Toru Nakabayashi

Autoimmunity associated with TGF-beta1-deficiency in mice is dependent on MHC class II antigen expression.

The progressive inflammatory process found in transforming growth factor beta1 (TGF-beta1)-deficient mice is associated with several manifestations of autoimmunity, including circulating antibodies to nuclear antigens, immune complex deposition, and increased expression of both class I and class II major histocompatibility complex (MHC) antigens. The contribution of MHC class II antigens to the genesis of this phenotype has been determined by crossing the TGF-beta1-null [TGF-beta1(-/-)] genotype into the MHC class II-deficient [MHC-II(-/-)] background. Mice homozygous for both the TGF-beta1 null allele and the class II null allele [TGF-beta1(-/-);MHC-II(-/-)] are without evidence of inflammatory infiltrates, circulating autoantibodies, or glomerular immune complex deposits. Instead, these animals exhibit extensive extramedullary hematopoiesis with progressive splenomegaly and adenopathy, surviving only slightly longer than TGF-beta1(-/-);MHC-II(+/+) mice. The role of CD4+ T cells, which are also absent in MHC class II-deficient mice, is directly demonstrated through the administration of anti-CD4 monoclonal antibodies in class II-positive, TGF-beta1(-/-) mice. The observed reduction in inflammation and improved survival emphasize the significance of CD4+ cells in the pathogenesis of the autoimmune process and suggest that the additional absence of class II antigens in TGF-beta1(-/-);MHC-II(-/-) mice may contribute to their extreme myeloid metaplasia. Thus, MHC class II antigens are essential for the expression of autoimmunity in TGF-beta1-deficient mice, and normally may cooperate with TGF-beta1 to regulate hematopoiesis.

FAS (CD95)-TRANSDUCED SIGNAL PREFERENTIALLY STIMULATES LUPUS PERIPHERAL T LYMPHOCYTES

Fas (CD95) is a cell surface receptor whose biological function in circulating peripheral T cells is not well understood. To address the question of abnormal T cell sensitivity to Fas stimulation in systemic lupus erythematosus (SLE), we studied Fas‐transduced stimulation and apoptosis in peripheral blood T cells from patients with SLE and normal control. Immobilized anti‐Fas monoclonal antibodies (mAb) (imCH‐11; IgM type) significantly stimulated SLE T cell proliferation compared to T cells from normal donors and patients with rheumatoid arthritis ( p < 0.003 and p < 0.005, respectively). The soluble form of CH‐11 and other immobilized anti‐Fas mAb (UB‐2, ZB‐4; IgG type) failed to stimulate lupus T cells while immobilized human Fas ligand did. Furthermore, imCH‐11 induced IL‐2 and IL‐6 mRNA expression. However, imCH‐11 activation failed to induce expression of the T cell activation surface molecules CD25 and CD69. Addition of exogenous ceramide, a second messenger for Fas‐mediated apoptosis signaling, also induced T cell proliferation in SLE and normal controls. Moreover, fumonisin B1, a specific ceramide synthase inhibitor, and caspase inhibitors markedly suppressed imCH‐11 induced T cell proliferation, suggesting that the ceramide pathway may be involved in Fas‐transduced stimulation signals in SLE T cells. These results show that SLE T cells have an alteration in the Fas signal transduction pathway leading to cell proliferation. This defect may be important in Fas‐mediated peripheral immune homeostasis.

New therapeutic option for thromboembolism – dabigatran etexilate

Background: Thrombin plays a key role in blood coagulation and haemostasis; thus its inhibition has been identified as a reasonable target to block the coagulation cascade. Direct thrombin inhibitors are potential prophylactic agents for venous thromboembolism and arterial thrombosis, which often accompany operative procedures and cardiac disease, especially orthopedic surgery and atrial fibrillation, respectively. New orally available anticoagulant agents with a wide therapeutic window are keenly anticipated because warfarin and heparins have some disadvantages, and recent progress in pharmaceutical techniques has led to the development of orally administered direct thrombin inhibitors. Objectives: In this review, we discuss the usefulness of dabigatran etexilate as a new therapeutic option for preventing thromboembolism, including chemistry, pharmacokinetics, and pharmacodynamics, from the results of recent clinical studies. Methods: We systematically focused on relevant published studies, as data from recent clinical studies were difficult to obtain owing to their ongoing status. Conclusions: Dabigatran etexilate is a promising new oral anticoagulant that offers greatly expanded therapeutic options for both patients and physicians.

Factor Xa inhibitors: new anti-thrombotic agents and their characteristics.

Factor Xa (FXa) is a key enzyme that is positioned at the convergence of the intrinsic and extrinsic pathways in the blood coagulation cascade, and inactivation by a specific FXa inhibitor effectively prevents the generation of thrombin. Various types of low molecular weight (LMW) heparin, which function as semi-selective and indirect FXa inhibitors, are replacing unfractionated heparin (UFH) as agents for the prevention and treatment of venous thromboembolism (VTE), as well as in initial treatment for coronary events. Of those, heparinoid has been shown to be safer and more effective for the prevention of postoperative VTE than UFH, especially for treatment of heparin-induced thrombocytopenia (HIT). Further, synthetic pentasaccharide has been found to offer advantages over current thromboprophylactic regimens in a number of patients undergoing major orthopedic surgery. Other studies have shown that pentasaccharide is more effective for overall VTE in comparison with LMW heparin, though it was also associated with an increased rate of major bleeding. Synthetic, selective, and direct inhibitors to FXa, such as DX-9065a, are highly potent and orally bioavailable antithrombotic agents that have demonstrated an improved side effect profile, probably by allowing sufficient thrombin to remain for platelet activation and normal hemostasis, while preventing pathological thrombus formation. For thrombosis therapy, the most desirable type of antithrombotic agent is an orally active drug that has a broad range of effective doses and no hemorrhagic side effects. Presently, many types of direct inhibitors are in various stages of clinical trials and expected to provide significant benefits as compared to currently utilized therapy strategies.

Increase in plasma thrombin-activatable fibrinolysis inhibitor may not contribute to thrombotic tendency in antiphospholipid syndrome because of inhibitory potential of antiphospholipid antibodies toward TAFI activation

The causes of thrombosis in antiphospholipid syndrome (APS) remain unknown, though several hypotheses in regard to hypofibrinolysis have been proposed. To clarify the mechanism, we measured plasma levels of thrombin-activatable fibrinolysis inhibitor (TAFI) in APS patients. Both the TAFI antigen (TAFI:Ag) level measured with an ELISA, and thrombin-thrombomodulin-dependent TAFI activity (TAFI:Ac) were elevated in 68 APS patients as compared with those in 66 healthy controls, though they were lower than those in 46 patients with autoimmune diseases. As for the influence of antiphospholipid antibodies (aPL) on TAFI levels, the mean TAFI:Ac level in 39 SLE patients positive for APS was significantly lower than that in 27 SLE patients without APS, whereas there was no difference in TAFI:Ag between those groups. Furthermore, purified IgG from patients positive for aPL, and monoclonal aPL (EY2C9 and 23-1D) inhibited the activation of TAFI in a concentration dependent manner. These results suggest that aPL inhibits TAFI activation by affecting the function of thrombomodulin-thrombin complex through phospholipids. Although TAFI in plasma is elevated in autoimmune diseases including APS, we concluded that an elevated level is not likely a risk factor for thrombosis in APS patients, because of the inhibition of TAFI activation by aPL.

Protein C Sapporo (protein C Glu 25 --> Lys): a heterozygous missense mutation in the Gla domain provides new insight into the interaction between protein C and endothelial protein C receptor.

Interaction of the gamma-carboxyglutamic acid (Gla) domain of protein C with endothelial protein C receptor (EPCR) is a critical step for efficient activation of protein C, though interactions by mutants in the Gla domain of protein C with EPCR have been rarely evaluated. We identified a 44-year-old Japanese woman with a history of recurrent thromboembolism as an inherited missense mutation, the first such case reported in Japan, which involved a protein C Gla 25 mutation. Total protein C antigen and Gla protein C antigen levels in the proband were normal. Protein C activity measured with an anticoagulant assay was reduced, whereas that measured with an amidolytic assay was normal. She was therefore phenotypically diagnosed as type IIb protein C deficiency. Direct sequencing of the PCR fragments revealed a heterozygous G to A transition at nucleotide position 1462 in exon 3, which predicted an amino acid substitution of Glu 25 by Lys. Her mother and one son were also heterozygous for this mutation. A molecular dynamics simulation of Gla 25-->Lys/EPCR complex in water suggested that the affinity between the molecules was decreased compared to the wild type Gla domain/EPCR complex. Since Gla 25 has been shown to play an important role in protein C function, not only in membrane phospholipid binding but also in binding to EPCR, our findings provide new insight into the mechanism by which the Glu 25-->Lys mutation induces type IIb protein C deficiency in individuals.

The inhibition of protein C anticoagulant activity by anti-β2-glycoprotein I (β2GPI) antibodies isolated from patients with antiphospholipid syndrome by chromatography methods

Abstract Antiphospholipid antibodies (aPL) are associated with an increased risk of thrombosis; however, the mechanism remains unknown. Recent studies have focused on the impediment of protein C anticoagulant activity by anti-β2-glycoprotein I (β2GPI) antibodies (aβ2GPI Ab). We purified IgG fractions containing a high concentration of aβ2GPI Ab from patients with antiphospholipid syndrome (APS) and then investigated the effect of purified aβ2GPI Ab on the activity of activated protein C (APC). Using a three-step chromatography method (DEAE-sepharose column, phosphatidylserine polyacrylamide gel column dependent on the presence of β2GPI, and protein G column chromatography), we successfully isolated anti-β2GPI IgG from nine patients with APS. Seven of nine samples inhibited APC activity in a concentration-dependent manner only in the presence of β2GPI, as observed by a chromogenic assay that was able to determine thrombin activity even in the presence of APC. The extent of APC inhibition by these fractions appeared to be related to aβ2GPI Ab titers of the purified IgG. However, the inhibitory effect of IgG from patients was not detected in the absence of β2GPI. IgG purified from three normal subjects did not affect APC activity. Herein, we show a useful method for the isolation of IgG containing a high concentration of aβ2GPI Ab. Moreover, the present findings indicate that inhibition by aβ2GPI Ab on APC anticoagulant activity could explain one of the mechanisms for the thrombotic state in APS.

TGF-β1 Null Mutation Leads to CD154 Upregulated Expression in Affected Tissues

The TGF-β1(–/–) mouse is a murine model for systemic autoimmune disease. The aim of this study is to elucidate the immunological mechanism that leads to multifocal tissue inflammation and autoantibody production in TGF-β1(–/–) mice. Heart, lung, liver, and salivary gland from TGF-β1(–/–) were assessed for CD154 expression by RT-PCR and immunohistochemistry. Compared to wild-type littermates, CD154 expression was elevated in all tissues studied. Furthermore, IL-12 mRNA was expressed in the salivary gland and heart of TGF-β1(–/–) mice and not in wild-type littermates. This suggests that the CD154 pathway is activated in these tissues. This shows that TGF-β1 regulates CD154 expression leading to spontaneous IL-12 production and autoimmunity.

Molecular dynamics calculations of wild type vs. mutant protein C: Relationship between binding affinity to endothelial cell protein C receptor and hereditary disease

Abstract Molecular dynamics simulations of the protein C γ-carboxyglutamic acid (Gla) domain and endothelial cell protein C receptor (EPCR) complex were performed to determine the effect of a hereditary disease, which results in a mutation (Gla 25 → Lys) in the protein C Gla domain. Our results suggest that the Gla 25 → Lys mutation causes a significant reduction in the binding force between protein C Gla domain and EPCR due to destabilization of the helix structure of EPCR and displacement of a Ca2+ ion.