity and its impact on extracellular matrix and immune landscape remodeling in cancer.

Tumor progression is marked by dense collagenous matrix accumulations that dynamically reorganize to accommodate a growing and invasive tumor mass. Cancer-associated fibroblasts (CAFs) play an essential role in matrix remodeling and influence other processes in the tumor microenvironment, including angiogenesis, immunosuppression, and invasion. These findings have spawned efforts to elucidate CAF functionality at the single-cell level. Here, we will discuss how those efforts have impacted our understanding of the ways in which CAFs govern matrix remodeling and the influence of matrix remodeling on the development of an immunosuppressive tumor microenvironment.

Identification of transforming growth factor beta induced (TGFBI) as an immune-related prognostic factor in clear cell renal cell carcinoma (ccRCC).

Clear cell renal cell carcinoma (ccRCC) is the most common subtype among kidney cancer, which has poor prognosis. The aim of this study was to screen out novel prognostic biomarkers and therapeutic targets for immunotherapy, and some novel molecule drugs for ccRCC treatment. Immune scores ranged from -1109.36 to 2920.81 and stromal scores ranged from -1530.11 to 1955.39 were firstly calculated by applying ESTIMATE algorithm. Then 17 DEGs associated with immune score and stromal score were further identified. 6 candidate hub genes were screened out by performing overall survival (OS) and disease-free survival analyses based on TCGA-KIRC data, one of which including TGFBI was further regarded as hub gene associated with prognosis by calculating the R2 (R2 = 0.011, P = 0.018) and AUC (AUC = 0.874). The prognostic value of TGFBI was validated by performing OS, CSS, and PFS analyses based on GSE29609 and E-MTAB-3267. CMap analysis suggested that 3 molecule drugs might be novel choice for ccRCC treatment. Further analysis demonstrated that CNVs of TGFBI was associated with OS of patients with ccRCC. TGFBI expression was also correlated with histologic grade, pathologic stage, and immune infiltration level, significantly. TGFBI was the most relevant gene with OS among the candidate hub genes, which might be novel DNA methylation biomarkers for ccRCC. In conclusion, our findings indicated that TGFBI was correlated with prognosis of patients with ccRCC, which might be novel prognostic biomarkers, and targets for immunotherapy in ccRCC. Three small molecule drugs were also identified, which showed strong potential for ccRCC treatment.

Antibodies against nephronectin ameliorate anti-type II collagen-induced arthritis in mice.

The extracellular matrix protein nephronectin (Npnt) is known to be critical for kidney development, but its function in inflammatory diseases is unknown. Here, we developed a new enzyme-linked immunosorbent assay system to detect Npnt in various autoimmune diseases, which revealed that plasma Npnt levels are increased in various mouse autoimmune models. We also report that antibodies against the α8ß1 integrin-binding region of Npnt protect mice from anti-type II collagen-induced arthritis, suggesting that Npnt may be a potential therapeutic target molecule for the prevention of autoimmune arthritis.

Fibroblasts Fuel Immune Escape in the Tumor Microenvironment.

Immune escape is central to the persistence of most, if not all, solid tumors and poses a critical obstacle to successful cancer (immuno)therapy. Cancer-associated fibroblasts (CAFs) constitute the most prevalent, yet heterogeneous, component of the tumor stroma, where they 'cool down' the immune microenvironment. The central role played by CAFs, both as a physical barrier and source of immunosuppressive molecules, sets them as a target to enhance immunotherapy of cancer. We outline the current understanding of how CAFs fuel immune escape, as well as their potential clinical applications. Whether these therapeutics really have clinically significant activity remains to be seen, but the outlook is positive.

A Case of Triple-Negative Myasthenia Gravis Lambert-Eaton Overlap Syndrome With Negative Agrin and LRP-4 Antibodies.

A case of triple-negative myasthenia gravis Lambert-Eaton overlap syndrome with negative Agrin and LRP-4 antibodies. Myasthenia gravis (MG) is an autoimmune disorder that shares similar features with Lambert-Eaton myasthenic syndrome. The combined clinical and electrophysiological findings of MG and Lambert-Eaton myasthenic syndrome have been reported, these cases represent the so-called "myasthenia gravis Lambert-Eaton overlap syndrome" (MLOS). A total of 55 MLOS cases have been identified, 13 cases were reported before the acetylcholine receptor (AChR) antibody (ab) testing era, 14 during the AChR-ab era, 26 during the voltage-gated calcium channel (VGCC)-ab era, and 2 cases have been reported during the muscle-specific kinase (MuSK)-ab era, of these; only 1 patient tested negative for all 3 antibodies. New immunological markers have been identified in the study of MG [Agrin and the low-density lipopro-tein receptor-related protein 4 (LRP-4)]. We present a patient with MLOS who tested negative for all 5 (AChR, MuSK, VGCC, Agrin, and LRP-4) serologic markers.

Cytoskeletal and extracellular matrix proteins resist the burning of bones.

Due the proteins from bone remains are highly resistant to pass of time and environmental conditions, they could tell us about the events that probably happened in the past. In the forensic and physical anthropology context, burnt bone remains are one of the most common pieces of recovered evidence and, generally, they are associated with funerary practices, criminal scenes or massive catastrophic events. In the present study, bone pieces of pigs were calcined at different calcination temperatures, and proteins were searched using biochemical, immunochemical and ultrastructure visualization under these experimentally conditions. For this purpose, it was successfully developed a non-demineralizing protein extraction method from burnt bone remains and the use of specific antibodies permitted the identification of different extracellular matrix and intracellular proteins. While collagen proteins type I and IV were identified and detected under middle and high calcination temperatures (300°C and 600°C); cytoskeletal proteins as actin, tubulin and, the microtubule associated protein Tau, were found under calcination process, even up high calcination temperatures. Under ultrastructural analysis, fibrous materials with a classical disposition of collagens were observed even at high calcination temperatures of the burnt bone remains. The protein identification and characterization in burnt bones as performed in present studies, is clearly demonstrating that using specific strategies for protein characterizations it is possible to found protein biomarkers in burnt bone remains and this strategy could be useful for forensic and anthropological purposes.

Site-specific absence of microfibrillar-associated protein 4 (MFAP4) from the internal elastic membrane of arterioles in the rheumatoid arthritis synovial membrane: an immunohistochemical study in patients with advanced rheumatoid arthritis versus osteoarthritis.

Microfibrillar-associated protein 4 (MFAP4) is a non-structural matrix protein with cell regulatory activities and a potential as seromarker for fibrosis. We aimed to study the occurrence of MFAP4 in the synovial membrane from patients with rheumatoid arthritis (RA) vs osteoarthritis (OA). Formaldehyde-fixed synovial tissue sections, from patients with RA (N = 6) and OA (N = 6) undergoing total hip arthroplasty, were deparaffinized and immunostained with monoclonal antibodies against MFAP4. Elastin was detected using ElastiKit. MFAP4 in serum (sMFAP4) and synovial fluid was measured by an immunoassay. MFAP4 was present in synovial biopsies from both RA and OA patients, particularly prominent in deep arterioles where it colocalized with elastin. Notably however, MFAP4 was absent from the internal elastic lamina in RA arterioles irrespective of disease duration and synovitis activity, while present although with irregular staining patterns in OA specimens. sMFAP4 was increased in RA and OA serum vs healthy controls: median (interquartile range) 29.8 (25.3-39.1) and 25.5 U/L (18.1-43.3) vs 17.7 U/L (13.7-21.2), p = 0.006 and p = 0.02, respectively The concentration of synovial fluid was lower than in serum in both RA and OA. These findings may suggest that MFAP4 is involved in adaptive vessel wall remodeling associated with chronic joint disease.

Pathway and network analysis of genes related to osteoporosis.

As a common degenerative disease, osteoporosis (OS) is characterized by reduced bone mass and microarchitectural deterioration of bone tissue. Both genetic and environmental factors are involved in OS development. To date, ~300 genes have been confirmed to be involved in the pathogenesis of OS, a large majority of which have been independently investigated. As OS is a polygenetic disease, a comprehensive analysis focusing on the biological functions and interactions of OS­related genes would provide valuable information. In this study, OS related research deposited in PubMed was retrieved and genes related to OS were catalogued. Pathways with an enriched biological function for these genes were extracted, and the crosstalk between the enriched pathways was analyzed. A comprehensive network was constructed, and a minimal network was extracted using the Steiner minimal network algorithm. In this study, a total of 294 genes in were retrieved from PubMed. Biological processes found to be enriched included those related to bone metabolism and the immune system. In total, 58 pathways were enriched. Furthermore, the comprehensive network consisting of 3,943 nodes and 7,976 edges was constructed, among which 631 nodes and 2,581 edges contributed to the OS­specific molecular network. In this network, in excess of 300 potential genes associated with OS and two modules were identified. Thus, this study provides a mechanistic insight into OS and suggests more than 300 potential OS­related genes for future research.