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We propose suitable ideas for non-formal deformation quantization of Frechet Poisson algebras. To deal with the convergence problem of deformation quantization, we employ Frechet algebras originally given by Gel’fand–Shilov. Ideas from deformation quantization are applied to expressions of elements of abstract algebras, which leads to a notion of “independence of ordering principle”. This principle is useful for the understanding of the star exponential functions and for the transcendental calculus in non-formal deformation quantization.

We encountered a male infant with infantile Alexander disease presenting with megalencephaly and hydrocephalus as a neonate and subtle seizures at 3 months of age. At 6 months of age, bulbar paralysis appeared. Brain magnetic resonance imaging (MRI) showed abnormal findings with white matter involvement and a characteristic periventricular rim, satisfying the diagnostic criteria proposed by van der Knaap, except for MRI contrast. R239H mutation of glial fibrillary acidic protein gene was identified, representing a common cause of infantile-type Alexander disease.

Although the efficacy of recombinant factor VIII (rFVIII) in the treatment of type 3 von Willebrand disease (VWD) has been reported, the mechanisms by which FVIII concentrates devoid of von Willebrand factor (VWF) induce improvements in hemostasis are poorly understood. To address the role of FVIII or intrinsic coagulation in the absence of VWF, we performed a hemostatic analysis. Blood samples were obtained before and after the administration of rFVIII to 2 patients with type 3 VWD. A rotating thromboelastometry assay was performed to examine global interactions in hemostasis. Studies of thrombin-and shear-induced platelet aggregation were also conducted to elucidate the effect on platelet activation. Furthermore, we assessed the rise in the thrombin-induced intracellular concentration of free calcium ([Ca2+]i). Addition of rFVIII to preinfusion blood in vitro corrected thromboelastometric parameters and thrombin-induced aggregation. In ex vivo studies, thromboelastometry analysis showed that rFVIII shortened the onset and progression of the coagulation process. Furthermore, rFVIII corrected low shear-induced and thrombin-induced platelet aggregation in platelet-rich plasma. In addition, rFVIII improved thrombin-induced [Ca2+]i flux in washed platelets. Our observations suggested that FVIII is incorporated into platelets to activate them, as well as to act directly in intrinsic coagulation in the absence of VWF. FVIII may play a critical role even in the absence of VWF.

Background: KL-6 in umbilical cord blood, including from premature infants, and perinatal factors that may affect these values have not yet been adequately discussed. The authors investigated factors affecting cord venous plasma levels of KL-6 in neonates, and to establish a normal range of values for KL-6 in neonatal cord blood. Methods: Cord plasma levels of KL-6 were measured in 75 neonates, and statistical analysis of factors affecting these levels was performed. A normal range for cord plasma KL-6 levels was calculated by statistical analysis methods based on guidelines by the National Committee for Clinical Laboratory Standards. Results: Data from a total of 75 neonates was analyzed. Gestational age ranged from 25 to 40 weeks (median, 33.4 weeks), and birthweight ranged from 776 to 3760 g (median, 1944 g). There was no statistically significant correlation between cord plasma KL-6 levels with gender, gestational age, birthweight, method of delivery, Apgar score 1 min, and postnatal respiratory distress. The normal range of values for cord plasma KL-6 was 44.3–148.2 U/mL (median, 73.0 U/mL). Conclusion: The cord plasma levels of KL-6 were about half the normal values reported in children and healthy adults and were not affected by various perinatal factors. The authors’ findings suggest that plasma KL-6 levels rise when respiratory function is established after birth and tend to further increase with age.

Plasmin not only functions as a key enzyme in the fibrinolytic system but also directly inactivates factor VIII and other clotting factors such as factor V. However, the mechanisms of plasmin-catalyzed factor VIII inactivation are poorly understood. In this study, levels of factor VIII activity increased ∼2-fold within 3 min in the presence of plasmin, and subsequently decreased to undetectable levels within 45 min. This time-dependent reaction was not affected by von Willebrand factor and phospholipid. The rate constant of plasmin-catalyzed factor VIIIa inactivation was ∼12- and ∼3.7-fold greater than those mediated by factor Xa and activated protein C, respectively. SDS-PAGE analysis showed that plasmin cleaved the heavy chain of factor VIII into two terminal products, A137–336 and A2 subunits, by limited proteolysis at Lys36, Arg336, Arg372, and Arg740. The 80-kDa light chain was converted into a 67-kDa subunit by cleavage at Arg1689 and Arg1721, identical to the pattern induced by factor Xa. Plasmin-catalyzed cleavage at Arg336 proceeded faster than that at Arg372, in contrast to proteolysis by factor Xa. Furthermore, breakdown was faster than that in the presence of activated protein C, consistent with rapid inactivation of factor VIII. The cleavages at Arg336 and Lys36 occurred rapidly in the presence of A2 and A3-C1-C2 subunits, respectively. These results strongly indicated that cleavage at Arg336 was a central mechanism of plasmin-catalyzed factor VIII inactivation. Furthermore, the cleavages at Arg336 and Lys36 appeared to be selectively regulated by the A2 and A3-C1-C2 domains, respectively, interacting with plasmin.

Prothrombin complex concentrates (PCC) have been used as bypassing agents for the treatment of haemophilia A patients with inhibitor as well as for replacement therapy in congenital and acquired deficiencies of vitamin-K-dependent clotting factors. The efficacy of PCC is variable, however, especially during long-term and high-dose use, and all currently available products of this nature contain heparin. We have examined the haemostatic properties of PCC using reconstituted whole blood made by mixing coagulation-factor-deficient plasma and washed blood cells. In rotation thromboelastometry (ROTEM), the recommended therapeutic dose of Proplex ST corrected the abnormal patterns. At higher concentrations, however, the ROTEM patterns regressed. In addition, specific assays of coagulation factors appeared unreliable in the presence of 2.5 U/ml Proplex ST; the abnormalities were corrected when protamine sulfate was added. The findings suggest that the presence of heparin in PCC might have a greater effect on global haemostasis. Careful attention to the anticoagulant effect as well as thrombogenicity of PCC is required. Monitoring therapy using such as ROTEM analysis could be highly informative.

This study examined the effects of low-intensity morning exercise (ME) on physiological response during unsteady workload evening exercise. Nine healthy men participated in the following 2 experimental conditions: 15 minutes of bicycle exercise at 40% maximum oxygen consumption (V[Combining Dot Above]O2max) in the morning (the ME condition) and rest (control [C] condition). Heart rate (HR), blood pressure (BP), temperature, oxygen uptake, and natural logarithm of high frequency, an index of cardiac parasympathetic modulation, were measured before evening exercises, which were performed for 32 minutes in 2 parts: The steady-state exercise test included three 4-min bouts of exercise at 20, 60, and 40% V[Combining Dot Above]O2max. The unsteady exercise test consisted of 4-min bouts of exercise with gradual increases and decreases in workload at 20 and 60% V[Combining Dot Above]O2max. Heart rate, BP, and oxygen uptake were measured in both experiments. Maximal and minimal values, amplitude, and phase lags were measured with each cycle of unsteady workload exercise. With steady-state exercise, HR and systolic BP at 60 and 40% V[Combining Dot Above]O2max were significantly lower in the ME condition than in the C condition. However, oxygen uptake was not significantly different between the 2 conditions. With unsteady exercise, the HR and oxygen uptake phase lags were significantly shorter and the amplitude of oxygen intake was significantly larger in the ME condition than in the C condition. There were no significant differences in physiological parameters between the conditions at rest or during recovery. The physiological response during evening exercise is enhanced by low-intensity ME, which might be an effective conditioning method on a sporting event day.

Current theories predict that Allee effects should be widespread in nature, but there is little consistency in empirical findings. We hypothesized that this gap can arise from ignoring spatial contexts (i.e. spatial scale and heterogeneity) that potentially mask an existing fitness–density relationship: a ‘cryptic’ Allee effect. To test this hypothesis, we analysed how spatial contexts interacted with conspecific density to influence the fertilization rate of the freshwater mussel Margaritifera laevis . This sessile organism has a simple fertilization process whereby females filter sperm from the water column; this system enabled us to readily assess the interaction between conspecific density and spatial heterogeneity (e.g. flow conditions) at multiple spatial levels. Our findings were twofold. First, positive density-dependence in fertilization was undetectable at a population scale (approx. less than 50.5 m 2 ), probably reflecting the exponential decay of sperm density with distance from the sperm source. Second, the Allee effect was confirmed at a local level (0.25 m 2 ), but only when certain flow conditions were met (slow current velocity and shallow water depth). These results suggest that spatial contexts can mask existing Allee effects.

We give a review on non formal star product and its star exponentials with concrete examples.

Factor (F)V is converted into its active form, FVa, by limited proteolysis. Thrombin-catalyzed activation of FV is essential for its full cofactor activation. Previously, we reported that thrombin was bound to the C2 domain in the light chain of FVIII. As FV has a similar domain structure to FVIII, we focused on the FV C2 domain as a possible binding region for thrombin. Kinetic parameters, measured by surface plasmon resonance, revealed that the K(d) values of anhydro-thrombin for FV, FVa, and the FV C2 domain were 66, 240, and 670 nmol L(-1), respectively. FV activation was increased by approximately 9-fold by the addition of thrombin. In the presence of the FV C2 domain, this increase of the FV activation was inhibited. However, FV activation was not inhibited by the addition of the FVIII C2 domain. FV was cleaved into a 105-kDa heavy chain and a 71/74-kDa light chain by thrombin-catalyzed proteolysis at Arg709, Arg1018 and Arg1545. In the presence of the FV C2 domain, the cleavage was inhibited at all sites. Proteolysis was not affected by the addition of the FVIII C2 domain. These results indicated that the FV C2 domain contains a major binding site for thrombin and that this domain is necessary for the proteolysis at all cleavage sites. Furthermore, the present results also suggested that thrombin has an independent binding site for FV different from that for FVIII.

8施設の血友病患者の入院医療コストを集計し, その特徴と診断群分類(DPC)点数表の導入に対する問題点を考察した. 2002年4月から2004年3月までの8施設における患者の各入院に対し, 血友病の種類, 体重, インヒビターの有無, 手術の有無, 入院日数, 請求点数などを集計した. さらにそれらのデータからそれぞれの因子により請求点数を解析し, どの因子が請求点数に関与しているか統計学的検定を行った. 入院日数, 請求点数は施設間にばらつきがみられ, 施設別1日あたりの平均医療コストは最少が124,110円, 最大が222,080円であった. 体重別では25kg未満127,280円, 25－50kg 210,720円, 50-75kg 280,861円, 75kg以上526,958円で有意差がみられた. インヒビターの有無(524,416円, 147,534円)でも有意差がみられた. 2004年度の血友病類縁疾患のDPC導入案と比較すると, 全施設で病院側の赤字になることが分かった. 以上より, 血友病患者の入院医療コストは患者の体重や状態により多種多様で, 安易にDPCを導入することは問題と思われた.

Adeno-associated virus (AAV) vectors carrying the B domain-deleted canine FVIII (BDD cFVIII) gene utilizing the beta-actin minimum promoter (167b) pseudotyped with serotype 1 (AAV1-beta-actin-cFVIII) and serotype 8 (AAV8-beta-actin-cFVIII) were developed to express cFVIII in hemophilia A mice. FVIII clotting activities measured by the APTT method increased in hemophilia A mice with intramuscular injection of AAV1-beta-actin-cFVIII in a dose-dependent manner. Therapeutic FVIII levels (2.9+/-1.0%) in hemophilia A mice with the AAV1-beta-actin-cFVIII dose of 1 x 10(12) gc/body were achieved, suggesting partial correction of the phenotype with AAV1-beta-actin-cFVIII vectors. FVIII clotting activity levels in hemophilia A mice with intravenous injection of AAV8-beta-actin-cFVIII also were increased dose-dependently, achieving therapeutic FVIII levels (5-90%) in hemophilia A mice with the AAV8-beta-actin-cFVIII doses of 1-3 x 10(11) gc/body and supernormal FVIII levels (180-670%) in hemophilia A mice with the AAV8-beta-actin-cFVIII dose of 1 x 10(12) gc/body. Transduction of the liver with AAV8-beta-actin-cFVIII is superior to transduction of skeletal muscles with AAV1cFVIII regarding the FVIII production and antibody formation. These data suggested that both AAV1 and AAV8 vectors carrying the FVIII gene utilizing a minimum promoter have a potential for hemophilia A gene therapy.

To elucidate differences in the antigenic structure of factor VIII (FVIII) among mammals, we evaluated cross-reactivities of well-defined antihuman FVIII antibodies with canine and other mammalian FVIII proteins. Monoclonal antibodies against human FVIII recognizing the A1 domain in the heavy chain and the A3 domain in the light chain showed 2.7% and 0% cross-reactivities, respectively, with canine FVIII. The cross-reactivities of 2 alloantibodies and a monoclonal antibody that recognized the A2 domain in the heavy chain were 10%, 38%, and 0%, respectively. On the other hand, 2 kinds of alloantibodies and a monoclonal antibody recognizing the C2 domain in the light chain showed 160%, 390%, and 130% cross-reactivity, respectively, with canine FVIII. The anti-C2 monoclonal antibody (NMC-VIII/5) showed a type 2 inactivating property when tested with canine and human plasma. Moreover, cross-reactivities of NMC-VIII/5 with simian and feline FVIII were 54.5% and 82.8%, respectively, while the cross-reactivities of the anti-A2 monoclonal antibody (JR8) with simian and feline FVIII were 1.3% and 0%, respectively. These findings suggest that the antigenic structure of the C2 epitope has remained relatively conserved throughout mammalian evolution in contrast to the A2 epitope and that a canine model of hemophilia A is useful for FVIII inhibitor experiments.

Ecthyma gangrenosum is a cutaneous infection associated most commonly with pseudomonal sepsis in the immunocompromised patient. We describe a previously healthy 4-year-old boy who developed ecthyma gangrenosum-like lesions secondary to antibiotic treatment for possible streptococcal infection. The skin, ears, and extremities were involved. This presentation emphasizes the importance of awareness of the rare complication of ecthyma gangrenosum-like lesions associated with non-Pseudomonas bacterial infection treated with antibiotics, even in a previously healthy child.

We report 10-year follow-up of a boy diagnosed with idiopathic pulmonary hemosiderosis (IPH). In early childhood, the primary etiology appeared viral, and frequent exacerbations occurred with respiratory infection. Progressive elevation of total IgE and graminaceous grass pollen–specific antibodies gradually become more evident. Furthermore, the flowering period of these plants had coincided with exacerbations in the previous couple of years. The most recent admission to our hospital followed relapse of IPH after an excursion to a nature park during the flowering period. Since the last hospitalization, relapse has not occurred for over a year during treatment with inhaled beclomethasone and leukotriene antagonist, with intravenous liposteroid initially administered on alternate days, tapering to every 6 days. Although these findings were observed from a single case, they suggest that exposure to plant allergens can trigger exacerbation of IPH. Pediatricians should be aware that relapse of IPH may be cause...

Sclerosing mediastinitis is a very rare benign disorder characterised by the development of dense fibrous tissue within the mediastinum. Affected patients are typically young adults with infant cases being uncommon especially in areas without endemic histoplasmosis. We report a Japanese boy with markedly elevated serum inflammatory markers for more than 1 year in the absence of any clinical manifestations. 67Ga-scintigraphy demonstrated an accumulation in the mediastinal region and an open biopsy revealed a hard fibrous mass in the anteriosuperior mediastinum. Thus, a diagnosis of idiopathic sclerosing mediastinitis was made. Conclusion:To the best of our knowledge, this case is the youngest patient reported with this disorder. In patients with mediastinal mass lesions the diagnosis of sclerosing mediastinitis should be considered as well as infectious, autoimmune or neoplastic disease even in children.

The cause of hemophilia is deficiency of coagulation factor VIII production in the liver, which can be cured by liver transplantation. Because the hepatic function of hemophilia patients is quite normal except for production of factor VIII, auxiliary partial orthotopic liver transplantation (APOLT) is beneficial in that patient survival is secured by preserving native liver even in the event of graft loss. However, it is not known whether the graft of APOLT would be enough to cure hemophilia. We evaluated the efficacy and feasibility of APOLT for hemophilia in a canine hemophilia A model that we established. Partial left liver graft was taken from the normal donor (blood factor VIII activity > 60%). The graft was transplanted to the hemophilia beagle dog (blood factor VIII activity < 5%) after resection of the left lobe preserving native right lobe. Changes in time of blood factor VIII activity and liver function parameters were observed after APOLT. APOLT and perioperative hemostatic management were successfully performed. The blood factor VIII activity increased to 30% after APOLT, and was sustained at least 6 weeks throughout the observation period without symptoms of bleeding. The result demonstrated sustained production of factor VIII in the hemophilia recipient after APOLT. Transplantation of approximately one third of whole liver resulted in cure of hemophilia. In conclusion, it is suggested that APOLT would be feasible as a curative treatment of hemophilia A to improve quality of life of the patients.

Liver tissue engineering using hepatocyte transplantation has been proposed as an alternative to whole-organ transplantation or liver-directed gene therapy to correct various types of hepatic insufficiency. Hepatocytes are not sustained when transplanted under the kidney capsule of syngeneic mice. However, when we transplanted hepatocytes with the extracellular matrix components extracted from Engelbreth-Holm-Swarm cells, hepatocytes survived for at least 140 days and formed small liver tissues. Liver engineering in hemophilia A mice reconstituted 5% to 10% of normal clotting activity, enough to reduce the bleeding time and have a therapeutic benefit. Conversely, the subcutaneous space did not support the persistent survival of hepatocytes with Engelbreth-Holm-Swarm gel matrix. We hypothesized that establishing a local vascular network at the transplantation site would reduce graft loss. To test this idea, we provided a potent angiogenic agent before hepatocyte transplantation into the subcutaneous space. With this procedure, persistent survival was achieved for the length of the experiment (120 days). To establish that these engineered liver tissues also retained their native regeneration potential in vivo, we induced two different modes of proliferative stimulus to the naive liver and confirmed that hepatocytes within the extrahepatic tissues regenerated with activity similar to that of naive liver. In conclusion, our studies indicate that liver tissues can be engineered and maintained at extrahepatic sites, retain their capacity for regeneration in vivo, and used to successfully treat genetic disorders.

Background: The proteinuria resulting from IgA nephropathy is secondary to altered charge-selective and/or size-selective properties of the glomerular capillary walls. However, the functional changes occurring within the glomerular capillary network which lead to proteinuria are still poorly understood. In this study, we analyzed the participation of charged components in the glomerular capillary and their role with respect to proteinuria in childhood IgA nephropathy. Methods: We examined glomerular anionic charge in renal biopsy specimens with confocal laser scanning microscopy using FITC-conjugated poly-L-lysine as a cationic tracer. The renal specimens investigated were from 9 children with IgA nephropathy (IgAN(+)) associated with detectable proteinuria in a morning urine specimen, 8 children with IgA nephropathy (IgAN(–)) and 11 children with thin basement membrane disease (TBMD) with no detectable proteinuria. Results: The labeling intensity of glomerular fixed anionic sites from IgAN(+) was significantly lower than that of IgAN(–) and TBMD. Staining the serial sections following methylation treatment revealed that the labeling intensity for fixed anionic sites in TBMD was significantly higher than that of both IgAN(+) and IgAN(–). On the other hand, saponification treatment resulted in significantly more intensive fluorescence in TBMD and IgAN(–) than in IgAN(+). Furthermore, statistical analysis demonstrated a significant correlation between 24-hour protein excretion and the intensity of fixed anionic sites in all patients with IgA nephropathy at pH 7.0 and following staining with saponification treatment. Conclusions: These findings suggest that a reduction of glomerular anionic charge might be causally associated with the development of proteinuria in childhood IgA nephropathy. Furthermore, sulfate components such as heparan sulfate proteoglycan might be involved initially followed by carboxyl components such as sialoglycoproteins in the glomeruli of patients with IgA nephropathy contributing to the occurrence of proteinuria. We suggest that this method represents a straightforward approach to dissect the participation of charged components in the pathogenesis of childhood IgA nephropathy and their relationship to the development of glomerular proteinuria.

Platelet aggregation is mediated by conformational change of integrin alpha(IIb)beta(3). Tyrosine-phosphorylation of cytoplasmic domain of beta(3) upon platelet activation has been demonstrated to play a critical role in this process. Recently, the adaptor protein ShcA has been shown to bind to the tyrosine-phosphorylated beta(3), while it remains open whether ShcA plays any role in platelet aggregation. Here, we show that ShcA bound to tyrosine-phosphorylated beta(3)-tail peptide through its phosphotyrosine-binding domain in vitro. Then, we examined the involvement of ShcA in platelet aggregation by a previously established in vitro assay using platelets permeabilized with streptolysin-O, where exogenous addition of platelet cytosol is required for reconstitution of the Ca(2+)-induced aggregation. When ShcA was specifically depleted with anti-ShcA antibody from the cytosol, this ShcA-depleted cytosol lost the aggregation-supporting activity, which was rescued by addition of purified recombinant ShcA. Thus, ShcA is essential for the Ca(2+)-induced platelet aggregation.

Haemostatic management of intraoral bleeding was investigated in patients with congenital alpha2-plasmin inhibitor (alpha2-PI) deficiency or congenital plasminogen activator inhibitor- 1 (PAI-1) deficiency. When extracting teeth from patients with congenital alpha2-PI deficiency, we advocate that 7.5-10 mg kg(-1) of tranexamic acid be administered orally every 6 h, starting 3 h before surgery and continuing for about 7 days. For the treatment of continuous bleeding, such as post-extraction bleeding, 20 mg kg(-1) of tranexamic acid should be administered intravenously, and after achieving local haemostasis 7.5 mg kg(-1) of tranexamic acid should be administered orally every 6 h for several days. In addition, when treating haematoma caused by labial or gingival laceration or buccal or mandibular contusion, haemostasis should be achieved by administering 7.5-10 mg kg(-1) of tranexamic acid every 6 h. Tranexamic acid can also be used for haemostatic management of intraoral bleeding in patients with congenital PAI-1 deficiency, but is less effective when compared with use in patients with congenital alpha2-PI deficiency. Continuous infusion of 1.5 mg kg(-1) h(-1) of tranexamic acid is necessary for impacted tooth extraction requiring gingival incision or removal of local bone.

Nuclear factor kappa beta (NF-κB) inhibits apoptosis in sensory, hippocampal, and striatal neurons of the central nervous system. Although several apoptotic stimuli have been shown to activate NF-κB in oligodendrocytes, the function of NF-κB in this cell type remains unknown. In this study, we introduced plasmids expressing either the p50- or p65-subunit of human NF-κB into Central Glia-4 (CG-4)—a rat oligodendrocyte precursor cell line—and determined the influence of NF-κB function on tumor necrosis factor-α (TNF-α)-induced apoptosis. Expression of NF-κB markedly prevented CG-4 apoptosis, with p50 being more effective than p65. This anti-apoptotic activity was repressed by IκB-α, an inhibitor of NF-κB. These results imply that NF-κB acts as a potent inhibitor of TNF-induced apoptosis in oligodendrocytes.

Summary. We report a case of haemophilia A with a high responding inhibitor of factor VIII (FVIII) who had a serious retroperitoneal haematoma caused by penetration of a duodenal ulcer. Inhibitor-bypassing therapy was commenced immediately on admission. On the 17th day of treatment with activated prothrombin complex concentrate (APCC; FEIBA®), re-bleeding occurred and thrombelastography (TEG) demonstrated resistance to therapy. Treatment was changed to recombinant activated factor VII (rFVIIa; NovoSeven®) and resulted in clinical improvement together with an improvement in TEG parameters. On the 10th day of continuous infusion with NovoSeven®, however, TEG again showed resistance to therapy. FEIBA® infusions were re-introduced and TEG results remained satisfactory for 7 days. On day 34, however, further retroperitoneal bleeding was evident and a decline in the haemostatic efficiency of FEIBA® was recorded by TEG. NovoSeven® was again successfully administered for 7 days. There were no laboratory findings to indicate disseminated intravascular coagulation (DIC), hypercoagulability or abnormal fibrinolysis. The plasma-based clotting tests did not show any additional prolongation on the occasions when the TEG demonstrated unresponsiveness to FEIBA® or NovoSeven®. These findings suggested that some component of whole blood, other than plasma might have governed the TEG data. The long-term use of APCC such as FEIBA® or rFVIIa, requires careful monitoring in terms of FVIII inhibitor bypassing activity as well as the tendency to DIC.

Summary Eight distinct and potentially causative mutations were identified in eight unrelated Japanese patients with protein S (PS) deficiency, by direct DNA sequencing of the protein Sa (PSa) gene-specific polymerase chain reaction products of all 15 exons and exon/intron boundaries. There were five missense mutations, including two novel mutations (Cys80Tyr and Arg314His), and three showed a major impact on the expected gene products: novel mutations of a 5-bp deletion (delCTCTG887:Cys206Stop) and a nonsense mutation (Glu208Stop), as well as a previously reported splice site (exon 10 +5 A fi G) mutation. One of the patients showed compound heterozygosity for delCTCTG887 and 732A fi G. Investigation for the cosegregation state of these two mutations with PS deficiency in the patient’s family suggested that the delCTCTG887 mutation was responsible for the abnormal phenotype and that the 732A fi G (Lys155Glu) mutation did not appear to play a key role. However, we also identified the same 732A fi G (Lys155Glu) mutation in an unrelated patient with apparent PS deficiency with severe pulmonary embolism, and found that this mutation seemed to cosegregate with a PS-deficient state in her family members. These data implied that unknown factor(s) other than the 732A fi G mutation itself might influence phenotypic expression of PS status in different individuals.

Hemophilia A is genetically very heterogeneous because disease-causing mutations involving deletions, point mutations, insertions, and inversions are scattered throughout the factor VIII gene. Of these mutations, inversions, which are intrachromosomal recombinations between int22h-1 (intron 22 homologous region 1) and 1 of 2 other extragenic copies located 500 kilobases upstream, are the more frequently found defects, especially in patients with severe hemophilia A. Reportedly, approximately half of all severe hemophilia A patients have inversions in intron 22. A group of unrelated patients from the middle of Japan with severe hemophilia A were screened by Southern blot analysis for gene inversions. Forty-two of 100 severely affected patients presented factor VIII gene rearrangements. Of these patients, 36 exhibited the distal type of inversion, and 6 exhibited the proximal type. No other variant type of recombination was observed. In this study, neither the prevalence of inhibitor development against factor VIII nor the frequency of sporadic cases in the group presenting gene inversions was significantly different from that in the group without chromosomal inversions. Southern blot analysis successfully detected a carrier in a hemophilia family for which no patient was available. Genetic counseling of patients with severe hemophilia A and their families will be considerably improved, because the inversions occur in 42% of the Japanese patients with severe hemophilia.

We describe a patient with haemophilia A and factor VIII inhibitor who underwent surgical excision of a large pseudotumour in the left femoral region. Haemostasis was successfully maintained with bolus doses of recombinant factor VIIa at 2-h intervals and anti-fibrinolytic therapy, and the pseudotumour was removed. Subsequently, the dose interval was gradually prolonged to 8 h for a total of 18 days. Although a spontaneous haemorrhage was observed on postoperative day 8, haemostasis was achieved by reducing the dosage interval. No adverse event occurred during the course of treatment.

Tooth extraction was performed in 1 patient with protein C deficiency and another with protein S deficiency — congenital hypercoagulation disorders. The clinical management of these patients is described.

Platelets play critical roles in hemostasis and thrombosis through their aggregation following activation of integrin alphaIIbbeta3. However, the molecular mechanism of the integrin activation inside platelets remains largely unknown. Pharmacological experiments have demonstrated that protein kinase C (PKC) plays an important role in platelet aggregation. Because PKC inhibitors can have multiple substrates and given that non-PKC-phorbol ester-binding signaling molecules have been demonstrated to play important roles, the precise involvement of PKC in cellular functions requires re-evaluation. Here, we have established an assay for analyzing the Ca2+-induced aggregation of permeabilized platelets. The aggregation of platelets was inhibited by the addition of the arginine-glycine-aspartate-serine peptide, an integrin-binding peptide inhibitor of alphaIIbbeta3, suggesting that the aggregation was mediated by the integrin. The aggregation was also dependent on exogenous ATP and platelet cytosol, indicating the existence of essential cytosolic factors required for the aggregation. To examine the role of PKC in the aggregation assay, we immunodepleted PKCalpha and beta from the cytosol. The PKC-depleted cytosol lost the aggregation-supporting activity, which was recovered by the addition of purified PKCalpha. Furthermore, the addition of purified PKCalpha in the absence of cytosol did not support the aggregation, whereas the cytosol containing less PKC supported it efficiently, suggesting that additional factors besides PKC would also be required. Thus, we directly demonstrated that PKCalpha is involved in the regulation of Ca2+-induced platelet aggregation.

One of haemorrhagic toxins present in snake venoms is L-amino acid oxidase (LAO), which catalyzes the oxidative deamination of L-amino acids with the generation of hydrogen peroxide. Although it is widely accepted that LAO alters platelet function, the effects of LAO on human blood coagulation remain largely unknown. The present study demonstrated, for the first time, that M-LAO, LAO purified from the venom of Agkistrodon halys blomhoffii (Japanese mamushi), possesses an anticoagulant activity. Thrombelastography (TEG) showed that M-LAO significantly delayed the onset and the progress of the coagulation process. In addition, the enzyme prolonged the activated partial thromboplastin time (aPTT) dose-dependently, but had little effect on the prothrombin time (PT), suggesting that its principal activity was mediated in the intrinsic coagulation pathway. Furthermore, M-LAO reduced factor IX procoagulant activity in a dose-dependent manner and did not affect other coagulation factors. These results indicate that M-LAO has an anticoagulant activity that impairs the intrinsic clotting by inhibiting factor IX.

Background There are few reports that demonstrate the chronologic changes in the functional integrity of the internal thoracic artery (ITA) wall after skeletonization. We investigated the impact of skeletonization on ITA wall integrity by immunohistochemical analyses in acute and chronic phases. Methods Nine mongrel dogs underwent bilateral ITA dissection with one skeletonized vessel and the other pedicled. The following studies were performed 1 week (acute phase, n=3) and 12 weeks (chronic phase, n=6) after ITA harvesting. All specimens of the ITAs were stained by antibodies against von Willebrand Factor (VWF), endothelial nitric oxide synthase (eNOS), inducible nitric oxide synthase (iNOS), and proliferating cell nuclear antigen (PCNA). After observation with confocal laser scanning microscopy, quantitative analyses of the staining signal for VWF and eNOS expressed on endothelial cells were performed. Results There were significantly more microvessels positive for VWF in the adventitia of skeletonized ITAs than in the adventitia of pedicled ITAs but the expression of PCNA in both groups was minimal, as in normal vessels. iNOS was not detected in any specimen. The intensity of VWF and eNOS expressed by endothelial cells had no significant differences between groups at either phase. Conclusions The functional integrity of skeletonized ITA was similar to that of pedicled ITA in both acute and chronic phases. Although skeletonization induced neovascularization in the adventitia it did not induce proliferation of smooth muscle cells in the media, which is supposed to be a feature of vascular remodeling.

To explore the mechanisms that underlie the bleeding tendency in type 2A and 2B von Willebrand disease (VWD), we analyzed the mural thrombus generation process on a collagen surface under physiologic blood flow in a perfusion chamber using whole blood from these VWD patients. At a low shear rate (50 s−1), thrombus generation in all type 2A and 2B VWD patients was comparable to that of healthy controls. At a high shear rate (1500 s−1), thrombus generation was impaired in all type 2A patients, whereas that in type 2B VWD patients varied from normal to significantly defective, as judged by epifluorescence microscopy of thrombus surface coverage. However, in type 2B patients who showed normal thrombus generation at 1500 s−1, the height and volume of thrombi was significantly reduced, albeit with the normal surface coverage, compared with control thrombi, and von Willebrand factor (VWF) was poorly distributed within the type 2B thrombus mass when analyzed in detail by confocal laser scanning microscopy. Addition of purified VWF to patient blood completely reversed the defective spatial thrombus growth in type 2B VWD. Thus, our results confirm the impaired thrombus generation in type 2B VWD, which has never been demonstrable in previous in vitro soluble-phase platelet aggregation assays, and point to the critical function of larger VWF multimers in the proper spatial growth of mural thrombi under high shear rate conditions.

SummaryReplacement therapy with factor VIII (FVIII) products causes immune abnormalities in human immunodeficiency virus (HIV)-seronegative hemophilia patients. However, the question remains why an absolute increase in the number of CD8+ T-cells and diminished proliferation responses of lymphocytes to antigen stimulation in vitro occurs in HIV-seronegative hemophilia patients.To examine whether the FVIII products induce skewing of T-cell receptor (TCR) repertoires, TCR variable region α-chain and β–chain repertoires were analyzed for peripheral blood mononuclear cells (PBMCs) from 15 hemophilia patients treated with heated and/or non-heated plasma-derived FVIII concentrates and 10 age-matched healthy adults. Also, T-cell clonality was compared between these groups using complementarity-determining region 3 (CDR3) size spectratyping. The skewing of TCR repertoires was significantly greater for hemophilia patients than healthy controls. The extent of T-cell clonality was greater for hemophilia patients than the controls, indicating that clonal T-cells frequently expanded in hemophilia patients. The skew in TCR usage and clonal expansion were primarily observed in patients treated with non-heated plasma-derived products.The spectratyping and sequencing of CDR3 regions revealed that the clonal expansion of T-cells was observed for CD8+ T-cells, but not CD4+ T-cells.These results suggest that extensive expansion of CD8+ T-cells is induced by some viruses other than HIV present in FVIII preparations, and the resulting accumulation of CD8+ T-cells is responsible for changes in peripheral T-cell population in HIV-seronegative hemophilia patients.

We demonstrate by reverse transcriptase-polymerase chain reaction and Southern blotting that an immortalized rat oligodendroglial cell line (CG-4) expresses the non-N-methyl-D-aspartate (non-NMDA) glutamate receptor (GluR) genes GluR2-7, KA-1, and KA-2 and that nonimmortalized cells of the rat oligodendroglial lineage express the GluR1-3, GluR5-7, KA-1, and KA-2 genes. Lactic dehydrogenase release assays show that both immortalized and nonimmortalized cells of the oligodendroglial lineage are damaged by a 24-h exposure to 500 microM kainate or 5 mM L-glutamate, but not by a 24-h exposure to up to 10 mM alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA). Damage is prevented by the non-NMDA GluR channel inhibitor 6-cyano-7-nitroquinoxaline-2,3-dione and is also averted if Ca2+ is removed from the culture medium. Cyclothiazide, which blocks desensitization of AMPA-preferring GluRs, increases cytotoxicity of kainate as well as inducing toxicity of AMPA. We conclude that cells of the oligodendroglial lineage express a population of AMPA-preferring and possibly also kainate-preferring GluR channels that are capable of mediating Ca(2+)-dependent excitotoxicity and that AMPA-induced cytotoxicity is blocked by desensitization of AMPA-preferring GluRs.

Hepatic veno-occlusive disease (VOD) is a life-threatening complication after stem cell transplantation (SCT), characterized by thrombus formation in hepatic venules leading to a symptom triad of hyperbilirubinemia, hepatomegaly, and ascites. Multifactorial defects in the hemostatic system may contribute to its pathogenesis, but its remains to be investigated. Unusually large VWF multimers (UL-VWFMs), produced in and released from vascular endothelial cells, are most biologically active in the interaction with platelets under a high shear stress. UL-VWFMs are cleaved and degraded into smaller VWFMs by a specific liver producing plasma protease, termed VWF-cleaving protease (VWF-CPase), which has recently been identified as a metalloprotease solely produced in liver, termed ADAMTS13. Herein, we studied the correlation between plasma VWF-CPase activity and UL-VWFMs in 21 patients who received SCT, seven patients with VOD and 14 patients without VOD. In non-VOD patients, activities (mean +/- 1s.d.) of VWF-CPase were 78 +/- 17% of the control before the conditioning regimen, 76 +/- 18% on day 0, 64 +/- 19% on day 7, 57 +/- 23% on day 14, 68 +/- 13% on day 21 and 79 +/- 19% on day 28 after SCT. The respective values in VOD patients were 32 +/- 19%, 27 +/- 15%, 18 +/- 11%, 22 +/- 18%, 26 +/- 22% and 12 +/- 4%. Thus, VWF-CPase activity was significantly reduced in VOD patients, even before the conditioning regimen, and such a difference was not found in other laboratory tests. However, despite such a clear difference, UL-VWFMs were present in plasmas of both patient groups, together with the increase of VWF antigen and ristocetin cofactor activity. These results indicate that the measurement of this enzyme activity is extremely useful in predicting the occurrence of VOD prior to a demonstration of its direct involvement in its pathogenesis.

Factor VIII (FVIII), an essential cofactor that accelerates the generation of factor Xa (FXa) in the tenase complex, is activated by proteolytic cleavage by thrombin or FXa. A strong relationship has been reported between high levels of FVIII activity and thrombosis. We have demonstrated previously that an anti-FVIII C2 antibody (ESH8) with a Val-2248-Gly-2285 epitope inhibited FXa-catalysed FVIII activation, and that a synthetic peptide designated EP-2 (residues 2253-2270) blocked C2 domain binding to FXa. We investigated the inhibitory effect of EP-2 on FXa-catalysed FVIII activation and its anticoagulant effect in the blood coagulation system. EP-2 inhibited FXa-catalysed activation in a clotting assay in a dose-dependent manner and reduced FXa generation in a chromogenic assay using FVIII, factor X, factor IXa and phospholipid. The peptide only inhibited FVIII binding to FXa. We also tested the anticoagulant effect of EP-2 in the plasma milieu. The peptide prolonged the activated partial thromboplastin time and activated clotting time in a dose-dependent manner, but not prothrombin time. Our results indicate that EP-2 mediates the anticoagulant effect by specific inhibition of FVIII and FXa interaction in the intrinsic pathway, and that FXa-catalysed FVIII activation plays a significant role in blood clotting. The peptide may provide the basis for the development of novel anticoagulant therapy.

Recent studies have revealed that the platelet adhesive process under flow is tightly regulated by multiple ligand-receptor interactions. However, platelet morphological changes during this process, particularly its physiological relevance, remain unknown under blood flow conditions. Using epifluorescence and scanning electron microscopy, we evaluated the real-time changes in platelet morphology during a platelet adhesive process on a von Willebrand factor-coated surface under physiological high shear flow in a perfusion chamber. Here, we show that dynamic platelet shape changes occurring during distinct phases of the adhesive process are precisely regulated by “inside-out” and “outside-in” integrin signals and are also a key regulatory element in successful platelet thrombogenesis opposing rapid blood flow in vivo.

Hemophilic dogs have an identical inheritance pattern and bleeding symptoms to human hemophilia A and therefore are a most suitable animal model of the human disease. The probability based on the pedigree data and the measurement of factor VIII coagulant activity (FVIII : C) have been used for carrier detection of canine hemophilia A. In order to improve the precision of carrier diagnosis, a ratio of FVIII : C to von Willebrand factor antigen (VWF : Ag) was used for the assessment of carrier status of canine hemophilia A. Two beagle dogs suspected to be hemophilic carriers by the reduction of FVIII : C to around 50 U/dl were tested for the ratio of FVIII : C/VWF : Ag. They were mated with a normal male beagle, and newborn puppies were tested for coagulation assays. One dog strongly suspected of being a carrier by the ratio was confirmed as a definite carrier by the production of a hemophilic puppy, which may be the first hemophilic dog in Japan resulting from a planned mating. The other dog suspected not to be a carrier by the ratio has never produced any hemophilic puppies. These findings suggest that the FVIII : C/VWF : Ag assessment is more reliable than FVIII : C alone for carrier detection of canine hemophilia A and is useful in the establishment of a canine hemophilic colony.

SummaryThe lower detection limit of the conventional one-stage aPTT based clotting assay for determining FVIII:C levels is generally 1.0-2.0 IU/dl. Consequently, it has been impossible to study the clinical significance of levels of FVIII:C less than 1.0 IU/dl. Using a photo-optical automated coagulation analyzer, the Organon Teknika MDA II®, we have performed qualitative and quantitative aPTT waveform analysis and measured FVIII:C levels by automated one-stage aPTT clotting assay in 36 severely affected Hemophilia A patients. Qualitative waveform analysis showed clear evidence of individual differences in the waveform profile suggesting differing coagulant activity from patient to patient. The FVIII:C level was less than 0.2 IU/dl in 23 cases and levels of FVIII:C between 0.2 and 1.0 IU/dl could be discriminated in 13 patients. The FVIII:C level in these patients was closely correlated with the minimum value of the second derivative of the aPTT waveform (Min2). This is a measure of the acceleration of change in optical transmission at the initiation of coagulation. Furthermore, the correlation of the aPTT (time and Min2) and the FVIII:C level, determined by a one-stage clotting assay on the same system, was high in plasmas with a range of levels of FVIII:C artificially prepared by the addition of various concentrations of rFVIII:C to FVIII deficient plasma. These data suggest that it is possible to segregate different levels of clotting activity in patients previously assigned to a single grouping, i.e. less than 1.0 IU/dl, by conventional one-stage assay. Thus clot waveform analysis may be useful for the investigation of the clinical phenotype of individual patients and their response to therapy.

Large-scale land abandonment and reconstruction activity has altered the ecosystem structure in the evacuation area for the Fukushima Daiichi power plant accident in 2011. Despite social concerns about changes in the avian assemblages that occurred after the accident, publicly accessible data are quite limited. We engaged in acoustic monitoring of birds using digital voice recorders from 2014 in and around the Fukushima evacuation zone. All monitoring sites were located within schoolyards (including those that had been converted to community centers) to examine the bird assemblages in the urban and rural landscapes that were heavily altered by land abandonment due to the nuclear plant accident. A digital voice recorder was installed at each monitoring site during May–July, and we recorded 20 min a day using timer-recording mode. We divided the audio data into 1-min segments and identified species occurred in sampled segments by experts. These data represent the presence-absence records from 52 sites monitored in 2014. In 2014, we identified the species for 7138 segments in total and 68 species occurred. We are continuing to monitor and intend to update the dataset with new observations hereafter. Our dataset will help people to recognize the status and dynamics of avian assemblage inside the evacuation zone, and will contribute to promote open science in avian ecological studies.

A 79-year-old man was admitted due to progressive weakness of both hands for two and a half years. Neurologically, he presented with weakness of the upper limbs, predominantly in the left distal portion, and hypoactive deep tendon reflexes. Nerve conduction studies were consistent with a motor and sensory demyelinating neuropathy. Sural nerve biopsy revealed a decrease of myelinated fibers with a predominance of larger diameter fibers. Widening of myelin lamellae and uncompacted myelin were detected on electron microscopy. Laboratory examinations showed IgM-kappa-type M-protein and anti-myelin-associated glycoprotein (MAG) antibody. He was diagnosed as having anti-MAG-associated demyelinating neuropathy based on the laboratory, electrophysiological, and pathological findings. While no bulbar symptoms or upper motor neuron signs were shown, the patient developed quadriparesis and respiratory failure after three years. Although anti-MAG-associated demyelinating neuropathy is usually characterized by sensory symptoms, particularly sensory ataxia, the present case indicates that motor symptoms such as quadriparesis and respiratory failure can be among the clinical manifestations of antiMAG-associated demyelinating neuropathy.

Hereditary diffuse leukoencephalopathy with spheroids (HDLS) is a young-adult-onset autosomal dominant white matter disease characterized by progressive cognitive dysfunction. We herein report the case of a 20-year-old woman who developed spastic hemiplegia. Brain magnetic resonance imaging revealed increased bilateral T2 signal intensity and bright diffusion-weighted imaging signals with a low apparent diffusion coefficient within the frontoparietal white matter. The lesion gradually expanded for over one year. The patient was initially diagnosed with multiple sclerosis (MS); however, she did not respond to immunosuppressive therapy. DNA sequencing showed a heterozygous c.2381T>C mutation in colony-stimulating factor 1 receptor. HDLS with a pure motor phenotype is sometimes difficult to differentiate from MS.