The PDE4 inhibitor tanimilast shows distinct immunomodulatory properties associated with a type 2 endotype and CD141 upregulation.

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.

STAT3 Gain-of-Function Mutations Underlie Deficiency in Human Nonclassical CD16+ Monocytes and CD141+ Dendritic Cells.

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 STAT3GOF CD14+ monocytes fail to differentiate into CD1a+ monocyte-derived DCs. Moreover, patients with STAT3GOF 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. STAT3GOF-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.

Selective targeting of nanomedicine to inflamed cerebral vasculature to enhance the blood-brain barrier.

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.

Coexpression of CD163 and CD141 identifies human circulating IL-10-producing dendritic cells (DC-10).

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.

Justification of specific genetic modifications in pigs for clinical organ xenotransplantation.

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.

Thrombomodulin Regulation of Mitogen-Activated Protein Kinases.

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.

Cardiac xenografts show reduced survival in the absence of transgenic human thrombomodulin expression in donor pigs.

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.

Role of Thrombin in Soluble Thrombomodulin-Induced Suppression of Peripheral HMGB1-Mediated Allodynia in Mice.

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.

Highly sensitive turn-on fluorescence detection of thrombomodulin based on fluorescence resonance energy transfer.

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 H2O2 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 H2O2.

Chimeric 2C10R4 anti-CD40 antibody therapy is critical for long-term survival of GTKO.hCD46.hTBM pig-to-primate cardiac xenograft.

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.

Xenotransplantation of Cells, Tissues, Organs and the German Research Foundation Transregio Collaborative Research Centre 127.

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.

Human thrombomodulin regulates complement activation as well as the coagulation cascade in xeno-immune response.

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.

Identifying the ERAD ubiquitin E3 ligases for viral and cellular targeting of MHC class I.

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.

Functional difference between membrane-bound and soluble human thrombomodulin.

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.

Recombinant thrombomodulin inhibits lipopolysaccharide-induced inflammatory response by blocking the functions of CD14.

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.

Genetically engineered pigs and target-specific immunomodulation provide significant graft survival and hope for clinical cardiac xenotransplantation.

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.

Elevated levels of full-length and thrombin-cleaved osteopontin during acute dengue virus infection are associated with coagulation abnormalities.

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.

A thrombomodulin mutation that impairs active protein C generation is detrimental in severe pneumonia-derived gram-negative sepsis (melioidosis).

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.