Postoperative D-dimer levels predict venous thromboembolisms detected with contrast-enhanced computerized tomography in patients undergoing anterior cruciate ligament reconstruction.

BACKGROUND: In the literature, factors associated with postoperative venous thromboembolisms (VTEs) after anterior cruciate ligament reconstruction (ACLR) are limited. This study aimed to investigate the incidence of venous thromboembolisms (VTEs) after anterior cruciate ligament reconstruction (ACLR) and to identify risk and predictive factors for VTEs. METHODS: This retrospective study included 136 patients who underwent arthroscopic ACLR with mechanical prophylaxis between April 2012 and July 2022. Contrast-enhanced computed tomography (CT) was applied to detect VTEs comprising deep venous thromboses and pulmonary embolisms 7 days after surgery. Data including age, sex, body mass index, concomitant treatments, graft types, smoking status, operative and tourniquet times, postoperative D-dimer levels, and other laboratory test results, were collected for analyses. The incidence of radiographically confirmed VTEs and the associated risk factors, such as age, sex, body mass index, concomitant treatments, graft types, smoking status, operative and tourniquet times, postoperative D-dimer levels, and other laboratory test results, were analyzed. RESULTS: The overall incidence of radiographic VTEs was 11.0% (15 cases) in 136 patients. There was one symptomatic patient who had Homan's sign. Multivariable analysis indicated that postoperative D-dimer level was an independent factor related to a radiographic VTE after ACLR, although there was no association between radiographic VTEs and preoperative status or operation status. The optimal cutoff value for postoperative D-dimer level was 2.8 µg/ml according to the receiver operating characteristic curve analysis, with a sensitivity of 80.0% and specificity of 83.5%. CONCLUSION: The incidence of ACLR-associated radiographical VTEs (deep venous thrombosis and pulmonary embolism) under mechanical prophylaxis was 11.0% in this study. An elevated D-dimer level at 7 days after surgery is an independent predictor of VTE in patients undergoing ACLR. The postoperative D-dimer level is a more reliable marker for identifying VTE in patients who underwent ACLR.

Peripheral endomorphins drive mechanical alloknesis under the enzymatic control of CD26/DPPIV.

BACKGROUND: Mechanical alloknesis (or innocuous mechanical stimuli-evoked itch) often occurs in dry skin-based disorders such as atopic dermatitis and psoriasis. However, the molecular and cellular mechanisms underlying mechanical alloknesis remain unclear. We recently reported the involvement of CD26 in the regulation of psoriatic itch. This molecule exhibits dipeptidyl peptidase IV (DPPIV) enzyme activity and exerts its biologic effects by processing various substances, including neuropeptides. OBJECTIVE: The aim of the present study was to investigate the peripheral mechanisms of mechanical alloknesis by using CD26/DPPIV knockout (CD26KO) mice. METHODS: We applied innocuous mechanical stimuli to CD26KO or wild-type mice. The total number of scratching responses was counted as the alloknesis score. Immunohistochemical and behavioral pharmacologic analyses were then performed to examine the physiologic activities of CD26/DPPIV or endomorphins (EMs), endogenous agonists of µ-opioid receptors. RESULTS: Mechanical alloknesis was more frequent in CD26KO mice than in wild-type mice. The alloknesis score in CD26KO mice was significantly reduced by the intradermal administration of recombinant DPPIV or naloxone methiodide, a peripheral µ-opioid receptor antagonist, but not by that of mutant DPPIV without enzyme activity. EMs (EM-1 and EM-2), selective ligands for µ-opioid receptors, are substrates for DPPIV. Immunohistochemically, EMs were located in keratinocytes, fibroblasts, and peripheral sensory nerves. Behavioral analyses revealed that EMs preferentially provoked mechanical alloknesis over chemical itch. DPPIV-digested forms of EMs did not induce mechanical alloknesis. CONCLUSION: The present results suggest that EMs induce mechanical alloknesis at the periphery under the enzymatic control of CD26/DPPIV.

Characterization of novel anti-IL-26 neutralizing monoclonal antibodies for the treatment of inflammatory diseases including psoriasis.

Interleukin (IL)-26, known as a Th17 cytokine, acts on various cell types and has multiple biological functions. Although its precise role still remains to be elucidated, IL-26 is suggested to be associated with the pathology of diverse chronic inflammatory diseases such as psoriasis, inflammatory bowel diseases and rheumatoid arthritis. To develop novel neutralizing anti-human IL-26 monoclonal antibodies (mAbs) for therapeutic use in the clinical setting, we immunized mice with human IL-26 protein. Hybridomas producing anti-IL-26 mAbs were screened for various in vitro functional assays, STAT3 phosphorylation and antibiotic assays. Although the IL-20RA/IL-10RB heterodimer is generally believed to be the IL-26 receptor, our data strongly suggest that both IL-20RA-dependent and -independent pathways are involved in IL-26-mediated stimulation. We also investigated the potential therapeutic effect of anti-IL-26 mAbs in the imiquimod-induced psoriasis-like murine model using human IL-26 transgenic mice. These screening methods enabled us to develop novel neutralizing anti-human IL-26 mAbs. Importantly, administration of IL-26-neutralizing mAb did not have an effect on the antimicrobial activity of IL-26. Taken together, our data strongly suggest that our newly developed anti-human IL-26 mAb is a potential therapeutic agent for the treatment of diverse chronic inflammatory diseases including psoriasis.

Development of novel monoclonal antibodies with specific binding affinity for denatured human CD26 in formalin-fixed paraffin-embedded and decalcified specimens.

A 110-kDa type II transmembrane glycoprotein with dipeptidyl peptidase IV (DPPIV) activity in its extracellular region, CD26 has a multitude of biological functions and plays an important role in the regulation of inflammatory responses and tumor biology. Our work has focused on CD26 as a novel therapeutic target for various tumors and immune disorders, and we have recently developed a humanized anti-CD26 monoclonal antibody (mAb), YS110, which has promising safety profile and clinical activity in patients with malignant pleural mesothelioma. The development of an anti-human CD26 mAb that can clearly and reliably detect the denatured CD26 molecule in formalin-fixed paraffin-embedded (FFPE) tissues in the clinical setting is therefore of the utmost importance. To develop novel anti-CD26 mAbs capable of binding to denatured CD26, we immunized mice with urea-treated CD26 protein. Hybridoma supernatants were screened for specific reactivity with human CD26 by immunostaining through the use of a set of FFPE human CD26-positive or negative tumor cell lines. This screening method enables us to develop novel anti-human CD26 mAbs suitable for immunohistochemical staining of CD26 in FFPE non-tumor and tumor tissue sections with reliable clarity and intensity. Specifically, these mAbs display strong binding affinity to denatured human CD26 rather than undenatured human CD26, and are capable of detecting denatured human CD26 in decalcified specimens. These novel anti-CD26 mAbs are potentially useful for the analysis of CD26 expression in cancer patients with bony metastasis, and may help decide the appropriateness of YS110 therapy for future cancer patients.

A novel role for CD26/dipeptidyl peptidase IV as a therapeutic target.

CD26 is a 110 kDa surface glycoprotein with intrinsic dipeptidyl peptidase IV activity that is expressed on numerous cell types and has a multitude of biological functions. The role of CD26 in immune regulation has been extensively characterized, with recent findings elucidating its linkage with signaling pathways and structures involved in T-lymphocyte activation as well as antigen presenting cell-T-cell interaction. In this paper, we will review emerging data on CD26-mediated immune regulation suggesting that CD26 may be an appropriate therapeutic target for the treatment of selected immune disorders as well as Middle East respiratory syndrome coronavirus. Moreover, we have had a long-standing interest in the role of CD26 in cancer biology and its suitability as a novel therapeutic target in selected neoplasms. We reported robust in vivo data on the anti-tumor activity of anti-CD26 monoclonal antibody in mouse xenograft models. We herein review significant novel findings and the early clinical development of a CD26-targeted therapy in selected immune disorders and cancers, advances that can lead to a more hopeful future for patients with these intractable diseases.

A possible role for CD26/DPPIV enzyme activity in the regulation of psoriatic pruritus.

BACKGROUND: Psoriasis (PSO) is one of the most common chronic inflammatory skin diseases, and pruritus affects approximately 60-90% of patients with PSO. However, the pathogenesis of pruritus in PSO remains unclear. Dipeptidyl peptidase IV (DPPIV) enzyme activity is involved in the regulation of peptide hormones, chemokines and neurotransmitters. OBJECTIVES: Our aim is to evaluate for a potential association between DPPIV and an increased risk of pruritus, and to identify possible underlying treatment targets in affected patients. METHODS: Utilizing clinical serum samples of PSO patients and in vivo experimental pruritus models, we evaluated for a potential association between DPPIV and an increased risk for pruritus, and attempted to identify possible underlying treatment targets in pruritus of PSO. RESULTS: We first showed that levels of DPPIV enzyme activity in sera of patients with PSO were significantly increased compared to those of healthy controls. We next evaluated levels of substance-P (SP), which is a neurotransmitter for pruritus and a substrate for DPPIV enzyme. Truncated form SP cleaved by DPPIV was significantly increased in sera of PSO. In an in vivo pruritus model induced by SP, scratching was decreased by treatment with a DPPIV inhibitor. Moreover, DPPIV-knockout mice showed attenuation of scratching induced by SP. Finally, scratching was decreased following the administration of a DPPIV inhibitor in an imiquimod-induced PSO model. On the other hand, scratching induced by imiquimod was increased in DPPIV overexpressing-mice. CONCLUSIONS: These results suggest that inhibition of DPPIV enzyme activity regulates pruritus in PSO.

Inhibition of VEGF-dependent angiogenesis by the anti-CD82 monoclonal antibody 4F9 through regulation of lipid raft microdomains.

CD82 (also known as KAI1) belongs to the tetraspanin superfamily of type III transmembrane proteins, and is involved in regulating cell adhesion, migration and proliferation. In contrast to these well-established roles of CD82 in tumor biology, its function in endothelial cell (EC) activity and tumor angiogenesis is yet to be determined. In this study, we show that suppression of CD82 negatively regulates vascular endothelial growth factor (VEGF)-induced angiogenesis. Moreover, we demonstrate that the anti-CD82 mAb 4F9 effectively inhibits phosphorylation of VEGF receptor 2 (VEGFR2), which is the principal mediator of the VEGF-induced angiogenic signaling process in tumor angiogenesis, by regulating the organization of the lipid raft microdomain signaling platform in human EC. Our present work therefore suggests that CD82 on EC is a potential target for anti-angiogenic therapy in VEGFR2-dependent tumor angiogenesis.

CD26-mediated induction of EGR2 and IL-10 as potential regulatory mechanism for CD26 costimulatory pathway.

CD26 is associated with T cell signal transduction processes as a costimulatory molecule, and CD26(+) T cells have been suggested to be involved in the pathophysiology of diverse autoimmune diseases. Although the cellular and molecular mechanisms involved in CD26-mediated T cell activation have been extensively evaluated by our group and others, potential negative feedback mechanisms to regulate CD26-mediated activation still remain to be elucidated. In the present study, we examine the expression of inhibitory molecules induced via CD26-mediated costimulation. We show that coengagement of CD3 and CD26 induces preferential production of IL-10 in human CD4(+) T cells, mediated through NFAT and Raf-MEK-ERK pathways. A high level of early growth response 2 (EGR2) is also induced following CD26 costimulation, possibly via NFAT and AP-1-mediated signaling, and knockdown of EGR2 leads to decreased IL-10 production. Furthermore, CD3/CD26-stimulated CD4(+) T cells clearly suppress proliferative activity and effector cytokine production of bystander T cells in an IL-10-dependent manner. Taken together, our data suggest that robust CD26 costimulatory signaling induces preferential expression of EGR2 and IL-10 as a potential mechanism for regulating CD26-mediated activation.

Angiomodulin, a marker of cancer vasculature, is upregulated by vascular endothelial growth factor and increases vascular permeability as a ligand of integrin αvß3.

Angiomodulin (AGM) is a member of insulin-like growth factor binding protein (IGFBP) superfamily and often called IGFBP-rP1 or IGFBP-7. AGM was originally identified as a tumor-derived cell adhesion factor, which was highly accumulated in blood vessels of human cancer tissues. AGM is also overexpressed in cancer-associated fibroblasts (CAFs) and activates fibroblasts. However, some studies have shown tumor-suppressing activity of AGM. To understand the roles of AGM in cancer progression, we here investigated the expression of AGM in benign and invasive breast cancers and its functions in cancer vasculature. Immunohistochemical analysis showed that AGM was highly expressed in cancer vasculature even in ductal carcinoma in situ (DCIS) as compared to normal vasculature, while its expression in CAFs was more prominent in invasive carcinomas than DCIS. In vitro analyses showed that AGM was strongly induced by vascular endothelial cell growth factor (VEGF) in vascular endothelial cells. Although AGM stimulated neither the growth nor migration of endothelial cells, it supported efficient adhesion of endothelial cells. Integrin αvß3 was identified as a novel major receptor for AGM in vascular endothelial cells. AGM retracted endothelial cells by inducing actin stress fibers and loosened their VE-cadherin-mediated intercellular junction. Consequently, AGM increased vascular permeability both in vitro and in vivo. Furthermore, AGM and integrin αvß3 were highly expressed and colocalized in cancer vasculature. These results suggest that AGM cooperates with VEGF to induce the aberrant functions of cancer vasculature as a ligand of integrin αvß3.

CD26-mediated regulation of periostin expression contributes to migration and invasion of malignant pleural mesothelioma cells.

Malignant pleural mesothelioma (MPM) is an aggressive malignancy arising from mesothelial lining of pleura. It is generally associated with a history of asbestos exposure and has a very poor prognosis, partly due to the lack of a precise understanding of the molecular mechanisms associated with its malignant behavior. In the present study, we expanded on our previous studies on the enhanced motility and increased CD26 expression in MPM cells, with a particular focus on integrin adhesion molecules. We found that expression of CD26 upregulates periostin secretion by MPM cells, leading to enhanced MPM cell migratory and invasive activity. Moreover, we showed that upregulation of periostin expression results from the nuclear translocation of the basic helix-loop-helix transcription factor Twist1, a process that is mediated by CD26-associated activation of Src phosphorylation. While providing new and profound insights into the molecular mechanisms involved in MPM biology, these findings may also lead to the development of novel therapeutic strategies for MPM.

Establishment of monoclonal anti-human CD26 antibodies suitable for immunostaining of formalin-fixed tissue.

BACKGROUND: A T cell costimulatory molecule with dipeptidyl peptidase IV (DPPIV) activity in its extracellular region, CD26 is a multifunctional molecule associated with various proteins such as adenosine deaminase, caveolin-1, CXCR4, collagen, and fibronectin, while playing an important role in the regulation of inflammatory responses and tumor biology. We have focused on CD26 as a novel therapeutic target for various tumors and immune disorders, and have developed a humanized anti-CD26 monoclonal antibody (mAb), YS110, which is currently being evaluated in a phase I clinical trial for patients with CD26-expressing tumors, including malignant mesothelioma. Since detection of tumor CD26 expression is required for determining potential eligibility for YS110 therapy, the development of anti-human CD26 mAb that can clearly and reliably detect the denatured CD26 molecule in the formalin-fixed paraffin-embedded tissues is critical. METHODS: To develop novel anti-CD26 mAbs capable of binding to the denatured CD26, we immunized mice with CD26 protein denatured in urea buffer. After the fusion of splenocytes and myeloma cells, the mAbs were screened for specific reactivity with human CD26 by flow cytometry, enzyme-linked immunosorbent assay, and immunohistochemistry. The binding competitiveness of novel anti-CD26 mAbs with the humanized anti-CD26 mAb YS110 was also examined. RESULTS: We have succeeded in developing novel anti-human CD26 mAbs suitable for immunohistochemical staining of CD26 in formalin-fixed tissue sections with reliable clarity and intensity. Importantly, some of these mAbs exhibit no cross-reactivity with the humanized anti-CD26 mAb. CONCLUSIONS: These novel mAbs are potentially useful as companion diagnostic agents to analyze CD26 expression in the clinical setting while advancing future CD26-related research. VIRTUAL SLIDES: The virtual slides for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/5987140221097729.

CD9 negatively regulates CD26 expression and inhibits CD26-mediated enhancement of invasive potential of malignant mesothelioma cells.

CD26/dipeptidyl peptidase IV is a cell surface glycoprotein which consists of multiple functional domains beside its ectopeptidase site. A growing body of evidence indicates that elevated expression of CD26 correlates with disease aggressiveness and invasive potential of selected malignancies. To further explore the molecular mechanisms involved in this clinical behavior, our current work focused on the interaction between CD26 and CD9, which were recently identified as novel markers for cancer stem cells in malignant mesothelioma. We found that CD26 and CD9 co-modulated and co-precipitated with each other in the malignant mesothelioma cell lines ACC-MESO1 and MSTO-211H. SiRNA study revealed that depletion of CD26 led to increased CD9 expression, while depletion of CD9 resulted in increased CD26 expression. Consistent with these findings was the fact that gene transfer of CD26 into CD26-negative MSTO-211H cells reduced CD9 expression. Cell invasion assay showed that overexpression of CD26 or gene depletion of CD9 led to enhanced invasiveness, while CD26 gene depletion resulted in reduced invasive potential. Furthermore, our work suggested that this enhanced invasiveness may be partly mediated by α5ß1 integrin, since co-precipitation studies demonstrated an association between CD26 and α5ß1 integrin. Finally, gene depletion of CD9 resulted in elevated protein levels and tyrosine phosphorylation of FAK and Cas-L, which are downstream of ß1 integrin, while depletion of CD26 led to a reduction in the levels of these molecules. Collectively, our findings suggest that CD26 potentiates tumor cell invasion through its interaction with α5ß1 integrin, and CD9 negatively regulates tumor cell invasion by reducing the level of CD26-α5ß1 integrin complex through an inverse correlation between CD9 and CD26 expression. Our results also suggest that CD26 and CD9 serve as potential biomarkers as well as promising molecular targets for novel therapeutic approaches in malignant mesothelioma and other malignancies.

Modulation of matrix metalloproteinase-9 secretion from tumor-associated macrophage-like cells by proteolytically processed laminin-332 (laminin-5).

Macrophages infiltrating tumor tissues (tumor-associated macrophages, TAM) affect the malignant behaviors of tumor cells. We previously reported that monocytes were differentiated into TAM-like cells secreting matrix metalloproteinase (MMP)-9 by co-culture with tumor cells, and that cell adhesion to extracellular matrix (ECM) proteins played a critical role in the differentiation. In this study, we found that the monocyte differentiation was promoted by laminin-332 (laminin-5), a major epithelial ECM component. We also demonstrated that the proteolytic processing of the γ2 chain of laminin-332 was essential for its activity but that the N-terminal short arm of the γ2 chain inhibited MMP-9 secretion. These results indicate that the activity of laminin-332 for monocyte differentiation is dynamically regulated by the proteolytic processing of the γ2 chain.

Amino-terminal fragments of laminin γ2 chain retract vascular endothelial cells and increase vascular permeability.

Laminin γ2 (Lmγ2) chain, a subunit of laminin-332, is a typical molecular marker of invading cancer cells, and its expression correlates with poor prognosis of cancer patients. It was previously found that forced expression of Lmγ2 in cancer cells promotes their invasive growth in nude mice. However, the mechanism of the tumor-promoting activity of Lmγ2 remains unknown. Here we investigated the interaction between Lmγ2 and vascular endothelial cells. When treated with an N-terminal proteolytic fragment of γ2 (γ2pf), HUVECs became markedly retracted or shrunken. The overexpression of Lmγ2 or treatment with γ2pf stimulated T-24 bladder carcinoma cells to invade into the HUVEC monolayer and enhanced their transendothelial migration in vitro. Moreover, γ2pf increased endothelial permeability in vitro and in vivo. As the possible mechanisms, γ2pf activated ERK and p38 MAPK but inactivated Akt in HUVECs. Such effects of γ2pf led to prominent actin stress fiber formation in HUVECs, which was blocked by a ROCK inhibitor. In addition, γ2pf induced delocalization of VE-cadherin and ß-catenin from the intercellular junction. As possible receptors, γ2pf interacted with heparan sulfate proteoglycans on the surface of HUVECs. Moreover, we localized the active site of γ2pf to the N-terminal epidermal growth factor-like repeat. These data suggest that the interaction between γ2pf and heparan sulfate proteoglycans induces cytoskeletal changes of endothelial cells, leading to the loss of endothelial barrier function and the enhanced transendothelial migration of cancer cells. These activities of Lmγ2 seem to support the aberrant growth of cancer cells.

Elevated expression of angiomodulin (AGM/IGFBP-rP1) in tumor stroma and its roles in fibroblast activation.

Angiomodulin (AGM/IGFBP-rP1), a glycoprotein of about 30 kDa, is overexpressed in tumor vasculature as well as some human cancer cell lines, but it has been suggested to be a tumor suppressor. To elucidate roles of angiomodulin (AGM) in tumor progression, we here examined distribution of AGM in three types of human cancer tissues by immunohistochemistry. The results showed that AGM was overexpressed in the stroma as well as the vasculature surrounding tumor cells in the human cancer tissues. AGM and α-smooth muscle actin (α-SMA) as an activated fibroblast marker were often colocalized in cancer-associated fibroblasts (CAFs). In vitro analysis indicated that transforming growth factor (TGF)-ß1 might be an important inducer of AGM in normal human fibroblasts. AGM strongly stimulated the expression of fibronectin and weakly that of α-SMA in normal fibroblasts. AGM significantly stimulated the proliferation and migration of fibroblasts. The AGM-induced expression of fibronectin and α-SMA was blocked by a TGF-ß signal inhibitor but neither the stimulation of cell growth nor migration. These results imply that AGM activates normal fibroblasts by TGF-ß-dependent and independent mechanisms. These findings also suggest that AGM and TGF-ß1 cooperatively or complementarily contribute to the stromal activation and connective tissue formation in human cancer tissues, contributing to tumor progression.

Downregulation of a newly identified laminin, laminin-3B11, in vascular basement membranes of invasive human breast cancers.

Laminins present in the basement membranes (BM) of blood vessels are involved in angiogenesis and other vascular functions that are critical for tumor growth and metastasis. Two major vascular laminins, the α4 (laminin-411/421) and α5 (laminin-511/521) types, have been well characterized. We recently found a third type of vascular laminin, laminin-3B11, consisting of the α3B, ß1 and γ1 chains, and revealed its biological activity. Laminin-3B11 potently stimulates vascular endothelial cells to extend lamellipodial protrusions. To understand the roles of laminin-3B11 in blood vessel functions and tumor growth, we examined localization of the laminin α3B chain in normal mammary glands and breast cancers, in comparison with the α4 and α5 laminins. In the immunohistochemical analysis, the α3B laminin was co-localized with the α4 and α5 laminins in the BM of venules and capillaries of normal breast tissues, but α3B was scarcely detected in vessels near invasive breast carcinoma cells. In contrast, the α4 laminin was overexpressed in capillaries of invasive carcinomas, where a large number of macrophages were found. The α5 laminin appeared to be weakly downregulated in cancer tissues, especially in capillary vessels. Furthermore, our in vitro analysis indicated that TNF-α significantly suppressed the laminin α3B expression in vascular endothelial cells, while it, as well as IL-1ß and TGF-α, upregulated the α4 expression. These results suggest that Lm3B11/3B21 may be required for normal mature vessels and interfere with tumor angiogenesis.