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Background: Tanimilast is a novel and selective inhaled inhibitor of phosphodiesterase-4 in advanced clinical development for chronic obstructive pulmonary disease (COPD). Tanimilast is known to exert prominent anti-inflammatory activity when tested in preclinical experimental models as well as in human clinical studies. Recently, we have demonstrated that it also finely tunes, rather than suppressing, the cytokine network secreted by activated dendritic cells (DCs). This study was designed to characterize the effects of tanimilast on T-cell polarizing properties of DCs and to investigate additional functional and phenotypical features induced by tanimilast., Methods: DCs at day 6 of culture were stimulated with LPS in the presence or absence of tanimilast or the control drug budesonide. After 24 h, DCs were analyzed for the expression of surface markers of maturation and activation by flow cytometry and cocultured with T cells to investigate cell proliferation and activation/polarization. The regulation of type 2-skewing mediators was investigated by real-time PCR in DCs and compared to results obtained in vivo in a randomized placebo-controlled trial on COPD patients treated with tanimilast., Results: Our results show that both tanimilast and budesonide reduced the production of the immunostimulatory cytokine IFN-γ by CD4 + T cells. However, the two drugs acted at different levels since budesonide mainly blocked T cell proliferation, while tanimilast skewed T cells towards a Th2 phenotype without affecting cell proliferation. In addition, only DCs matured in the presence of tanimilast displayed increased CD86/CD80 ratio and CD141 expression, which correlated with Th2 T cell induction and dead cell uptake respectively. These cells also upregulated cAMP-dependent immunosuppressive molecules such as IDO1, TSP1, VEGF-A and Amphiregulin. Notably, the translational value of these data was confirmed by the finding that these same genes were upregulated also in sputum cells of COPD patients treated with tanimilast as add-on to inhaled glucocorticoids and bronchodilators., Conclusion: Taken together, these findings demonstrate distinct immunomodulatory properties of tanimilast associated with a type 2 endotype and CD141 upregulation in DCs and provide a mechanistic rationale for the administration of tanimilast on top of inhaled corticosteroids., (© 2022. The Author(s).)

Genetic analysis of human inborn errors of immunity has defined the contribution of specific cell populations and molecular pathways in the host defense against infection. The STAT family of transcription factors orchestrate hematopoietic cell differentiation. Patients with de novo activating mutations of STAT3 present with multiorgan autoimmunity, lymphoproliferation, and recurrent infections. We conducted a detailed characterization of the blood monocyte and dendritic cell (DC) subsets in patients with gain-of-function (GOF) mutations across the gene. We found a selective deficiency in circulating nonclassical CD16 + and intermediate CD16 + CD14 + monocytes and a significant increase in the percentage of classical CD14 + monocytes. This suggests a role for STAT3 in the transition of classical CD14 + monocytes into the CD16 + nonclassical subset. Developmentally, ex vivo-isolated STAT3 GOF CD14 + monocytes fail to differentiate into CD1a + monocyte-derived DCs. Moreover, patients with STAT3 GOF mutations display reduced circulating CD34 + hematopoietic progenitors and frequency of myeloid DCs. Specifically, we observed a reduction in the CD141 + DC population, with no difference in the frequencies of CD1c + and plasmacytoid DCs. CD34 + hematopoietic progenitor cells from patients were found to differentiate into CD1c + DCs, but failed to differentiate into CD141 + DCs indicating an intrinsic role for STAT3 in this process. STAT3 GOF -differentiated DCs produced lower amounts of CCL22 than healthy DCs, which could further explain some of the patient pathological phenotypes. Thus, our findings provide evidence that, in humans, STAT3 serves to regulate development and differentiation of nonclassical CD16 + monocytes and a subset of myeloid DCs., (Copyright © 2021 by The American Association of Immunologists, Inc.)

Drug targeting to inflammatory brain pathologies such as stroke and traumatic brain injury remains an elusive goal. Using a mouse model of acute brain inflammation induced by local tumor necrosis factor alpha (TNFα), we found that uptake of intravenously injected antibody to vascular cell adhesion molecule 1 (anti-VCAM) in the inflamed brain is >10-fold greater than antibodies to transferrin receptor-1 and intercellular adhesion molecule 1 (TfR-1 and ICAM-1). Furthermore, uptake of anti-VCAM/liposomes exceeded that of anti-TfR and anti-ICAM counterparts by ∼27- and ∼8-fold, respectively, achieving brain/blood ratio >300-fold higher than that of immunoglobulin G/liposomes. Single-photon emission computed tomography imaging affirmed specific anti-VCAM/liposome targeting to inflamed brain in mice. Intravital microscopy via cranial window and flow cytometry showed that in the inflamed brain anti-VCAM/liposomes bind to endothelium, not to leukocytes. Anti-VCAM/LNP selectively accumulated in the inflamed brain, providing de novo expression of proteins encoded by cargo messenger RNA (mRNA). Anti-VCAM/LNP-mRNA mediated expression of thrombomodulin (a natural endothelial inhibitor of thrombosis, inflammation, and vascular leakage) and alleviated TNFα-induced brain edema. Thus VCAM-directed nanocarriers provide a platform for cerebrovascular targeting to inflamed brain, with the goal of normalizing the integrity of the blood-brain barrier, thus benefiting numerous brain pathologies., Competing Interests: Competing interest statement: O.A.M.-C., H.P., V.V.S., D.W., and V.R.M. are inventors on a patent filed on some aspects of this work. Those interests have been fully disclosed to the University of Pennsylvania.

Tolerogenic dendritic cells (DCs) are key players in maintaining immunological homeostasis, dampening immune responses, and promoting tolerance. DC-10, a tolerogenic population of human IL-10-producing DCs characterized by the expression of HLA-G and ILT4, play a pivotal role in promoting tolerance via T regulatory type 1 (Tr1) cells. Thus far, the absence of markers that uniquely identify DC-10 has limited in vivo studies. By in vitro gene expression profiling of differentiated human DCs, we identified CD141 and CD163 as surface markers for DC-10. The coexpression of CD141 and CD163 in combination with CD14 and CD16 enables the ex vivo isolation of DC-10 from the peripheral blood. CD14 + CD16 + CD141 + CD163 + cells isolated from the peripheral blood of healthy subjects (ex vivo DC-10) produced spontaneously and upon activation of IL-10 and limited levels of IL-12. Moreover, in vitro stimulation of allogeneic naive CD4 + T cells with ex vivo DC-10 induced the differentiation of alloantigen-specific CD49b + LAG-3 + Tr1 cells. Finally, ex vivo DC-10 and in vitro generated DC-10 exhibited a similar transcriptional profile, which are characterized by an anti-inflammatory and pro-tolerogenic signature. These results provide new insights into the phenotype and molecular signature of DC-10 and highlight the tolerogenic properties of circulating DC-10. These findings open the opportunity to track DC-10 in vivo and to define their role in physiological and pathological settings.

Xenotransplantation research has made considerable progress in recent years, largely through the increasing availability of pigs with multiple genetic modifications. We suggest that a pig with nine genetic modifications (ie, currently available) will provide organs (initially kidneys and hearts) that would function for a clinically valuable period of time, for example, >12 months, after transplantation into patients with end-stage organ failure. The national regulatory authorities, however, will likely require evidence, based on in vitro and/or in vivo experimental data, to justify the inclusion of each individual genetic modification in the pig. We provide data both from our own experience and that of others on the advantages of pigs in which (a) all three known carbohydrate xenoantigens have been deleted (triple-knockout pigs), (b) two human complement-regulatory proteins (CD46, CD55) and two human coagulation-regulatory proteins (thrombomodulin, endothelial cell protein C receptor) are expressed, (c) the anti-apoptotic and "anti-inflammatory" molecule, human hemeoxygenase-1 is expressed, and (d) human CD47 is expressed to suppress elements of the macrophage and T-cell responses. Although many alternative genetic modifications could be made to an organ-source pig, we suggest that the genetic manipulations we identify above will all contribute to the success of the initial clinical pig kidney or heart transplants, and that the beneficial contribution of each individual manipulation is supported by considerable experimental evidence., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The multifaceted role of mitogen-activated protein kinases (MAPKs) in modulating signal transduction pathways in inflammatory conditions such as infection, cardiovascular disease, and cancer has been well established. Recently, coagulation factors have also emerged as key players in regulating intracellular signaling pathways during inflammation. Among coagulation factors, thrombomodulin, as a high affinity receptor for thrombin on vascular endothelial cells, has been discovered to be a potent anti-inflammatory and anti-tumorigenic signaling molecule. The protective signaling function of thrombomodulin is separate from its well-recognized role in the clotting cascade, which is to function as an anti-coagulant receptor in order to switch the specificity of thrombin from a procoagulant to an anti-coagulant protease. The underlying protective signaling mechanism of thrombomodulin remains largely unknown, though a few published reports link the receptor to the regulation of MAPKs under different (patho)physiological conditions. The goal of this review is to summarize what is known about the regulatory relationship between thrombomodulin and MAPKs.

A combination of genetic manipulations of donor organs and target-specific immunosuppression is instrumental in achieving long-term cardiac xenograft survival. Recently, results from our preclinical pig-to-baboon heterotopic cardiac xenotransplantation model suggest that a three-pronged approach is successful in extending xenograft survival: (a) α-1,3-galactosyl transferase (Gal) gene knockout in donor pigs (GTKO) to prevent Gal-specific antibody-mediated rejection; (b) transgenic expression of human complement regulatory proteins (hCRP; hCD46) and human thromboregulatory protein thrombomodulin (hTBM) to avoid complement activation and coagulation dysregulation; and (c) effective induction and maintenance of immunomodulation, particularly through co-stimulation blockade of CD40-CD40L pathways with anti-CD40 (2C10R4) monoclonal antibody (mAb). Using this combination of manipulations, we reported significant improvement in cardiac xenograft survival. In this study, we are reporting the survival of cardiac xenotransplantation recipients (n = 3) receiving xenografts from pigs without the expression of hTBM (GTKO.CD46). We observed that all grafts underwent rejection at an early time point (median 70 days) despite utilization of our previously reported successful immunosuppression regimen and effective control of non-Gal antibody response. These results support our hypothesis that transgenic expression of human thrombomodulin in donor pigs confers an independent protective effect for xenograft survival in the setting of a co-stimulation blockade-based immunomodulatory regimen., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

High mobility group box 1 (HMGB1), a nuclear protein, once released into the extracellular space under pathological conditions, plays a pronociceptive role in redox-dependent distinct active forms, all-thiol HMGB1 (at-HMGB1) and disulfide HMGB1 (ds-HMGB1), that accelerate nociception through the receptor for advanced glycation endproducts (RAGE) and Toll-like receptor 4 (TLR4), respectively. Thrombomodulin (TM), an endothelial membrane protein, and soluble TM, known as TMα, promote thrombin-mediated activation of protein C and also sequester HMGB1, which might facilitate thrombin degradation of HMGB1. The present study aimed at clarifying the role of thrombin in TMα-induced suppression of peripheral HMGB1-dependent allodynia in mice. Thrombin-induced degradation of at-HMGB1 and ds-HMGB1 was accelerated by TMα in vitro. Intraplantar (i.pl.) injection of bovine thymus-derived HMGB1 in an unknown redox state, at-HMGB1, ds-HMGB1 or lipopolysaccharide (LPS), known to cause HMGB1 secretion, produced long-lasting mechanical allodynia in mice, as assessed by von Frey test. TMα, when preadministered i.pl., prevented the allodynia caused by bovine thymus-derived HMGB1, at-HMGB1, ds-HMGB1 or LPS, in a dose-dependent manner. The TMα-induced suppression of the allodynia following i.pl. at-HMGB1, ds-HMGB1 or LPS was abolished by systemic preadministration of argatroban, a thrombin-inhibiting agent, and accelerated by i.pl. co-administered thrombin. Our data clearly indicate that TMα is capable of promoting the thrombin-induced degradation of both at-HMGB1 and ds-HMGB1, and suppresses the allodynia caused by either HMGB1 in a thrombin-dependent manner. Considering the emerging role of HMGB1 in distinct pathological pain models, the present study suggests the therapeutic usefulness of TMα for treatment of intractable and/or persistent pain.

As an integral glycoprotein on the surface of endothelial cells, thrombomodulin (TM) has very high affinity for thrombin. TM has been regarded to be a marker of endothelial damage since it can be released during endothelial cell injury. In this work, a highly sensitive fluorescence method for the quantitative detection of TM was developed. TM antibody (Ab) and bovine serum albumin (BSA) were bound on gold nanoparticles (AuNPs) to construct BSA-AuNPs-Ab nanocomposites and they were characterized by transmission electron microscope and UV-vis spectrophotometry. The fluorescence of acridine orange (AO) was quenched by the prepared gold nanocomposites based on fluorescence resonance energy transfer (FRET). In the presence of TM, the fluorescence was turned on due to the effective separation of AO from the surface of gold nanocomposites. Under optimum conditions, the enhanced fluorescence intensity displayed a linear relationship with the logarithm of the TM concentration from 0.1pgmL -1 to 5ngmL -1 with a low detection limit of 12fgmL -1 . The release of soluble thrombomodulin (sTM) by the injured HUVEC-C cells in the presence of H 2 O 2 was investigated using the proposed method. The released sTM content in the growth medium was found to be increased with the enhancement of contact time of the cells with H 2 O 2 ., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Preventing xenograft rejection is one of the greatest challenges of transplantation medicine. Here, we describe a reproducible, long-term survival of cardiac xenografts from alpha 1-3 galactosyltransferase gene knockout pigs, which express human complement regulatory protein CD46 and human thrombomodulin (GTKO.hCD46.hTBM), that were transplanted into baboons. Our immunomodulatory drug regimen includes induction with anti-thymocyte globulin and αCD20 antibody, followed by maintenance with mycophenolate mofetil and an intensively dosed αCD40 (2C10R4) antibody. Median (298 days) and longest (945 days) graft survival in five consecutive recipients using this regimen is significantly prolonged over our recently established survival benchmarks (180 and 500 days, respectively). Remarkably, the reduction of αCD40 antibody dose on day 100 or after 1 year resulted in recrudescence of anti-pig antibody and graft failure. In conclusion, genetic modifications (GTKO.hCD46.hTBM) combined with the treatment regimen tested here consistently prevent humoral rejection and systemic coagulation pathway dysregulation, sustaining long-term cardiac xenograft survival beyond 900 days.

The human cytomegalovirus (HCMV) US2 and US11 gene products hijack mammalian ER-associated degradation (ERAD) to induce rapid degradation of major histocompatibility class I (MHC-I) molecules. The rate-limiting step in this pathway is thought to be the polyubiquitination of MHC-I by distinct host ERAD E3 ubiquitin ligases. TRC8 was identified as the ligase responsible for US2-mediated MHC-I degradation and shown to be required for the cleavage-dependent degradation of some tail-anchored proteins. In addition to MHC-I, plasma membrane profiling identified further immune receptors, which are also substrates for the US2/TRC8 complex. These include at least six α integrins, the coagulation factor thrombomodulin and the NK cell ligand CD112. US2's use of specific HCMV-encoded adaptors makes it an adaptable viral degradation hub. US11-mediated degradation is MHC-I-specific and genetic screens have identified TMEM129, an uncharacterised RING-C2 E3 ligase, as responsible for US11-mediated degradation. In a unique auto-regulatory loop, US11 readily responds to changes in cellular expression of MHC-I. Free US11 either rebinds more MHC-I or is itself degraded by the HRD1/SEL1L E3 ligase complex. While virally encoded US2 and US11 appropriate mammalian ERAD, the MHC-I complex also undergoes stringent cellular quality control and misfolded MHC-I is degraded by the HRD1/SEL1L complex. We discuss the identification and central role of E3 ubiquitin ligases in ER quality control and viral degradation of the MHC-I chain., (Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: With the introduction of the α1, 3-galactosyltransferase gene-knockout (GT-KO) pig and its pivotal role in preventing hyperacute rejection (HAR), coagulation remains a considerable obstacle yet to be overcome in order to provide long-term xenograft survival. Thrombomodulin (TBM) plays a critical anticoagulant and anti-inflammatory role in its part of the protein C pathway. Many studies have demonstrated the strong anticoagulant effects of TBM in xenotransplantation, but its complement regulatory effects have not been appropriately examined. Here, we investigate whether TBM can regulate complement activation as well as coagulation in response to xenogeneic stimuli., Methods: We transfected porcine endothelial cells (MPN-3) with adenovirus vectors containing the human TBM gene (ad-hTBM), or a control gene containing GFP (ad-GFP). The expression level of ad-hTBM was measured by flow cytometry. To confirm the anticoagulant effect of TBM, coagulation time was measured after treatment with recalcified human plasma in ad-hTBM-transfected MPN-3, and a thrombin activity assay was performed after treatment with 50% human serum in ad-hTBM-infected MPN-3., Results: Thrombin generation was significantly decreased in a dose-dependent manner in ad-TBM group, and coagulation time was increased in the ad-hTBM group when compared to the ad-GFP group. Complement-dependent serum toxicity assays were performed after treatment with 20% human serum or heat-inactivated human serum by LDH assay. Complement-dependent toxicity was significantly attenuated in the ad-hTBM group, but complement-independent toxicity was not attenuated in the ad-hTBM group. These results suggest that human thrombomodulin (hTBM) has complement regulatory effects as well as anticoagulant effects. To further investigate the mechanisms of complement regulation by hTBM, we deleted the EGF5, 6 domains that are involved in thrombin generation or the lectin-like domain involved in inflammation of TBM and functional tests were performed using these modified forms. We showed that the EGF5, 6 domain of TBM principally inhibits complement activation rather than the lectin domain., Conclusion: The EGF5, 6 domains of TBM appear to be the major domains for down-regulating the complement system rather than the lectin-like domain during xenogenic stimuli. The role of EGF5, 6 domains of hTBM may be due to inhibition of thrombin as thrombin can cleave C3a and C5a directly and hTBM may also be involved in complement regulation. Clearly then human TBM has complement regulatory effects as well as anticoagulant effects in xeno-immune response, and it is a promising target for attenuating xenograft rejection., (© 2015 The Authors Xenotransplantation Published by John Wiley & Sons Ltd.)

Background: For successful xenotransplantation, in addition to α1,3-galactosyltransferase gene-knockout and human complement regulatory protein (CD46, CD55, CD59) gene insertion, cloned pigs expressing human thrombomodulin (hTM) have been produced to solve the problem of molecular incompatibility in their coagulation system. Recombinant soluble hTM (S-hTM) which has been recently approved for treatment of disseminated intravascular coagulation might be potentially available. The purpose of this study is to examine the functional difference in endothelial cells between membrane-bound hTM (MB-hTM) and S-hTM and to elucidate effective strategy using both types of hTM., Methods: The following factors regarding coagulation and inflammation were compared between hTM-expressing pig aortic endothelial cells (PAEC) derived from cloned pig and nontransgenic PAEC in the presence of S-hTM under tumor necrosis factor-α-activated conditions; (i) clotting time (ii) pig tissue factor (TF), (iii) pig E-selectin, (iv) direct prothrombinase activity, (v) activated protein C (APC), and (vi) prothrombinase activity., Results: The MB-hTM significantly suppressed the expression of pig TF and E-selectin and direct prothrombinase activity in tumor necrosis factor-α-activated PAEC, suggesting strong anti-inflammatory effect, compared to S-hTM. In contrast, S-hTM had more potent capacity to inhibit thrombin generation and to produce APC than MB-hTM, although MB-hTM had the same level of capacity as human endothelial cells., Conclusions: It was speculated that S-hTM treatment would be of assistance during high-risk periods for excessive thrombin formation (e.g., ischemia reperfusion injury or severe infection/rejection). Considering the properties of MB-hTM exhibiting anti-inflammatory function as well as APC production, hTM-expressing cloned pigs might be indispensible to long-term stabilization of graft endothelial cells.

CD14, a multiligand pattern-recognition receptor, is involved in the activation of many TLRs. Thrombomodulin (TM), a type I transmembrane glycoprotein, originally was identified as an anticoagulant factor that activates protein C. Previously, we showed that the recombinant TM lectin-like domain binds to LPS and inhibits LPS-induced inflammation, but the function of the recombinant epidermal growth factor-like domain plus serine/threonine-rich domain of TM (rTMD23) in LPS-induced inflammation remains unknown. In the current study, we found that rTMD23 markedly suppressed the activation of intracellular signaling pathways and the production of inflammatory cytokines induced by LPS. The anti-inflammatory activity of rTMD23 was independent of activated protein C. We also found that rTMD23 interacted with the soluble and membrane forms of CD14 and inhibited the CD14-mediated inflammatory response. Knockdown of CD14 in macrophages suppressed the production of inflammatory cytokines induced by LPS, and rTMD23 inhibited LPS-induced IL-6 production in CD14-knockdown macrophages. rTMD23 suppressed the binding of LPS to macrophages by blocking the association between monocytic membrane-bound TM and CD14. The administration of rTMD23 in mice, both pretreatment and posttreatment, significantly increased the survival rate and reduced the inflammatory response to LPS. Notably, the serine/threonine-rich domain is essential for the anti-inflammatory activity of rTMD23. To summarize, we show that rTMD23 suppresses the LPS-induced inflammatory response in mice by targeting CD14 and that the serine/threonine-rich domain is crucial for the inhibitory effect of rTMD23 on LPS-induced inflammation., (Copyright © 2015 by The American Association of Immunologists, Inc.)

Human organ transplantation is the therapy of choice for end-stage organ failure. However, the demand for organs far exceeds the donation rate, and many patients die while waiting for a donor. Clinical xenotransplantation using discordant species, particularly pigs, offers a possible solution to this critical shortfall. Xenotransplantation can also increase the availability of cells, such as neurons, and tissues such as cornea, insulin producing pancreatic islets and heart valves. However, the immunological barriers and biochemical disparities between pigs and primates (human) lead to rejection reactions despite the use of common immunosuppressive drugs. These result in graft vessel destruction, haemorrhage, oedema, thrombus formation, and transplant loss. Our consortium is pursuing a broad range of strategies to overcome these obstacles. These include genetic modification of the donor animals to knock out genes responsible for xenoreactive surface epitopes and to express multiple xenoprotective molecules such as the human complement regulators CD46, 55, 59, thrombomodulin and others. We are using (new) drugs including complement inhibitors (e.g. to inhibit C3 binding), anti-CD20, 40, 40L, and also employing physical protection methods such as macro-encapsulation of pancreatic islets. Regarding safety, a major objective is to assure that possible infections are not transmitted to recipients. While the aims are ambitious, recent successes in preclinical studies suggest that xenotransplantation is soon to become a clinical reality.

Objectives: Cardiac transplantation and available mechanical alternatives are the only possible solutions for end-stage cardiac disease. Unfortunately, because of the limited supply of human organs, xenotransplantation may be the ideal method to overcome this shortage. We have recently seen significant prolongation of heterotopic cardiac xenograft survival from 3 to 12 months and beyond., Methods: Hearts from genetically engineered piglets that were alpha 1-3 galactosidase transferase knockout and expressed the human complement regulatory gene, CD46 (groups A-C), and the human thrombomodulin gene (group D) were heterotropically transplanted in baboons treated with antithymocyte globulin, cobra venom factor, anti-CD20 antibody, and costimulation blockade (anti-CD154 antibody [clone 5C8]) in group A, anti-CD40 antibody (clone 3A8; 20 mg/kg) in group B, clone 2C10R4 (25 mg/kg) in group C, or clone 2C10R4 (50 mg/kg) in group D, along with conventional nonspecific immunosuppressive agents., Results: Group A grafts (n = 8) survived for an average of 70 days, with the longest survival of 236 days. Some animals in this group (n = 3) developed microvascular thrombosis due to platelet activation and consumption, which resulted in spontaneous hemorrhage. The median survival time was 21 days in group B (n = 3), 80 days in group C (n = 6), and more than 200 days in group D (n = 5). Three grafts in group D are still contracting well, with the longest ongoing graft survival surpassing the 1-year mark., Conclusions: Genetically engineered pig hearts (GTKOhTg.hCD46.hTBM) with modified targeted immunosuppression (anti-CD40 monoclonal antibody) achieved long-term cardiac xenograft survival. This potentially paves the way for clinical xenotransplantation if similar survival can be reproduced in an orthotopic transplantation model., (Copyright © 2014 The American Association for Thoracic Surgery. All rights reserved.)

Introduction: Dengue virus (DENV) is transmitted by the mosquito vector, and causes a wide range of symptoms that lead to dengue fever (DF) or life-threatening dengue hemorrhagic fever (DHF). The host and viral correlates that contribute to DF and DHF are complex and poorly understood, but appear to be linked to inflammation and impaired coagulation. Full-length osteopontin (FL-OPN), a glycoprotein, and its activated thrombin-cleaved product, trOPN, integrate multiple immunological signals through the induction of pro-inflammatory cytokines., Materials and Method: To understand the role of OPN in DENV-infection, we assessed circulating levels of FL-OPN, trOPN, and several coagulation markers (D-dimer, thrombin-antithrombin complex [TAT], thrombomodulin [TM], and ferritin in blood obtained from 65 DENV infected patients in the critical and recovery phases of DF and DHF during a dengue virus epidemic in the Philippines in 2010., Results: Levels of FL-OPN, trOPN, D-dimer, TAT, and TM were significantly elevated in the critical phase in both the DF and DHF groups, as compared with healthy controls. During the recovery phase, FL-OPN levels declined while trOPN levels increased dramatically in both the DF and DHF groups. FL-OPN levels were directly correlated with D-dimer and ferritin levels, while the generation of trOPN was associated with TAT levels, platelet counts, and viral RNA load., Conclusion: Our study demonstrated the marked elevation of plasma levels of FL-OPN and thrombin-cleaved OPN product, trOPN, in DENV-infection for the first time. Further studies on the biological functions of these matricellular proteins in DENV-infection would clarify its pathogenesis., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Background: During severe (pneumo)sepsis inflammatory and coagulation pathways become activated as part of the host immune response. Thrombomodulin (TM) is involved in a range of host defense mechanisms during infection and plays a pivotal role in activation of protein C (PC) into active protein C (APC). APC has both anticoagulant and anti-inflammatory properties. In this study we investigated the effects of impaired TM-mediated APC generation during melioidosis, a common form of community-acquired Gram-negative (pneumo)sepsis in South-East Asia caused by Burkholderia (B.) pseudomallei., Methodology/principal Findings: (WT) mice and mice with an impaired capacity to activate protein C due to a point mutation in their Thbd gene (TMpro/pro mice) were intranasally infected with B. pseudomallei and sacrificed after 24, 48 or 72 hours for analyses. Additionally, survival studies were performed. When compared to WT mice, TMpro/pro mice displayed a worse survival upon infection with B. pseudomallei, accompanied by increased coagulation activation, enhanced lung neutrophil influx and bronchoalveolar inflammation at late time points, together with increased hepatocellular injury. The TMpro/pro mutation had limited if any impact on bacterial growth and dissemination., Conclusion/significance: TM-mediated protein C activation contributes to protective immunity after infection with B. pseudomallei. These results add to a better understanding of the regulation of the inflammatory and procoagulant response during severe Gram-negative (pneumo)sepsis.

The use of targeted therapeutics to replenish pathologically deficient proteins on the luminal endothelial membrane has the potential to revolutionize emergency and cardiovascular medicine. Untargeted recombinant proteins, like activated protein C (APC) and thrombomodulin (TM), have demonstrated beneficial effects in acute vascular disorders, but have failed to have a major impact on clinical care. We recently reported that TM fused with an scFv antibody fragment to platelet endothelial cell adhesion molecule-1 (PECAM-1) exerts therapeutic effects superior to untargeted TM. PECAM-1 is localized to cell-cell junctions, however, whereas the endothelial protein C receptor (EPCR), the key co-factor of TM/APC, is exposed in the apical membrane. Here we tested whether anchoring TM to the intercellular adhesion molecule (ICAM-1) favors scFv/TM collaboration with EPCR. Indeed: i) endothelial targeting scFv/TM to ICAM-1 provides ~15-fold greater activation of protein C than its PECAM-targeted counterpart; ii) blocking EPCR reduces protein C activation by scFv/TM anchored to endothelial ICAM-1, but not PECAM-1; and iii) anti-ICAM scFv/TM fusion provides more profound anti-inflammatory effects than anti-PECAM scFv/TM in a mouse model of acute lung injury. These findings, obtained using new translational constructs, emphasize the importance of targeting protein therapeutics to the proper surface determinant, in order to optimize their microenvironment and beneficial effects.

The complement system contains a great deal of biological "energy". This is demonstrated by the atypical hemolytic uremic syndrome (aHUS), which is a thrombotic microangiopathy (TMA) characterized by endothelial and blood cell damage and thrombotic vascular occlusions. Kidneys and often also other organs (brain, lungs and gastrointestinal tract) are affected. A principal pathophysiological feature in aHUS is a complement attack against endothelial cells and blood cells. This leads to platelet activation and aggregation, hemolysis, prothrombotic and inflammatory changes. The attacks can be triggered by infections, pregnancy, drugs or trauma. Complement-mediated aHUS is distinct from bacterial shiga-toxin (produced e.g. by E. coli O:157 or O:104 serotypes) induced "typical" HUS, thrombotic thrombocytopenic purpura (TTP) associated with ADAMTS13 (an adamalysin enzyme) dysfunction and from a recently described disease related to mutations in intracellular diacylglycerol kinase ε (DGKE). Mutations in proteins that regulate complement (factor H, factor I, MCP/CD46, thrombomodulin) or promote (C3, factor B) amplification of its alternative pathway or anti-factor H antibodies predispose to aHUS. The fundamental defect in aHUS is an excessive complement attack against cellular surfaces. This can be due to 1) an inability to regulate complement on self cell surfaces, 2) hyperactive C3 convertases or 3) complement activation and coagulation promoting changes on cell surfaces. The most common genetic cause is in factor H, where aHUS mutations disrupt its ability to recognize protective polyanions on surfaces where C3b has become attached. Most TMAs are thus characterized by misdirected complement activation affecting endothelial cell and platelet integrity., (Copyright © 2013 European Federation of Internal Medicine. Published by Elsevier B.V. All rights reserved.)

Sepsis results from the host hyperinflammatory response to bacterial infection, causing multiple organ failure and high mortality. We previously demonstrated that LPS binds to monocytic membrane-bound thrombomodulin (TM), but the role of monocytic TM in LPS-induced inflammation remains unknown. In this study, we demonstrated that TM knockdown in human monocytic cells attenuated LPS-induced signaling pathways and cytokine production. Coimmunoprecipitation and immunofluorescence assays showed that monocytic TM interacted with the LPS receptors, CD14 and TLR4/myeloid differentiation factor-2 (MD-2) complex, indicating that it binds to LPS and triggers an LPS-induced inflammatory response by interacting with the CD14/TLR4/MD-2 complex. We also found that monocytic TM knockdown reduced cytokine production induced by gram-negative bacteria Klebsiella pneumoniae, suggesting that monocytic TM plays an important role in gram-negative bacteria-induced inflammation. To further investigate the function of monocytic TM in vivo, myeloid-specific TM-deficient mice were established and were found to display improved survival that resulted from the attenuation of septic syndrome, including reduced systemic inflammatory response and resistance to bacterial dissemination, after K. pneumoniae infection or cecal ligation and puncture surgery. The inhibition of bacterial dissemination in mice with a deficiency of myeloid TM may be caused by the early increase in neutrophil infiltration. Therefore, we conclude that monocytic TM is a novel component in the CD14/TLR4/MD-2 complex and participates in the LPS- and gram-negative bacteria-induced inflammatory response.

Introduction: Factor V Leiden mutation (FV(Leiden)) is associated with impaired down-regulation of activated FV procoagulant activity and loss of FV anticoagulant function that result in an increased risk of venous thromboembolism. As the downstream effects of FV(Leiden) on clot formation and fibrinolyis have only partially been revealed, we investigated its effect on the activation of factor XIII (FXIII) and the cross-linking of fibrin., Methods: In the plasma samples of fifteen healthy individuals with known FV genotypes coagulation was initiated by recombinant human tissue factor and phospholipids with or without recombinant human thrombomodulin (rhTM). FV deficient plasma supplemented with purified wild type FV or FV(Leiden) were also investigated. Clots were recovered and analyzed by SDS-PAGE and quantitative densitometric evaluation of Western blots., Results: rhTM considerably delayed the activation of FXIII in the plasma from FV wild type individuals. This effect of rhTM was significantly impaired in the plasma from FV(Leiden) carriers. The results were confirmed in experiments with FV deficient plasma supplemented by FV prepared from wild type individuals or FV(Leiden) homozygotes. Fibrin γ-chain dimerization was also considerably delayed by rhTM in plasma samples from individuals without Leiden mutation, but not in plasma samples from FV(Leiden) heterozygotes or homozygotes. The difference between heterozygotes and homozygotes was not statistically significant., Conclusion: The highly diminished delaying effect of TM on FXIII activation and on the cross-linking of fibrin in FV(Leiden) carriers might represent a novel mechanism contributing to the increased thrombosis risk of these individuals., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The goal is to provide an extensive review of the physiologic role of thrombomodulin (TM) in maintaining vascular homeostasis, with a focus on its anti-inflammatory properties. Data were collected from published research. TM is a transmembrane glycoprotein expressed on the surface of all vascular endothelial cells. Expression of TM is tightly regulated to maintain homeostasis and to ensure a rapid and localized hemostatic and inflammatory response to injury. By virtue of its strategic location, its multidomain structure and complex interactions with thrombin, protein C (PC), thrombin activatable fibrinolysis inhibitor (TAFI), complement components, the Lewis Y antigen, and the cytokine HMGB1, TM exhibits a range of physiologically important anti-inflammatory, anti-coagulant, and anti-fibrinolytic properties. TM is an essential cofactor that impacts on multiple biologic processes. Alterations in expression of TM and its partner proteins may be manifest by inflammatory and thrombotic disorders. Administration of soluble forms of TM holds promise as effective therapies for inflammatory diseases, and infections and malignancies that are complicated by disseminated intravascular coagulation.

Accelerated atherosclerosis represents a major problem in both systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) patients, and endothelial damage is a key feature of atherogenesis. We aimed to assess early endothelial changes in SLE and RA female patients (127 SLE and 107 RA) without previous CV events. Biomarkers of endothelial cell activation (intercellular adhesion molecule-1 (sICAM-1), vascular cell adhesion molecule-1 (sVCAM-1), thrombomodulin (TM), and tissue factor (TF)) were measured and endothelial function was assessed using peripheral artery tonometry. Reactive hyperemia index (RHI), an indicator of microvascular reactivity, and augmentation index (AIx), a measure of arterial stiffness, were obtained. In addition, traditional CV risk factors, disease activity and medication were determined. Women with SLE displayed higher sICAM-1 and TM and lower TF levels than women with RA (p = 0.001, p<0.001 and p<0.001, respectively). These differences remained significant after controlling for CV risk factors and medication. Serum levels of vascular biomarkers were increased in active disease and a moderate correlation was observed between sVCAM-1 levels and lupus disease activity (rho = 0.246) and between TF levels and RA disease activity (rho = 0.301). Although RHI was similar across the groups, AIx was higher in lupus as compared to RA (p = 0.04). Also in active SLE, a trend towards poorer vasodilation was observed (p = 0.06). In conclusion, women with SLE and RA present with distinct patterns of endothelial cell activation biomarkers not explained by differences in traditional CV risk factors. Early vascular alterations are more pronounced in SLE which is in line with the higher CV risk of these patients.

Acute graft-versus-host disease (GVHD) is a major complication of allogeneic stem cell transplantation (SCT) and can be readily controlled by systemic high-dose steroids in many patients. However, patients whose GVHD is refractory to this therapy have a poor prognosis. Refractory patients have ongoing end-organ damage despite effective immunosuppression with second-line regimens, suggesting pathomechanisms independent from the initiating T-cell attack. To explore whether endothelial damage might contribute to GVHD refractoriness and to study the role of angiopoietin-2 (ANG2) in this process, we have compared kinetics of T-cell activation markers and markers of endothelial dysfunction in the serum of patients with sensitive (n = 23) and refractory GVHD (n = 25). Longitudinal measurements of soluble FAS ligand along with other immune markers demonstrate that refractory patients are not exposed to an overwhelming or unresponsive T-cell attack. However, in contrast to sensitive GVHD, refractory GVHD was associated with rising thrombomodulin levels and high ANG2/ vascular endothelial-derived growth factor ratios. Patients with refractory GVHD showed significantly increased ANG2 levels already before SCT. These results suggest that endothelial cell vulnerability and dysfunction, rather than refractory T-cell activity, drives treatment refractoriness of GVHD and opens new avenues for prediction and control of this devastating condition.

Purpose: There is growing interest in delivery of drugs and radioisotopes with carriers designed to target molecular receptors in the vascular space. In contrast to targets outside of blood vessels, vascular receptors are easily accessed and tracer binding is not significantly impacted by the size of the targeted drug carrier. Although it is accepted that vascular targeting is very efficient, the kinetics of target binding in the vascular space immediately after injection has not been evaluated. MAb 201B accumulation in mouse lung, by virtue of its binding to thrombomodulin on lung endothelium, has been used as a model for imaging and vascular targeting., Procedures: MAb 201B was radioiodinated with either (125)I comparing two different methods of radioiodination to assess label stability. MAb accumulation and retention were monitored after iv injection in normal BALB/c mice by standard biodistribution experiments, SPECT/CT. MAb radiolabeled with positron emitting (124)I was evaluated by dynamic microPET., Results: The data show that lung uptake and kinetics of loss (t1/2≈40 hrs) of (125)I MAb 201B was similar with two different radio-iodination methods. Loss of radioiodine was detected in sample radiolabeled using the chloramines T method, but only at later time points(24hrs). For very short time evaluations the more efficient chloramine T method was adopted for PET studies with (124)I. Lung uptake of (124)I MAb 201B occurred within seconds of injection as observed in dynamic microPET analysis with little (124)I MAb ever detected in the peripheral circulation. In contrast, distribution kinetics of control (124)I MAb 14 or (124)I MAb 201B that had been diluted with excess cold MAb demonstrated equilibration throughout the vascular space., Conclusion: Accumulation of MAb targeted to epitopes in the vascular space occurs very rapidly, is highly specific and very efficient. Drug delivery with vascular targeting agents can accommodate fast acting therapeutic agents including short half lived radioisotopes.

Cross-species transplantation (xenotransplantation) has immense potential to solve the critical need for organs, tissues and cells for clinical transplantation. The increasing availability of genetically engineered pigs is enabling progress to be made in pig-to-nonhuman primate experimental models. Potent pharmacologic immunosuppressive regimens have largely prevented T-cell rejection and a T-cell-dependent elicited antibody response. However, coagulation dysfunction between the pig and primate is proving to be a major problem, and this can result in life-threatening consumptive coagulopathy. This complication is unlikely to be overcome until pigs expressing a human 'antithrombotic' or 'anticoagulant' gene, such as thrombomodulin, tissue factor pathway inhibitor or CD39, become available. Progress in islet xenotransplantation has been more encouraging, and diabetes has been controlled in nonhuman primates for periods in excess of 6 months, although this has usually been achieved using immunosuppressive protocols that might not be clinically applicable. Further advances are required to overcome the remaining barriers.

Background: Problems of coagulation disorder remain to be resolved in pig-to-primate xenotransplantation. Molecular incompatibilities in the coagulation systems between pigs and humans, such as the thrombomodulin (TM)-protein C system or direct prothrombinase activity, have been suggested as possible causes. Coagulation and complement activation are closely related to each other. The purpose of this study was to elucidate the protective effects on the coagulation system of the expression of human TM and decay accelerating factor (hDAF) (for inhibition of complement activation) in pig endothelial cells., Methods: Human aortic endothelial cells (HAEC), porcine aortic endothelial cells (PAEC), hDAF-expressing PAEC (hDAF-PAEC), hDAF/Endo-beta-galactosidase C-expressing PAEC (hDAF/EndoGalC-PAEC), hTM-expressing PAEC (hTM-PAEC), hDAF/hTM expressing-PAEC (hDAF/hTM-PAEC), and hDAF/EndoGalC/hTM-expressing PAEC (hDAF/EndoGalC/hTM-PAEC) were used in this study. Coagulation activity was examined by clotting, activated protein C (APC), and thrombin generation assay., Results: A large difference was observed in clotting time of human plasma when exposed to PAEC (170 s) and HAEC (1020 s). hTM expression on PAEC was proven to produce a comparable level of APC to that produced by HAEC, which prolonged the clotting time, though not to the level of HAEC. Pretreatment with human sera considerably shortened the clotting time in PAEC (80 s). hDAF-PAEC significantly inhibited such a shortening of clotting time by reductions in tissue factor expression and thrombin generation. Thrombin generation through direct prothrombinase activity, which was detected only in PAEC, could be suppressed by hTM expression. Suppression of antibody binding and complement activation improved clotting time not in PAEC, but in PAEC expressing hTM., Conclusions: In addition to effective suppression of antibody-induced complement activation, hTM expression in PAEC may be essential for regulating procoagulant activity in xenotransplantation.

Background: Dendritic cells (DCs) are important in allergic diseases such as asthma, although little is known regarding the mechanisms by which DCs induce T(H)2-polarized responses in atopic individuals. It has been suggested that intrinsic properties of allergens can directly stimulate T(H)2 polarizing functions of DCs, but little is known of the underlying mechanisms., Objective: To identify novel genes expressed by house dust mite (HDM) allergen-exposed DCs., Methods: We screened for allergen-induced gene expression by microarray, and validated differentially expressed genes at the mRNA and protein levels., Results: Thrombomodulin (CD141, blood dendritic cell antigen 3) expression by microarray was higher on HDM-stimulated DCs from atopic (relative to nonatopic) individuals. These findings were confirmed at both the mRNA and protein levels in an independent group. Purified thrombomodulin(+) DCs induced a strongly T(H)2-polarized cytokine response by allergen-specific T cells compared with DCs lacking thrombomodulin. In vivo, thrombomodulin(+) circulating DCs were significantly more frequent in subjects with HDM allergy and asthma, compared with control subjects. Furthermore, thrombomodulin expression in blood leukocytes was higher in children with acute asthma than at convalescence 6 weeks later., Conclusion: Thrombomodulin expression on DCs may be involved in the pathogenesis of atopy and asthma.

Introduction: Nanoparticles (NP) have potential as carriers for drugs and radioisotopes. Quantitative measures of NP biodistribution in vivo are needed to determine the effectiveness of these carriers. We have used a model system of radiolabeled quantum dots to document the competition between efficient vascular targeting and interaction of the NP with the reticuloendothelial (RE) system., Methods: We have prepared (125m)Te-labeled CdTe NP that are capped with ZnS. Te-125m has a half-life and decay characteristics very similar to those for (125)I. The synthesized particles are stable in aqueous solution and are derivatized with mercaptoacetic acid and then conjugated with specific antibody. To evaluate specific targeting, we used the monoclonal antibody MAb 201B that binds to murine thrombomodulin expressed in the lumen of lung blood vessels. The MAb-targeted NP were tested for targeting performance in vivo using single-photon emission computed tomography (SPECT)/computed tomography (CT) imaging, tissue autoradiography and standard organ biodistribution techniques. Biodistribution was also determined in mice that had been depleted of phagocytic cells by use of clodronate-loaded liposomes., Results: Cd(125m)Te/ZnS NP coupled with MAb 201B retained radioisotope and antibody activity and accumulated in lung (>400% injected dose [ID]/g) within 1 h of intravenous injection. Control antibody-coupled NP did not accumulate in lung (<10% ID/g) but accumulated in liver and spleen. Images from microSPECT/CT and autoradiography studies of the targeted NP document this specific uptake and demonstrate uniform distribution in lung with minor accumulation in liver and spleen. Within a few hours, a large fraction of lung-targeted NP redistributed to spleen and liver or was excreted. We hypothesized that NP attract phagocytic cells that engulfed and removed them from circulation. This was confirmed by comparing biodistribution of targeted NP in normal mice versus those depleted of phagocytic cells. In mice treated with clodronate liposomes, accumulation of NP in liver was reduced by fivefold, while accumulation in lung at 1 h was enhanced by approximately 50%. By 24 h, loss of the targeted NP from lung was inhibited by several-fold, while accumulation in liver and spleen remained constant. Thus, the treated mice had a much larger accumulation and retention of the NP at the target site and a decrease in dose to other organs except spleen., Conclusion: Nanoparticles composed of CdTe, labeled with (125m)Te and capped with ZnS, can be targeted with MAb to sites in the lumen of lung vasculature. In clodronate-treated mice, which have a temporary depletion of phagocytic cells, accumulation in liver was reduced dramatically, whereas that in spleen was not. The targeting to lung was several-fold more efficient in clodronate-treated mice due to larger initial accumulation and better retention of the MAb-targeted NP at that site. This model system indicates that targeting of NP preparations is a competition between the effectiveness of the targeting agent and the natural tendency for RE uptake of the particles. Temporary inhibition of the RE system may enhance the usefulness of NP for drug and radioisotope delivery.

CD93 belongs to a newly described family of transmembrane glycoproteins which also includes endosialin and thrombomodulin. These cell surface proteins are grouped into a family based on similar ectodomain architecture consisting of a C-type lectin-like domain, a series of EGF-like repeats and a highly glycosylated mucin-like domain. However, recent studies suggest overlapping functions in the regulation of processes involving innate immunity and inflammation. CD93 regulates phagocytosis of apoptotic cells in vivo, a function critical to development, tissue repair and maintenance of tissue homeostasis. In addition, in vitro studies have demonstrated a role for CD93 in phagocytosis of antibody and complement opsonized particles and also in leukocyte and endothelial cell adhesion. Analysis of CD93 expression on endothelial cells in the developing mouse embryo correlates with the remodeling of blood vessels suggesting a role for CD93 in regulating angiogenesis, a process tightly linked to acute and chronic inflammation and also required for tumor metastasis. Endosialin has been characterized as a tumor specific antigen and functions in angiogenesis. Thrombomodulin is best characterized as a natural anticoagulant; however, more recent reports have illuminated the importance of thrombomodulin in the regulation of inflammation. This review discusses similarities and differences in the family members, and focuses on known and speculated functions in regulation of innate immunity.

Vascular drug targeting may improve therapies, yet a thorough understanding of the factors that regulate effects of drugs directed to the endothelium is needed to translate this approach into the clinical domain. To define factors modulating the efficacy and effects of endothelial targeting, we used a model enzyme (glucose oxidase, GOX) coupled with monoclonal antibodies (anti-TM(34) or anti-TM(201)) to distinct epitopes of thrombomodulin, a surface determinant enriched in the pulmonary endothelium. GOX delivery results in conversion of glucose and oxygen into H(2)O(2) leading to lung damage, a clear physiologic endpoint. Results of in vivo studies in mice showed that the efficiency of cargo delivery and its effect are influenced by a number of factors including: 1) The level of pulmonary uptake of the targeting antibody (anti-TM(201) was more efficient than anti-TM(34)); 2) The amount of an active drug delivered to the target; 3) The amount of target antigen on the endothelium (animals with suppressed TM levels showed less targeting); and, 4) The substrate availability for the enzyme cargo in the target tissue (hyperoxia augmented GOX-induced injury). Therefore, both activities of the conjugates and biological factors control targeting and effects of enzymatic cargo. Understanding the nature of such "modulating biological factors" will hopefully allow optimization and ultimately applications of drug targeting for "individualized" pharmacotherapy.

To produce specific monoclonal antibody (McAb) against human thrombomodulin (hTM), the full-length hTM cDNA-expressing plasmid pThr402 was transfected into CHO cells by Lipofectamine 2000 reagent. The hTM-expressing CHO cells, which was confirmed by flow cytometry and Western blot, were obtained by G418 selection. Then the McAb against hTM was prepared with classic hybridoma technique. A cell line of CHO-TM5 with high level of hTM was used to immunize female Balb/c mice 3 times at an interval of 4 weeks. On the third day after the third immunization, mice were sacrificed and spleen cells were harvested to prepare hybridoma cells with SP2/0 cells at the ratio of 10 to 1. Hybridoma cells were then cultured at 96-well plates for screening. Cellular enzyme-linked immunoabsorbent assay (CELISA) was applied twice. The first CELISA was done with polythene ELISA plate with a monolayer of CHO-TM5 cells. The positive clones from the first screen were then selected by reacting with similar screening ELISA plate but with CHO cell monolayer instead. Only clones that were positive for the first screening and negative for the second screening were kept, and called as CHO-TM5(+)CHO(-) hybridoma cells. Balb/c mice were intraperitoneally injected with the selected hybridoma cells. Ascites were collected and monoclonal antibodies were purified using FPLC, and its Ig class, subclass, and titer were then determined respectively. The specificity of the yielded McAb was identified with CELISA, flow cytometry, ABC immunohistochemistry and immunoblotting. One line of hybridoma cells with high expression of specific McAb against hTM, NH-1, was obtained. The Ig subclass of the McAb was IgG1 and the titer of ascitic McAb was 1x10(-6). Flow cytometry, CELISA and Western blot assays demonstrated that McAb NH-1 could specifically recognize hTM expressed in CHO-TM5 cells and human umbilical vascular endothelial cells. Meanwhile, the tissue specificity of antigen recognized by McAb NH-1 was identified by immunohistochemical ABC staining. NH-1 can specifically recognize the natural hTM expressed mainly in vascular endothelial cells, which will potentially be useful for investigation of the functions and clinic values of hTM.

Objective: To investigate the immunopathological changes of micro-vessels in dermatomyositis., Methods: Twelve patients with dermatomyositis, 5 males and 7 females, aged 40.8 (6 - 72), underwent muscle biopsy of biceps muscle of arm (n = 11) or deltoid muscle (n = 1). The specimens underwent routine histological, enzyme histochemical, and immunohistochemical staining and microscopy. Ten biopsy specimens from patients with other diseases were used as controls., Results: Perifascicular atrophy of muscular fibers and inflammatory infiltration in perimysium were seen in all patients with dermatomyositis. Non-specific esterase staining showed deep staining of capillaries and micro vascular endothelium among the muscular fibers. Immunohistochemistry showed remarkable reduction of capillaries positive in von Willebrand factor (vWF), thrombomodulin (TM), and endothelial cell nitric oxide synthase (eNOS) in the perifascicular region, and low expression of .eNOS and TM in the microvascular endothelium in the perimysium., Conclusion: The vascular lesions of dermatomyositis are located not only in capillaries, but also in other microvessels. Lower expression of TM and eNOS in vascular endothelium suggests the reduction of anticoagulation and vasodilation functions of vascular endothelium. Dermatomyositis is an inflammatory vascular endothelial disease.

Objective: To clarify whether mouse monoclonal antibodies (mAb) against human thrombomodulin (TM), which react with human TM present on the endothelial cell (EC) surface, have anti-endothelial cell antibody (AECA) activity and influence antiinflammatory properties of human TM expressed on the EC surface in vitro., Methods: Three preparations of mouse mAb against human TM that react with different sites of the human TM epidermal growth factor-like domain were tested for their ability to 1) bind to ECs, 2) modulate cytokine secretion from ECs, EC adhesion molecule expression, and neutrophil adhesion to ECs, and 3) stimulate nuclear translocation of NF-kappaB through the degradation of cytoplasmic IkappaB in ECs. Recombinant human interleukin-1beta (IL-1beta) was used as a positive control, and mouse IgG1 and mouse IgG2a were used as negative controls., Results: The 3 preparations of mouse mAb against human TM that bind to unfixed EC monolayers enhanced IL-6 and IL-8 secretion from ECs, up-regulated expression of endothelial leukocyte adhesion molecule 1, vascular cell adhesion molecule 1, and intercellular adhesion molecule 1 on EC monolayers, and enhanced neutrophil adhesion to ECs to a degree similar to that observed with IL-1beta stimulation, but they did not induce the secretion of tumor necrosis factor alpha or IL-1beta from ECs throughout the incubation period. The 3 preparations stimulated nuclear translocation of NF-kappaB through the degradation of cytoplasmic IkappaB. Mouse IgG1 and mouse IgG2a did not exhibit such effects., Conclusion: These results suggest the possibility that AECA can react with antigens such as TM that are present on the EC surface and activate ECs. Such events on ECs may lead to vascular inflammation and damage in patients with connective tissue diseases and vasculitis in which AECA are present.

We evaluated the sensitivity and specificity of 10 monoclonal and two polyclonal antibodies for distinguishing epithelioid mesothelioma from adenocarcinoma (AdCA) using immunohistochemistry (IHC). The antibodies were directed against the mesothelial-associated antigens mesothelin, calretinin, cytokeratin 5, thrombomodulin, Wilms' tumor-1 (WT-1) gene product and HBME-1, and the nonmesothelial antigens Lewis-Y blood group (antibody BG8), MOC-31, BerEp4, CD15, and carcinoembryonic antigen (CEA) family. The 133 tumors evaluated included 65 malignant epithelioid mesotheliomas, 22 lung AdCAs, 27 ovarian serous carcinomas, 24 breast carcinomas, and five gastric carcinomas. Diagnoses were based on clinical, histologic, ultrastructural, and/or IHC findings. Calretinin had the best sensitivity for mesothelioma (95%), followed by HBME-1 (84%), WT-1 (78%), cytokeratin 5 (76%), mesothelin (75%), and vimentin and thrombomodulin (68%). Thrombomodulin had the best specificity for mesothelioma (92%), followed by cytokeratin 5 (89%), calretinin (87%) vimentin (84%), and HBME-1 (45%). When ovarian carcinomas were excluded from the analysis, the specificity of mesothelin and WT-1 for the diagnosis of mesothelioma increased to 90 and 81%, respectively. The sensitivity of the nonmesothelial antigens for AdCA was organ dependent, with BG8 performing best in the breast cancer group (96%), and BerEp4, BG8, MOC-31 performing best in the lung cancer group (100%). The specificity of the nonmesothelial antigens for AdCA was 98% for BG8 and CEA, 97% for CD15, 95% for BerEp4, and 87% for MOC-31. A novel statistical analysis technique employing logic regression analysis identified a three-antibody immunohistochemical panel including calretinin, BG8, and MOC-31, which provided over 96% sensitivity and specificity for distinguishing epithelioid mesothelioma from AdCA.

Activation of inflammatory and coagulation pathways is important in the pathogenesis of vascular disease. There is ample evidence that extensive cross-talk between these two systems exists, whereby inflammation not only leads to activation of coagulation, but coagulation also markedly affects inflammatory activity. The main interfaces linking coagulation and inflammation are the tissue factor pathway, thrombin, the protein C system and the fibrinolytic (or plasminogen-plasmin) system. Proinflammatory cytokines and chemokines can affect all these coagulation mechanisms, and vice versa, activated coagulation proteases and physiological anticoagulants or components of the plasminogen-plasmin system can modulate inflammation by specific cell receptors. The intricate relationship between inflammation and coagulation may not only be relevant for vascular thrombotic disease but also has major consequences in the pathogenesis of microvascular failure and subsequent multiple organ failure in the setting of severe infection. This review focuses on the present understanding of the bidirectional relationship between inflammation and coagulation.

Thrombomodulin (TM) is an endothelial cell surface molecule, capable of specific binding for thrombin. The thrombin/TM complex promotes activation of plasma anticoagulant protein C (PC) and negatively regulates blood coagulation. Along with anticoagulant function, TM has been shown to have additional physiological functions such as regulation of fibrinolysis, cell adhesion, tumor growth, and embryonic development. The extracellular region of TM contains a lectin domain and six epidermal growth factor (EGF)-like domains, which are required for the various functions. To analyze the functions, we established a panel of monoclonal antibodies (MAbs) reactive to each functional domain. We obtained MAbs that react to the lectin domain or the front half of EGF domains from the first to the third using the antigen of a transfected cell line expressing full-length TM. We also obtained MAbs that reacted to the bottom half of the EGF domain from the fourth to the sixth using the antigen of a transfected cell line expressing truncated form of TM lacking the lectin domain and the EGF domains from the first to the third. All obtained MAbs could be used for Western blotting. Endothelial cell function for PC activation can be mimicked by transfected cells positive for TM and the endothelial cell protein C receptor (EPCR). Effects of the established MAbs on thrombin-dependent PC activation on the transfected cells were examined. Strong inhibition was demonstrated by three MAbs, which reacted to the fourth or fifth EGF domain, but not by MAbs to the other domains. The fourth EGF domain is known as the interaction site for PC, and the fifth domain is known to be required for thrombin binding. The sixth EGF domain also has been shown to be required for thrombin binding. An MAb against the domain strongly inhibited thrombin-binding. However, the MAb demonstrated little effect on thrombin dependent PC activation. The contradictory results demonstrated with the MAb to the sixth EGF domain suggest an unknown molecular mechanism for PC activation on the cell surface. A panel of MAbs reactive to each domain could be useful for analyzing the multifunctional molecule thrombomodulin.

Endothelial cell membrane-bound thrombomodulin (TM) plays a critical role as a cofactor in the protein C pathway, important in regulating coagulation as well as inflammation. Heterogeneous soluble TM fragments circulate in the plasma and are found at increased levels in various diseases such as cardiovascular disease and diabetes, and in ischemic and/or inflammatory endothelial injuries. The anticoagulant function of these soluble fragments has not been measured in healthy individuals or in patients. Using an immobilized monoclonal antibody against TM and a microtiter plate format, an assay was designed to capture the soluble TM fragments in plasma and measure their cofactor activity in the thrombin-mediated activation of protein C. In addition, soluble TM antigen levels were measured by enzyme-linked immunosorbent assay. Both assays were used to investigate a group of healthy blood donors. TM fragments released into plasma were shown to retain significant cofactor activity, and reference intervals for healthy men and women were established. Furthermore, a statistically significant correlation was observed between soluble TM antigen levels and soluble TM cofactor activity. This notwithstanding, soluble TM activity only accounted for a minor part of all variation in soluble TM antigen levels (R2 = 22% in men and R2 = 16% in women).

Background: Various endothelial markers are available for the evaluation of vascular tumors and malformations, including anti CD34, anti-CD31, von Willebrand factor (vWF), and anti-thrombomodulin (anti-TM) antibodies. All have their limitations, and we sought to compare the utility of anti-TM antibody as a marker for several types of vascular neoplasms vs. previously established endothelial markers., Methods: We examined immunostaining profiles of 30 capillary hemangiomas, 10 pyogenic granulomas, five tufted angiomas, 17 Kaposi's sarcomas, and nine angiosarcomas. Immunostains for TM, CD34, and vWF were carried out using a labeled streptavidin-biotin peroxidase detection system., Results: Anti-TM antibody showed moderately intense immunostaining in 89% of benign and malignant vascular neoplasms. Anti-CD34 antibody showed moderate to diffuse immunostaining in 98% of vascular neoplasms, and vWF showed weak focal staining in 84% of all vascular neoplasms examined., Conclusion: Anti-TM antibody proved to be a sensitive marker for both benign and malignant vascular neoplasms. While not as sensitive as anti-CD34, it may have some advantages in specificity that would make it a more reliable vascular tumor marker in certain situations.

Thrombomodulin (TM) is a cell surface endothelial glycoprotein having anticoagulant properties. It inhibits thrombin, and activates protein C, leading to the inhibition of activated factors V and VIII. TM autoantibodies could theoretically predispose to thrombosis. We have tested 83 unselected patients with deep venous thrombosis, 36 males and 47 females aged from 1 to 70 years [mean +/- standard deviation (SD), 34.2 +/- 14.5 years] for the presence of IgG anti-TM in their plasmas. Tests were performed by enzyme-linked immunosorbent assay (ELISA) using recombinant human TM kindly provided by PAION GmbH (Aachen, Germany). Results are expressed as the optical density (OD) differences between coated and un-coated wells. Plasmas from 83 normal volunteer donors were used to define the cut-off value as the mean of absorbance of the control group + 3 SDs. The median OD of normal controls group was 0.024 (mean, 0.034; SD, 0.066; range, -0.048 to 0.309). The median OD obtained with plasmas from patients was 0.048 (mean, 0.114; SD, 0.215; range, -0.039 to 1.312) and was significantly higher than that of the control group (P < 0.0001). Choosing a cut-off value of 0.232 (mean OD of the control group + 3 SDs), 11 patients are considered as positive for IgG autoantibodies to TM and three normal controls are weakly positive. Selected plasma with IgG anti-TM and purified IgG were further tested by dot blot using recombinant purified TM and were found positive. Purified IgG of positive plasmas inhibits protein C activation by TM and thrombin, suggesting that anti-TM antibodies have a procoagulant effect. Interestingly, in our study, anti-TM antibodies are found in three of six patients with Budd-Chiari syndrome and four of eight patients with cerebral venous thrombosis. In conclusion, thrombomodulin autoantibodies could be a new interesting marker of thrombophilia easily detected by ELISA.

Oxidant stress is a well known cause of damage in the atherosclerotic process. Vitamin E is one of the most promising natural antioxidants. In this study we investigated if a vitamin E-coated dialyzer was able to reduce the plasma levels of auto-antibodies against oxidized-LDL, von Willebrand factor (vWf) and thrombomodulin (TM) as markers of endothelial damage. In this controlled 6-month prospective study, we investigated these markers in two matched groups (n=16 each) of patients on regular hemodialysis not yet diagnosed for atherosclerosis cardiovascular disease (ACVD) (mean age=58.3+/-7.0 yrs, mean dialysis age=30.1+/-10.0 months), in which cellulosic (CLS) and vitamin E-modified dialyzers (CLE) were compared. At inclusion all the patients were treated with CLS. Then, the study group was shifted to CLE for 6 months. At baseline the patients showed normal levels of vitamin E and high levels of oxLDL-Ab, vWf and TM compared to healthy subjects. In the CLE group oxLDL-Ab and vWf, but not TM levels, decreased progressively (from 472+/-287 to 264+/-199 mU/mL, p<0.0001 and from 101.1+/-7.5% to 76.7+/-18.5%; p<0.001, respectively), and vitamin E increased from 4.40+/-0.81 to 7.81+/-1.16 microg/mg of cholesterol. At the end of the study, 8 of the patients treated with CLE were randomly selected and went back to the membrane without Vitamin E for six months. They showed an significant increase in OxLDL-Ab and vWf levels and a significant reduction in tocoferol levels. In conclusion, CLE compared to cellulosic dialyzers can lower some indices of damage to LDL and endothelial cells.

Activated protein C (APC), a natural anticoagulant, is formed from protein C by the action of the thrombin-thrombomodulin (TM) complex on the endothelial cell surface. Endothelial protein C receptor augments the activation of protein C by the thrombin/TM system. APC inactivates the activated form of coagulation factors V and VIII in the presence of protein S. Administration of APC reduced the pulmonary vascular injury and hypotension as well as the coagulation abnormalities by inhibiting production of the tumor necrosis factor-alpha (TNF-alpha) in rats given endotoxin (ET). These therapeutic effects of APC could not be attributed to its anticoagulant effects. APC inhibited ET-induced TNF-alpha production in human monocytes by inhibiting activation of nuclear factor K-B and activator protein-1 in vitro. Administration of the human plasma-derived APC ameliorated coagulation abnormalities without any adverse effects in patients with disseminated intravascular coagulation (DIC). Recombinant APC was reported to reduce the mortality of patients with severe sepsis, and the therapeutic effect was more marked in such patients with overt DIC than those without it. These observations strongly suggest that APC plays important roles in the regulation of inflammation as well as coagulation. Both anti-inflammatory and anticoagulant properties of APC might contribute to the therapeutic usefulness in patients with severe sepsis.

The antiphospholipid syndrome (APS) is a multi-system prothrombotic disorder associated with circulating auto-antibodies directed against various phospholipid-binding proteins. The major clinical manifestations are recurrent arterial or venous thrombosis, but due to its heterogeneity, atypical presentations can obscure the diagnosis. Decisions regarding when to attribute complications to aPL are difficult. The most established tests are lupus anticoagulant (LA) detected by clotting assays and anticardiolipin (aCL) detected by ELISA. Although LA and aCL assays are clinically useful, these tests do not clearly differentiate antibodies with different specificities. Antibodies to beta2GPI are associated with thrombosis in the APS. Although these antibodies are detected by aCL assay (e.g. beta2GPI-dependent aCL), some aCL are not associated with the syndrome (e.g. beta2GPI-independent aCL). Regarding LAs, more studies are needed to determine if it is clinically important to differentiate specificities against beta2GPI or prothrombin. The role of aPLs in the pathogenesis of thrombosis requires further and intensive investigation. If autoantibodies to particular phospholipid binding proteins are shown to be associated with different clinical presentations or to confer different risks, the availability of more accurate diagnostic techniques will be required for the recognition of pathogenic aPLs. By now, clinical judgement, careful exclusion of other etiologies and serial aPL levels are helpful in this regard.

Objective: To determine if anti-endothelial cell antibodies (AECA) and plasma markers of endothelial cell function are related to disease severity in systemic lupus erythematosus (SLE)., Methods: We measured AECA by human umbilical vein endothelial cell binding, endothelial markers von Willebrand factor, soluble thrombomodulin, and soluble E-selectin by ELISA, and disease severity by SLEDAI and SLICC/ACR in 35 patients with SLE., Results: Despite high levels of IgG AECA (p = 0.001) and von Willebrand factor (p = 0.0007) compared to 21 healthy controls, we found a positive correlation only between IgG AECA and the SLEDAI index (r = 0.393, p = 0.021)., Conclusion: IgG AECA seem to be related to disease activity in SLE, possibly in a pathogenic role. Conversely, plasma markers of endothelial cell damage seem to be an epiphenomenon and may simply be related to excess inflammation.

Oxidative endothelial stress, leukocyte transmigration, and pulmonary thrombosis are important pathological factors in acute lung injury/acute respiratory distress syndrome (ALI/ARDS). Vascular immunotargeting of the H(2)O(2)-generating enzyme glucose oxidase (GOX) to the pulmonary endothelium causes an acute oxidative lung injury in mice.(1) In the present study we compared the pulmonary thrombosis and leukocyte transmigration caused by GOX targeting to the endothelial antigens platelet-endothelial cell adhesion molecule (PECAM) and thrombomodulin (TM). Both anti-PECAM and anti-TM delivered similar amounts of (125)I-GOX to the lungs and caused a dose-dependent, tissue-selective lung injury manifested within 2 to 4 hours by high lethality, vascular congestion, polymorphonuclear neutrophil (PMN) sequestration in the pulmonary vasculature, severe pulmonary edema, and tissue oxidation, yet at an equal dose, anti-TM/GOX inflicted more severe lung injury than anti-PECAM/GOX. Moreover, anti-TM/GOX-induced injury was accompanied by PMN transmigration in the alveolar space, whereas anti-PECAM/GOX-induced injury was accompanied by PMN degranulation within vascular lumen without PMN transmigration, likely because of PECAM blockage. Anti-TM/GOX caused markedly more severe pulmonary thrombosis than anti-PECAM/GOX, likely because of TM inhibition. These results indicate that blocking of specific endothelial antigens by GOX immunotargeting modulates important pathological features of the lung injury initiated by local generation of H(2)O(2) and that this approach provides specific and robust models of diverse variants of human ALI/ARDS in mice. In particular, anti-TM/GOX causes lung injury combining oxidative, prothrombotic, and inflammatory components characteristic of the complex pathological picture seen in human ALI/ARDS.