The landscape of prognostic and immunological role of myosin light chain 9 (MYL9) in human tumors.

INTRODUCTION: Recent studies have shown that myosin light chain 9 (MYL9) plays a vital role in immune infiltration, tumor invasion, and metastasis; however, the prognostic and immunological role of MYL9 has not been reported. The purpose of this study was to explore the potential prognostic and immunological roles of MYL9 in human cancers by public datasets mainly including the cancer genome atlas (TCGA) and Gene expression omnibus. METHODS: The expression pattern and prognostic value of MYL9 were analyzed across multiple public datasets in different cancer. The correlations between MYL9 expression and immune infiltration among multiple cancers were analyzed by using the TIMER2.0. The MYL9-related gene enrichment analysis was implemented by mainly using KEGG and GO datasets. RESULTS: MYL9 was lowly expressed in most cancers, such as breast cancer, lung adenocarcinoma and squamous cell carcinoma, and stomach adenocarcinoma; but it was highly expressed in several cancers, such as cholangiocarcinoma, head and neck squamous cell carcinoma, and liver hepatocellular carcinoma. Furthermore, MYL9 expression was distinctively associated with prognosis in adrenocortical carcinoma, colon adenocarcinoma, brain glioma, lung cancer, ovarian cancer, gastric cancer, breast cancer, blood cancer, and prostate cancer patients. The expressions of MYL9 were significantly associated with the infiltration of cancer-associated fibroblasts, B cell, CD8+ T cell, CD4+ T cell, macrophage, neutrophil, dendritic cell in different tumors as well as immune markers. In addition, we found that the functional mechanisms of MYL9 involved muscle contraction and focal adhesion. CONCLUSION: MYL9 can serve as a prognostic signature in pan-cancer and is associated with immune infiltration. This pan-cancer study is the first to show a relatively comprehensive understanding of the prognostic and immunological roles of MYL9 across different cancers.

CD4+ T cells in inflammatory diseases: pathogenic T-helper cells and the CD69-Myl9 system.

CD4+ T cells not only direct immune responses against infectious micro-organisms but are also involved in the pathogenesis of inflammatory diseases. In the last two to three decades, various researchers have identified and characterized several functional CD4+ T-cell subsets, including T-helper 1 (Th1), Th2, Th9 and Th17 cells and regulatory T (Treg) cells. In this mini-review, we introduce the concept of pathogenic Th cells that induce inflammatory diseases with a model of disease induction by a population of pathogenic Th cells: the 'pathogenic Th population disease-induction model'. We will focus on Th2 cells that induce allergic airway inflammation-pathogenic Th2 cells (Tpath2 cells)-and discuss the nature of Tpath2 cells that shape the pathology of chronic inflammatory diseases. Various Tpath2-cell subsets have been identified and their unique features are summarized in mouse and human systems. Second, we will discuss how Th cells migrate and are maintained in chronic inflammatory lesions. We propose a model known as the 'CD69-Myl9 system'. CD69 is a cell surface molecule expressed on activated T cells and interaction with its ligand myosin light chain 9 (Myl9) is required for the induction of inflammatory diseases. Myl9 molecules in the small vessels of inflamed lungs may play a crucial role in the migration of activated T cells into inflammatory lesions. Emerging evidence may provide new insight into the pathogenesis of chronic inflammatory diseases and contribute to the development of new therapeutic strategies for intractable inflammatory disorders.

Expression and epitope identification of myosin light chain isoform 1, an allergen in Procambarus clarkii.

Myosin light chain isoform 1 (MLC1) is reported to be a novel allergen in crayfish (Procambarus clarkii). However, little information is available about its allergic epitopes. In this study, recombinant crayfish MLC1 (rMLC1) was expressed and confirmed by mass spectrometry. Circular dichroic analysis and serological test were performed for the measuring of structural and immunological properties of rMLC1. Specific-protein-A-enriched IgG raised in rabbits against purified rMLC1 was used to screen a phage display random peptide library. Nine MLC1 mimotope clones were identified among 16 random clones after biopanning. Five conformational epitopes were identified with the program LocaPep, and mapped into 3 epitope regions at the antibody-binding interface of MLC1. MLC1 of crayfish showed high primary and secondary structure identity to MLC of other allergenic species, its epitopes were located in the structure conserved regions, and its cross-reactivity among related species was indicated by immunological assays.

Immunization with the recombinant myosin regulatory light chain (FhrMRLC) in Adjuplex® adjuvant elicits a Th1-biased immune response and a reduction of parasite burden in Fasciola hepatica infected rats.

The aim of this study was to assess the immune response and the protective efficacy elicited by the vaccination with the recombinant Fasciola hepatica myosin regulatory light chain (FhrMRLC) in Adjuplex® adjuvant against the infection with F. hepatica in rats. Four groups of 15 animals each were used for the study, one group was immunized with the recombinant F. hepatica MRLC in Adjuplex® adjuvant and the other groups remained as adjuvant, positive and negative control groups. The parasitological study showed that a statistically significant reduction of 65.1% and 82.1% in fluke burden and fecal egg count, respectively, was detected in vaccinated animals. In addition, vaccination with FhrMRLC induced a well-defined humoral and cellular immune response characterized by a significant production of specific IgG and IL-2, IL-12, TNF-α and IFN-γ; which confirms the immunogenic capacity of the FhrMRLC.

Conservation Analysis of B-Cell Allergen Epitopes to Predict Clinical Cross-Reactivity Between Shellfish and Inhalant Invertebrate Allergens.

Understanding and predicting an individual's clinical cross-reactivity to related allergens is a key to better management, treatment and progression of novel therapeutics for food allergy. In food allergy, clinical cross-reactivity is observed in patients reacting to unexpected allergen sources containing the same allergenic protein or antibody binding patches (epitopes), often resulting in severe allergic reactions. Shellfish allergy affects up to 2% of the world population and persists for life in most patients. The diagnosis of shellfish allergy is however often challenging due to reported clinical cross-reactivity to other invertebrates including mites and cockroaches. Prediction of cross-reactivity can be achieved utilizing an in-depth analysis of a few selected IgE-antibody binding epitopes. We combined available experimentally proven IgE-binding epitopes with informatics-based cross-reactivity prediction modeling to assist in the identification of clinical cross-reactive biomarkers on shellfish allergens. This knowledge can be translated into prevention and treatment of allergic diseases. To overcome the problem of predicting IgE cross-reactivity of shellfish allergens we developed an epitope conservation model using IgE binding epitopes available in the Immune Epitope Database and Analysis Resource (http://www.iedb.org/). We applied this method to a set of four different shrimp allergens, and successfully identified several non-cross-reactive as well as cross-reactive epitopes, which have been experimentally established to cross-react. Based on these findings we suggest that this method can be used for advanced component-resolved-diagnosis to identify patients sensitized to a specific shellfish group and distinguish from patients with extensive cross-reactivity to ingested and inhaled allergens from invertebrate sources.

Contaminating reactivity of a monoclonal CCAAT/Enhancer Binding Protein ß antibody in differentiating myoblasts.

OBJECTIVE: CCAAT/Enhancer Binding proteins (C/EBPs) are transcription factors involved in the regulation of a variety of cellular processes. We used the Abcam Recombinant Anti-C/EBP beta antibody (E299) to detect C/EBPß expression during myogenesis. Though the antibody is monoclonal, and the immunogen used is highly specific to C/EBPß, we identified an intense band at 23 kDa on western blot that did not correspond to any of the known isoforms of C/EBPß, or family members predicted to cross-react. Absent in myoblast cells overexpressing C/EBPß, the band was present when C/EBPß was knocked down, confirming specificity for a protein other than C/EBPß. The objective of this work was to identify the contaminating reactivity. RESULTS: We performed immunoprecipitation followed by mass spectrometry to identified myosin light chain 4 (MYL4) as the unknown band, suggesting that the Abcam monoclonal antibody directed against C/EBPß is not pure, but contains a contaminating antibody against MYL4. Caution should be used when working in cells lines that express MYL4 to not confound the detection of MYL4 with that of C/EBPß isoforms.

Cloning, Expression, and Epitope Identification of Myosin Light Chain 1: An Allergen in Mud Crab.

Mud crab (Scylla paramamosain) is a commonly consumed seafood as a result of its high nutritional value; however, it is associated with food allergy. The current understanding of crab allergens remains insufficient. In the present study, an 18 kDa protein was purified from crab muscle and confirmed to be myosin light chain 1 (MLC1) by matrix-assisted laser desorption/ionization-tandem time-of-flight mass spectrometry. Total RNA was isolated and amplified to obtain a MLC1 open reading frame of 462 bp, encoding 154 amino acids. A structural analysis revealed that recombinant MLC1 (rMLC1) expressed in Escherichia coli contained α-helix and random coil. Moreover, rMLC1 displayed strong immunoactivity by dot blot and a basophil activation test. Furthermore, seven allergenic epitopes of MLC1 were predicted, and five critical epitope regions were identified by an inhibition enzyme-linked immunosorbent assay and human mast cell degranulation assay. This comprehensive research of an allergen helps to conduct component-resolved diagnoses and immunotherapies related to crab allergies.

A new therapeutic target: the CD69-Myl9 system in immune responses.

CD69 is an activation marker on leukocytes. Early studies showed that the CD69+ cells were detected in the lung of patients with asthmatic and eosinophilic pneumonia, suggesting that CD69 might play crucial roles in the pathogenesis of such inflammatory diseases, rather than simply being an activation marker. Intensive studies using mouse models have since clarified that CD69 is a functional molecule regulating the immune responses. We discovered that Myosin light chain 9, 12a, 12b (Myl9/12) are ligands for CD69 and that platelet-derived Myl9 forms a net-like structure (Myl9 nets) that is strongly detected inside blood vessels in inflamed lung. CD69-expressing activated T cells attached to the Myl9 nets can thereby migrate into the inflamed tissues through a system known as the CD69-Myl9 system. In this review, we summarize the discovery of the CD69-Myl9 system and discuss how this system is important in inflammatory immune responses. In addition, we discuss our recent finding that CD69 controls the exhaustion status of tumor-infiltrating T cells and that the blockade of the CD69 function enhances anti-tumor immunity. Finally, we discuss the possibility of CD69 as a new therapeutic target for patients with intractable inflammatory disorders and tumors.

An update on shellfish allergy.

PURPOSE OF REVIEW: Shellfish is an important cause of food allergy worldwide, and a major cause of food-triggered anaphylaxis. Despite the wide variety of shellfish, there is considerable serological and clinical cross-reactivity of major shellfish allergens, and accurate diagnosis remains a challenge in the management of shellfish allergy. RECENT FINDINGS: Novel minor allergens have been discovered and characterized, and advances in component resolved diagnostics have provided insights into the prevalence of sensitization and their clinical importance in shellfish allergy. The extensive cross-reactivity between tropomyosin of house-dust mite and crustacean shellfish has been postulated to be the cause of a proposed mite-shellfish oral allergy syndrome. SUMMARY: More studies in food challenge-proven patients are required to establish the true prevalence and natural history of shellfish allergy. Refinement of component resolved diagnostics and testing for minor allergens may be helpful in developing more precise species-specific tests. Further investigation into the role of tropomyosin in house-dust mite and shellfish allergies may provide novel immunotherapeutic approaches for shellfish allergy.

Anthocyanins inhibit tumor necrosis alpha-induced loss of Caco-2 cell barrier integrity.

An increased permeability of the intestinal barrier is proposed as a major event in the pathophysiology of conditions characterized by chronic gut inflammation. This study investigated the capacity of pure anthocyanins (AC), and berry and rice extracts containing different types and amounts of AC, to inhibit tumor necrosis alpha (TNFα)-induced permeabilization of Caco-2 cell monolayers. Caco-2 cells differentiated into intestinal epithelial cell monolayers were incubated in the absence/presence of TNFα, with or without the addition of AC or AC-rich plant extracts (ACRE). AC and ACRE inhibited TNFα-induced loss of monolayer permeability as assessed by changes in transepithelial electrical resistance (TEER) and paracellular transport of FITC-dextran. In the range of concentrations tested (0.25-1 µM), O-glucosides of cyanidin, and delphinidin, but not those of malvidin, peonidin and petunidin protected the monolayer from TNFα-induced decrease of TEER and increase of FITC-dextran permeability. Cyanidin and delphinidin acted by mitigating TNFα-triggered activation of transcription factor NF-κB, and downstream phosphorylation of myosin light chain (MLC). The protective actions of the ACRE on TNFα-induced TEER increase was positively correlated with the sum of cyanidins and delphinidins (r2 = 0.83) content in the ACRE. However, no correlation was observed between TEER and ACRE total AC, malvidin, or peonidin content. Results support a particular capacity of cyanidins and delphinidins in the protection of the intestinal barrier against inflammation-induced permeabilization, in part through the inhibition of the NF-κB pathway.

Immune protection conferred by recombinant MRLC (myosin regulatory light chain) antigen in TiterMax Gold® adjuvant against experimental fasciolosis in rats.

Protection against experimental fasciolosis in rats immunized with recombinant myosin regulatory light chain (MRLC) in TiterMax Gold® adjuvant was assessed. The experimental trial consisted of four groups of 15 animals; group 1 was unimmunized and infected, group 2 was immunized with MRLC in adjuvant and infected, group 3 was infected and immunized with adjuvant only and group 4 was unimmunized and uninfected. Immunization with MRLC in TiterMax Gold® adjuvant (group 2) induced a reduction in fluke burdens of 51.0% (p<0.001) when compared with the adjuvant control group, and 61.5% (p<0.001) when compared with the unimmunized infected controls. There was a reduction in fecal egg output in group 2 of 44.8% and 37.3% compared with group 1 and group 3, respectively; although this difference was not statistically significant. Measurement of cytokine levels revealed higher levels of TNF-alpha and IL-2 as well as lower levels of IL-4 in group 2 during the chronic stage of infection (p<0.05), along with higher levels of IFN-gamma during early stages of infection (p<0.05). These results suggest a mixed Th1/Th2 phenotype immune response; however predominance of Th1 cytokines was observed. Levels of anti-MRLC serum IgG in group 2 were significantly higher than controls at the time of euthanasia (p<0.05). This is the first report of immunization with recombinant MRLC in rats, demonstrating that this antigen significantly reduces fluke burdens, increases the Th1 immune response and encourages further studies to improve the vaccine's efficacy.

Role of connexin 43 in vascular hyperpermeability and relationship to Rock1-MLC20 pathway in septic rats.

Connexin (Cx)43 has been shown to participate in several cardiovascular diseases. Increased vascular permeability is a common and severe complication in sepsis or septic shock. Whether or not Cx43 takes part in the regulation of vascular permeability in severe sepsis is not known, and the underlying mechanism has not been described. With cecal ligation and puncture-induced sepsis in rats and lipopolysaccharide (LPS)-treated vascular endothelial cells (VECs) from pulmonary veins, the role of Cx43 in increased vascular permeability and its relationship to the RhoA/Rock1 pathway were studied. It was shown that vascular permeability in the lungs, kidneys, and mesentery in sepsis rats and LPS-stimulated monolayer pulmonary vein VECs was significantly increased and positively correlated with the increased expression of Cx43 and Rock1 in these organs and cultured pulmonary vein VECs. The connexin inhibitor carbenoxolone (10 mg/kg iv) and the Rock1 inhibitor Y-27632 (2 mg/kg iv) alleviated the vascular leakage of lung, mesentery, and kidney in sepsis rats. Overexpressed Cx43 increased the phosphorylation of 20-kDa myosin light chain (MLC20) and the expression of Rock1 and increased the vascular permeability and decreased the transendothelial electrical resistance of pulmonary vein VECs. Cx43 RNA interference decreased the phosphorylation of MLC20 and the expression of Rock1 and decreased LPS-stimulated hyperpermeability of cultured pulmonary vein VECs. The Rock1 inhibitor Y-27632 alleviated LPS- and overexpressed Cx43-induced hyperpermeability of monolayer pulmonary vein VECs. This report shows that Cx43 participates in the regulation of vascular permeability in sepsis and that the mechanism is related to the Rock1-MLC20 phosphorylation pathway.

Purification, Characterization, and Analysis of the Allergenic Properties of Myosin Light Chain in Procambarus clarkii.

Myosin light chain (MLC) plays a vital role in cell and muscle functions and has been identified as an allergen in shrimp. In this study, MLC with a molecular mass of 18 kDa was purified from crayfish (Procambarus clarkii) muscle. Its physicochemical characterization showed that the purified MLC is a glycoprotein with 4.3% carbohydrate, highly stable to heat, acid-alkali, and digestion, and weakly retains IgE-binding activity when its secondary structure was altered. Serological assays suggested that conformational epitopes predominate over linear epitopes in the purified MLC. Two isoforms of the MLC gene (MLC1 and MLC2) were cloned, and the purified MLC was identified as MLC1. Analysis of the secondary and tertiary structures of the MLCs indicated that MLC1 has four conformational epitopes and three linear epitopes, whereas MLC2 had a major conformational epitope and three linear epitopes. These results are significant for understanding hypersensitization of humans to crayfish.

Identification of 4-Trimethylaminobutyraldehyde Dehydrogenase (TMABA-DH) as a Candidate Serum Autoantibody Target for Kawasaki Disease.

Kawasaki disease (KD), an acute vasculitis that preferentially affects coronary arteries, is still the leading cause of acquired heart disease in children. Although the involvement of immune system malfunction in the onset of KD is suggested, its etiology still remains to be clarified. We investigated autoantibodies in KD patients, which are frequently found in sera from patients with autoimmune diseases, vasculitides and arteritides. We performed two-dimensional western blotting and LC-MS/MS to analyze the antigens of autoantibodies, detected two protein spots with 4 out of 24 sera from KD patients but not with 6 control sera, and identified the antigens as 4-trimethylaminobutyraldehyde dehydrogenase (TMABA-DH). A slot blot analysis with TMABA-DH as an antigen also revealed higher reactivities of patients' sera than control sera (positive rates: 18/43 vs 3/41). Using an enzyme-linked immunosorbent assay (ELISA), we found that the reactivity of anti-TMABA-DH antibodies in sera from KD patients was significantly higher than that in sera from age-matched controls. The optimal cut-off value of 0.043 had a sensitivity of 83.7% and a specificity of 80.0% in detecting KD patients (positive rates: 37/43 for KD patients, 9/41 for controls). Immunohistochemistry performed on thin sections of rat heart revealed that TMABA-DH colocalized with myosin light chains in cardiac myocytes. Patient sera with high reactivity gave similar immunostaining pattern. These results suggest that the detection of anti-TMABA-DH autoantibody could be a potential strategy for a diagnosis of KD.

Bispecific antibodies, nanoparticles and cells: bringing the right cells to get the job done.

Pre-arming therapeutic cells with bispecific antibodies (BiAbs) before infusion can home the cells to specific tissue antigens in the body. With the development of nanotechnology, we developed a novel strategy, namely magnetic bispecific cell engager (MagBICE), that combines BiAbs with biodegradable iron nanoparticles. Compared to conventional BiAbs, the latter enables magnetic targeting and imaging. This editorial discusses current knowledge of BiAbs and their applications in targeting activated T cells to cancerous tissues or targeting bone marrow-derived stem cells to myocardial infarction. We will also discuss the fabrication of MagBICE and its application in treating rodents with myocardial infarction.

Shellfish Allergy: a Comprehensive Review.

Shellfish allergy is of increasing concern, as its prevalence has risen in recent years. Many advances have been made in allergen characterization. B cell epitopes in the major allergen tropomyosin have been characterized. In addition to tropomyosin, arginine kinase, sarcoplasmic calcium-binding protein, and myosin light chain have recently been reported in shellfish. All are proteins that play a role in muscular contraction. Additional allergens such as hemocyanin have also been described. The effect of processing methods on these allergens has been studied, revealing thermal stability and resistance to peptic digestion in some cases. Modifications after Maillard reactions have also been addressed, although in some cases with conflicting results. In recent years, new hypoallergenic molecules have been developed, which constitute a new therapeutic approach to allergic disorders. A recombinant hypoallergenic tropomyosin has been developed, which opens a new avenue in the treatment of shellfish allergy. Cross-reactivity with species that are not closely related is common in shellfish-allergic patients, as many of shellfish allergens are widely distributed panallergens in invertebrates. Cross-reactivity with house dust mites is well known, but other species can also be involved in this phenomenon.

Anti-ß2GPI antibodies stimulate endothelial cell microparticle release via a nonmuscle myosin II motor protein-dependent pathway.

The antiphospholipid syndrome is characterized by thrombosis and recurrent fetal loss in patients with antiphospholipid antibodies (APLAs). Most pathogenic APLAs are directed against ß2-glycoprotein I (ß2GPI), a plasma phospholipid binding protein. One mechanism by which circulating antiphospholipid/anti-ß2GPI antibodies may promote thrombosis is by inducing the release of procoagulant microparticles from endothelial cells. However, there is no information available concerning the mechanisms by which anti-ß2GPI antibodies induce microparticle release. In seeking to identify proteins phosphorylated during anti-ß2GPI antibody-induced endothelial activation, we observed phosphorylation of nonmuscle myosin II regulatory light chain (RLC), which regulates cytoskeletal assembly. In parallel, we observed a dramatic increase in the formation of filamentous actin, a two- to fivefold increase in the release of endothelial cell microparticles, and a 10- to 15-fold increase in the expression of E-selectin, intercellular adhesion molecule 1, vascular cell adhesion molecule 1, and tissue factor messenger RNA. Microparticle release, but not endothelial cell surface E-selectin expression, was blocked by inhibiting RLC phosphorylation or nonmuscle myosin II motor activity. These results suggest that distinct pathways, some of which mediate cytoskeletal assembly, regulate the endothelial cell response to anti-ß2GPI antibodies. Inhibition of nonmuscle myosin II activation may provide a novel approach for inhibiting microparticle release by endothelial cells in response to anti-ß2GPI antibodies.

Characterization of myosin light chain gene up-regulated in the large yellow croaker immunity by interaction with RanGTPase.

RanGTPases are highly conserved in eukaryotes from yeast to human and have been implicated in many aspects of nuclear structure and function. In our previous study, it was revealed that the RanGTPase was up-regulated in large yellow croaker challenged by pathogen. However, the mechanism of RanGTPase in immunity remains unclear. In this investigation, on the basis of protein interaction, it was found that RanGTPase interacted with myosin light chain (designated as LycMLC), a crucial protein in the process of phagocytosis. Furthermore, it was found and characterized in this marine fish for the first time. The full-length cDNA of LycMLC was 771bp, including a 5'-terminal untranslated region (UTR) of 36bp, 3'-terminal UTR of 279bp and an open reading frame (ORF) of 456bp encoding a polypeptide of 151 amino acids. RT-PCR analysis indicated that LycMLC gene was constitutively expressed in the 9 tissues examined, including kidney, liver, gill, muscle, spleen, skin, heart, intestine and blood. The result of quantitative real-time PCR analysis revealed the highest expression in muscle and the weakest expression in skin. Time course analysis showed that LycMLC expression was obviously up-regulated in blood after immunization with either poly I:C or formalin-inactive Gram-negative bacteria Vibrio parahaemolyticus. It indicated that the highest expression was 4.5 times (at 24h) as much as that in the control (P<0.05) challenged by poly I:C and 5.0 times (at 24h) challenged by bacteria. These results suggested that LycMLC might play an important role in large yellow croaker defense against the pathogen infection. Therefore our study revealed a novel pathway concerning immunity of RanGTPase by the direct interaction with the cytoskeleton protein, which would help to better understand the molecular events in immune response against pathogen infection in fish.

Identification of novel autoantigens by a triangulation approach.

High titer autoantibodies, which are often associated with specific clinical phenotypes, are useful diagnostically and prognostically in systemic autoimmune diseases. In several autoimmune rheumatic diseases (e.g. myositis and Sjogren's syndrome), 20-40% of patients are autoantibody negative as assessed by conventional assays. The recent discovery of new specificities (e.g., anti-MDA5) in a subset of these autoantibody-negative subjects demonstrates that additional specificities await identification. In this manuscript, we describe a rapid multidimensional method to identify new autoantigens. A central foundation of this rapid approach is the use of an antigen source in which a pathogenic pathway active in the disease is recapitulated. Additionally, the method involves a modified serological proteome analysis strategy which allows confirmation that the correct gel plug has been removed prior to sending for sequencing. Lastly, the approach uses multiple sources of information to enable rapid triangulation and identification of protein candidates. Possible permutations and underlying principles of this triangulation strategy are elaborated to demonstrate the broad utility of this approach for antigen discovery.

Synthesis and evaluation of an anti-MLC1 × anti-CD90 bispecific antibody for targeting and retaining bone-marrow-derived multipotent stromal cells in infarcted myocardium.

A key issue regarding the use of stem cells in cardiovascular regenerative medicine is their retention in target tissues. Here, we have generated and assessed a bispecific antibody heterodimer designed to improve the retention of bone-marrow-derived multipotent stromal cells (BMMSC) in cardiac tissue damaged by myocardial infarction. The heterodimer comprises an anti-human CD90 monoclonal antibody (mAb) (clone 5E10) and an anti-myosin light chain 1 (MLC1) mAb (clone MLM508) covalently cross-linked by a bis-arylhydrazone. We modified the anti-CD90 antibody with a pegylated-4-formylbenzamide moiety to a molar substitution ratio (MSR) of 2.6 and the anti-MLC1 antibody with a 6-hydrazinonicotinamide moiety to a MSR of 0.9. The covalent modifications had no significant deleterious effect on mAb epitope binding. Furthermore, the binding of anti-CD90 antibody to BMMSCs did not prevent their differentiation into adipo-, chondro-, or osteogenic lineages. Modified antibodies were combined under mild conditions (room temperature, pH 6, 1 h) in the presence of a catalyst (aniline) to allow for rapid generation of the covalent bis-arylhydrazone, which was monitored at A(354). We evaluated epitope immunoreactivity for each mAb in the construct. Flow cytometry demonstrated binding of the bispecific construct to BMMSCs that was competed by free anti-CD90 mAb, verifying that modification and cross-linking were not detrimental to the anti-CD90 complementarity-determining region. Similarly, ELISA-based assays demonstrated bispecific antibody binding to plastic-immobilized recombinant MLC1. Excess anti-MLC1 mAb competed for bispecific antibody binding. Finally, the anti-CD90 × anti-MLC1 bispecific antibody construct induced BMMSC adhesion to plastic-immobilized MLC1 that was resistant to shear stress, as measured in parallel-plate flow chamber assays. We used mAbs that bind both human antigens and the respective pig homologues. Thus, the anti-CD90 × anti-MLC1 bispecific antibody may be used in large animal studies of acute myocardial infarction and may provide a starting point for clinical studies.