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Congenital factor XIII (FXIII) deficiency is a rare autosomal recessive bleeding disorder. To date 33 patients with FXIII deficiency have been reported in Japan. we examined the genetic defects in two unrelated Japanese families in which three patients had been diagnosed as congenital FXIII A subunit deficiency. Exons I-XV of the gene encoding the FXIII A subunit were individually amplified by polymerase chain reaction (PCR) and the PCR products were directly sequenced by the dideoxy termination method. The sequence analysis revealed a substitution from G to A at position 1468 in exon XI resulting in Gly461Arg in Family A, and a substitution from C to T at position 2068 in exon XIV resulting in Arg661stop in Family B. One patient of Family A was homozygote of the Gly461Arg mutation and his daughter and grandchild were heterozygote. Since this mutation did not generate or lose any restriction endonuclease recognition sites, we designed a mutagenic primer (F13E11Mut) that serves to generate a new EcoN restriction site in the amplified DNA from affected individuals, when 100 normal alleles were amplified by PCR using the F13E11Mut primer and digested by EcoN, the G to A mutation was not found in any of the normal alleles, and no other mutation was found in the remaining region of the gene encoding the FXIII A subunit. This suggests that Gly461Arg would cause a FXIII A subunit deficiency in Family A. Two patients in Family B were homozygote of the mutation Arg661stop and three other members of this family were heterozygote. This mutation has previously been reported by Mikkola et al. They observed this mutation in six of eight Finnish families and one Swedish family with congenital FXIII deficiency. Since the mutation is due to CpG dinucleotide change to TpG, a hot spot mutation site, the Arg661stop is considered to be a common cause of FXIII A subunit deficiency.

This report describes life-threatening spontaneous splenic rupture in two brothers with congenital afibrinogenemia.Two brothers, aged 11 and 14 years, had intra-abdominal bleeding due to splenic rupture confirmed by ECHO ultrasonography and computed tomography scans.Splenectomy was performed after administration of heat- and solvent-detergent treated fibrinogen concentrates.Hemostatic treatment for splenic rupture using heat- and solvent detergent-treated fibrinogen concentrates was effective. Careful attention must be paid to the risk of splenic rupture during the growth spurt in physically active children with this rare coagulation disorder.

Neonatal lupus erythematosus (NLE) is an antibody-mediated disorder of infants characterized by two major clinical manifestations; cutaneous lupus lesions and congenital heart block (CHB). The disease is associated with placentally transferred maternal anti-Ro/SSA and/or La/SSB antibodies. There is a tendency for the same disease expression to occur within a sibship. To reveal a possible association of class II MHC genes with maternal anti-Ro/SSA autoimmune responses and neonatal outcomes in NLE with a relatively homogeneous ethnic background, haplotype, and allele distributions were analyzed based on the PCR-RFLP results in 26 Japanese anti-Ro/SSA-positive mothers from three groups defined by neonatal outcomes. The results were as follows: (i) maternal HLA-DR5 haplotype DRB1*1101-DQA1*0501-DQB1*0301 and individual class II alIeles making up this haplotype were significantly associated with neonatal cutaneous lupus but not CHB. Conversely, maternal HLA-DQB1*0602 carried on HLA-DR2 haplotypes was associated with CHB but not cutaneous NLE; (ii) HLA-DQA1 alleles with glutamine at position 34 of the first domain, which have reportedly been associated with the autointmune responses to Ro/SSA antigens in other ethnic groups, were increased in the mothers of infants with cutaneous involvement; and (iii) there was no particular class II HLA profile that distinguished the disease manifestations in infants. These findings suggest that specific maternal MHC class II genes might correlate with specific neonatal outcomes in NLE.

High shear stress facilitates von Willebrand factor (vWF) binding to platelet glycoprotein (GP) Ib/IX, causing activation of GPIIb/IIIa to induce platelet aggregation. Here we report that activated GPIIb/IIIa, even occupied by ligands, is not sufficient to mediate platelet aggregation under high shear stress conditions when vWF binding to GPIb/IX is blocked. Platelet rich plasma or washed platelet suspension supplemented with purified human fibrinogen at a concentration of 2 mg/mL were treated with an anti-vWF monoclonal antibody NMC-4 which blocks the binding of vWF to GPIb/IX. After addition of 10 μmol/L ADP, aggregation was continuously monitored under various shear stress conditions (0-108 dyne/cm 2 ) using a cone-plate type aggregometer previously described (Ikeda Y et al J Clin Invest 1991; 87:1234). The extent of maximal aggregation of agonist-stimulated platelets in the presence of NMC-4 correlated inversely with the level of shear stress applied, with the virtual absence of aggregation at 108 dyne/cm 2 . Once aggregated by 10 μmol/L ADP under low shear stress (12 dyne/cm 2 ), platelets could be disaggregated, in part, by the application of high shear stress (108 dyne/cm 2 ), and reaggregated when shear stress was returned to 12 dyne/cm 2 . Flow cytometric analysis revealed that platelets stimulated with 10 μmol/L ADP at 108 dyne/cm 2 bound fluorescein isothiocyanate (FITC)-labeled fibrinogen, although aggregation was absent in this experimental condition. These results demonstrate the dual effect of shear stress on platelet functions; a pro-aggregating activity that induces vWF-GPIb/IX interaction leading to platelet activation, and an anti-aggregating force to prevent the growth of platelet thrombi. It is suggested that the efficacy of vWF blockade is greater under high shear than low shear stress conditions, and that a selective inhibition of platelet functions can be possible.

With the use of the principal neutralizing determinant (PND) peptide-based ELISA to measure anti-PND antibodies that specifically bound synthetic peptides derived from HIVIIIB, HIVMN, HIVRF, HIVSC, HIVWJM-2, HIVAf1l.con, or HIVAf2.con, type-specific antibodies to the HIVMN peptide were studied in 350 serum specimens from Japanese with hemophilia A who had been injected with known unheated factor VIII concentrates until 1985 and had been infected with HIV-1 subtype B. These antibodies were not found in any of the seronegative sera of hemophiliacs, patients with autoimmune diseases, or normal healthy controls. Further, all hemophiliacs rapidly progressing to AIDS and death among the 95 hemophiliacs in a restricted Nara area had antibody titers of less than 20 and their low levels preceded the rapid progression to the disease state. In contrast, slowly progressing hemophiliacs maintained an antibody titer of more than 100 from the initial stages of viral infection and remained asymptomatic. Sequence analysis of the V3 regions of HIV-1 indicated that the hemophiliacs who maintained a high anti-PNDMN antibody level showed a conserved MN sequence. In contrast, the HIV-infected hemophiliacs with nonreactivity in the ELISA showed sequence changes in the neutralizing epitopes of HIVMN. The dynamic of the serum anti-PNDMN antibody titer appear to be a characteristic indicator of the progression of the HIV-infected status in Japanese hemophiliacs seropositive for HIV-1.

Preschool sarcoidosis occurring in children less than 6 years old is rare and characterized by the triad of skin, joint and eye manifestations without any pulmonary lesion. Because of similar clinical manifestations, the diagnosis of preschool sarcoidosis and juvenile rheumatoid arthritis (JRA) is confusing. A girl with preschool sarcoidosis, initially diagnosed and treated as having JRA, is reported here. Ophthalmologic examinations revealed posterior involvement of the eye. A gallium scintigram of the head showed panda appearance. Biopsy of the cutaneous lesion demonstrated non-caseating granuloma. Gallium scanning may be an important clue to correct diagnosis.

Background: Neonatal lupus erythematosus (NLE) is a syndrome characterized by dermatitis and congenital heart block. The disease is mostly associated with transplacental passage of maternal anti-Ro(SS-A) or anti-La(SS-B) antibodies. Maternal HLA-DR3 and DQ2 alleles are associated with NLE in white and North American black populations. Objective: We sought evidence of a potential genetic disposition to NLE in mothers with a relatively homogeneous ethnic background. Methods: Class II human major histocompatibility complex HLA-DRB1, DQA1, DQB1, and DPB1 alleles were determined by polymerase chain reaction–restriction fragment length polymorphism in anti-Ro(SS-A)–positive mothers as well as in infants from seven Japanese families with siblings concordant or discordant for disease expression of NLE. Results: All seven mothers had two or three DQ alleles of DQA1 and DQB1 possessing specific amino acid residues, which are reportedly associated with anti-Ro(SS-A) autoantibody response in white and black populations. There was no class II HLA profile that distinguished disease manifestations of NLE in infants. Conclusion: The HLA class II allele associations with anti-Ro(SS-A) autoantibodies that have been noted in other ethnic groups were also found in Japanese anti-Ro(SS-A)–positive mothers whose infants had NLE, suggesting shared susceptibility factors across racial barriers in maternal predisposition to Ro(SS-A) autoimmune response. (J Am Acad Dermatol 1997;36:186-90.)

SummaryThe structural and functional properties of plasma and platelet vWF were studied in 8 patients (5 unrelated families) with vWD demonstrating a mutation at position 611 (R611C or R611H). Following reduction, electrophoresis and immunoblotting with a polyclonal anti-reduced vWF antibody, abnormal proteolysis of vWF was demonstrated in plasma and to a lesser extent in platelets from all patients, leading to the formation of a unique 209 kDa fragment undetectable in control as well as in type 2A, 2B or 2N vWF. Immunoblotting with MoAbs to reduced vWF showed that the C-terminal end of the 209 kDa fragment was located beyond residue 1744 of the subunit and that its N-terminus was between residues 523 and 1114. Multimeric analysis of patients vWF showed an abnormal pattern in both plasma and platelets, with a moderate decrease of the HMW multimers together with a significant increase of the lowest MW forms. The specific sensitivity of vWF R611C and vWF R611H to proteolysis was further evidenced using V-8 protease. In all patient’s samples the enzyme produced a unique monomeric 80 kDa fragment, absent in V-8 digested normal vWF, which overlapped the N-terminal part of the subunit. The functional analysis of vWF showed a markedly decreased affinity of mutated plasma vWF for platelet GPIb in the presence of ristocetin. Infusion of DDAVP in two of these patients did not lead to significant platelet count change. It induced a limited increase of the HMW multimers in plasma together with a poor correction of the vWF binding to platelet GPIb. In conclusion, our data demonstrate that in addition to a normal proteolysis, vWF mutated at position 611 undergoes a specific cleavage in plasma and platelets. In contrast to the increased proteolysis observed in type 2A and 2B patients’ plasma, this additional cleavage produced a unique 209 kDa species but maintained a HMW multimer-like structure of vWF R611C and R611H.

Introduction. The notion of deformation quantization was introduced by F.Bayen, M.Flato et al. in [1]. The basic idea is to formally deform the pointwise commutative multiplication in the space of smooth functions C∞(M) on a symplectic manifold M to a noncommutative associative multiplication, whose first order commutator is proportional to the Poisson bracket. It is of interest to compute this quantization for naturally occuring cases. In this paper, we discuss deformations of contact algebras and give a definition of deformations of algebras slightly different from the deformation quantization of Poisson algebras. Since the standard 3-sphere is a basic example of a contact manifold, we study the properties of the noncommutative 3-sphere obtained by this reduction. We remark that the parameter of the deformation of a contact algebra is not in the center, while the deformation quantization of Poisson algebras is given by algebras of formal power series of functions on a manifold; in particular, the deformation parameter is a central element. Details and related results will appear in [6] and [7].

Sister Mary Joseph's nodule (SMJN) is a rare umbilical nodule that develops secondary to metastatic cancer. Primary malignancies are located in the abdomen or pelvis. Patients with SMJN have a poor prognosis. An 83-year-old woman presented to our hospital with a 1-month history of a rapidly enlarging umbilical mass. Endoscopic findings revealed advanced transverse colon cancer. computer tomography and fluorodeoxyglucose-positron emission tomography revealed tumors of the transverse colon, umbilicus, right inguinal lymph nodes, and left lung. The feeding arteries and drainage veins for the SMJN were the inferior epigastric vessels. Imaging findings of the left lung tumor allowed for identification of the primary lung cancer, and a diagnosis of advanced transverse colon cancer with SMJN and primary lung cancer was made. The patient underwent local resection of the SMNJ and subsequent single-site laparoscopic surgery involving right hemicolectomy and paracolic lymph node dissection. Intra-abdominal dissemination to the mesocolon was confirmed during surgery. Histopathologically, the transverse colon cancer was confirmed to be moderately differentiated tubular adenocarcinoma. We suspect that SMJN may occur via a hematogenous pathway. Although chemotherapy for colon cancer and thoracoscopic surgery for the primary lung cancer were scheduled, the patient and her family desired home hospice. Seven months after surgery, she died of rapidly growing lung cancer.

In January 1985, a single protocol consisting of cyclophosphamide, vincristine, tetrahydropyranyl adriamycin, and cis-platinum for the treatment of advanced neuroblastoma was begun nationwide in Japan and was found to improve clinical results significantly in terms of 2- or 3-year survival rate. Between January 1985 and December 1988, 113 eligible patients (7 infants younger than 12 months of age with stage IVA disease and 106 patients aged 12 months or older with stage III or IV disease) were enrolled and followed up for 5 years or more after initiation of treatment, as of March 1994. In this study, the usefulness of the protocol for the treatment of advanced neuroblastoma was evaluated with survival rates in relation to age, tumor site, stage, and N-myc amplification for patients surviving more than 5 years after initiation of the protocol. Fifty of the 113 patients were alive 5 years or more after initiation of the treatment, 39 without any episodes of disease recurrence. Fourteen (70%) of 20 patients with stage III, 6 (50%) of 12 with stage IVB, and 24 (30%) of 81 with stage IVA disease were alive and disease-free 5 years after initiation of the protocol. Twenty (56%) of 36 patients without N-myc amplification were alive at 5 years after initiation of the protocol. Only one patient who was alive without evidence of the disease at 5 years had recurrence afterward.

To know the role and initiation mechanism of the interaction of glycoprotein (GP) Ib with surface-immobilized von Willebrand factor (vWF), we examined the effect of shear stress levels on platelet binding to vWF-coated plates using a cone-and-plate type viscometer capable of loading various levels of shear stress. The extent of platelet binding to immobilized vWF reached a plateau at the shortest period tested (20 seconds) under high shear stress (90 dyne/cm2), whereas 9 to 12 minutes was necessary for saturable platelet binding under static conditions. This shear effect, which was found to be dependent on the vWF-GP Ib interaction, was observed even under the lowest shear stress (1.5 dyne/cm2) examined. In contrast with the high shear effect previously reported to initiate the interaction of GP Ib with soluble vWF, these results indicate that relatively low levels of shear stress can promote the interaction of GP Ib with surface- immobilized vWF. This effect of shear stress was observed regardless of the manner in which vWF was immobilized, suggesting that immobilization itself and not, as previously hypothesized, a conformational change in vWF induced by direct adsorption to the surface is responsible for the enhanced GPIb binding. Thus, the present findings suggest that the vWF- GP Ib interaction contributes optimally to rapid platelet cohesion on a thrombogenic surface when vWF is in a static state and when platelets are moved by an appropriate rheological force such as low shear stress.

Oligodendrocyte-like cells (OLD) derived from the rat oligodendroglial precursor line, CG-4, express Ca2+-permeable non-methyl-D-aspartate glutamate receptor channels (GluR). Exposure to kainate, an L-glutamate analogue, markedly elevates OLC Ca2+ influx and cytosolic [Ca2+], and results in damage to both OLC plasma membrane and OLC nuclear DNA. Two observations indicate that kainate-induced OLC internucleosomal DNA nicking is not simply a delayed consequence of cell necrosis: 1) there is no temporal lag between onset of plasma membrane injury and of DNA nicking; and 2) aurintricarboxylic acid, an endonuclease inhibitor, blocks kainate-induced damage to the plasma membrane. N-acetyl-L-cysteine also inhibits OLC kainate injury, suggesting that reactive oxygen species participate in OLC excitotoxicity. Kainate-induced OLC Ca2+ influx and excitotoxicity are blocked by α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA), indicating that these kainate effects are mediated by AMPA-GluR. AMPA and L-glutamate fail to elicit OLC damage unless cyclothiazide, an AMPA-GluR desensitization blocker, is present. OLC express both the “flip” and “flop” forms of GluR2, GluR3, and GluR4 mRNAs, but neither flip nor flop GluR1 mRNA. These data, together with the restriction of the desensitization-blocking activity of cyclothiazide to GluR containing flip-encoded GluR subunits, and the sharply diminished Ca2+ permeability of GluR containing edited GluR2, suggest OLC excitotoxicity is mediated by AMPA-GluR that contain flip GluR3 and/or flip GluR4 protein subunits, but neither flip nor flop GluR2 protein subunits. Rapid desensitization of these GluR is likely to be important in protecting cells of the oligodendroglial lineage from excitotoxicity. © 1996 Wiley-Liss, Inc.

We evaluated expression of N-methyl-D-aspartate (NMDA) and non-NMDA glutamate receptor (GluR) genes by reverse transcriptase-polymerase chain reaction (RT-PCR) and Southern blotting in nine established cell lines: rat CG-4 (oligodendroglial lineage) and RINm5F insulinoma cells; human CHP134, SMS-KCNR, SKNSH, and Nb69 neuroblastoma cells; and human D384Med, D425Med, and D458Med medulloblastoma cells. CG-4 expressed mRNAs encoding GluR2–7, KA-1, and KA-2 non-NMDA GluR (Yoshioka et al.: J Neurochem 64:2442–2448, 1995) and NR1 (NMDAR1) and NR2D NMDA GluR. After differentiation to oligodendrocyte-like cells, CG-4 also expressed NR2B mRNA. Rat insulinoma cells expressed GluR5, and KA-2 non-NMDA and NR1 and NR2D NMDA GluR mRNAs. The four human neuroblastoma lines all expressed mRNAs encoding GluR2–4, 6, 7 and KA-1 non-NMDA and NR1 NMDA GluR, and the three human medulloblastoma cell lines all expressed mRNAs encoding GluR1, 6 and KA-1, but none of the NMDA GluRs. Whereas CG-4 is susceptible to kainate excitotoxicity, treatment of insulinoma, neuroblastoma, and medulloblastoma lines with L-glutamate, kainate, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), or NMDA failed to cause cell damage or to augment 45Ca2+ influx. Thus, despite expressing a variety of non-NMDA and NMDA GluR genes, the human neuroblastoma and medulloblastoma and rat insulinoma lines failed to assemble Ca2+-permeable NMDA or non-NMDA GluR channels. This failure confers protection against excitotoxicity and may contribute to progression of tumors of these types. © 1996 Wiley-Liss, Inc.

Congenital deficiency of α2-plasmin inhibitor is a very rare disease that is associated with severe bleeding problems. To our knowledge, the condition was first reported in 1978 by Koie et al., who described a twenty-five-year-old man who had symptoms similar to those of severe hemophilia. Since then, the cases of at least eight patients, from six families, who had homozygous deficiency have been reported3,7,8,10,12,17. The main symptoms were umbilical bleeding, bleeding in the central nervous system, epistaxis, bleeding gums, hemothorax, hypermenorrhea, hemarthrosis, and excessive loss of blood after minor trauma. These patients often needed to be hospitalized for long periods because of the tendency for severe bleeding that had begun in early childhood and was clinically similar to hemophilia. The episodes of bleeding were thought to be characterized by hemostatic plugs that had formed normally but disappeared rapidly because of unrestrained fibrinolysis. Thus, routine laboratory studies revealed no important abnormality of coagulation or platelet function except the shortened times of lysis of whole blood and euglobulin. The diagnosis usually was made on the basis of assay of the level of antigen or the activity of α2-plasmin inhibitor. We first reported15 multiple intramedullary hematomas, a form of bleeding, in 1991. This condition developed in the long bones of three sisters who had a congenital deficiency of α2-plasmin inhibitor. The episodes of bleeding in these sisters were described in that report15 and in a previous one17. The long-term clinical features of the lesions and the results of successful operative treatment are described in the present report. The concentration of α2-plasmin inhibitor in the plasma of these patients was assayed with functional and immunological methods14, and it …

SUMMARY. Hepatitis C virus (HCV) RNA was tested for, and HCV genotypes determined, in 96 patients with haemophilia A in Japan. Of 88 patients aged ≥ 10 years, 74 (84%) were positive for HCV RNA at a frequency higher than that in patients aged less than 10 years (one of eight, 13%P

Using enzyme immunoassay, we measured the binding ability of artificial gamma-carboxyglutamic acid (Gla)-domainless-Factor VII (FVII) to tissue factor (TF) or of Factor VII in 44 patients stabilized by long term treatment with warfarin. Purified FVII digested with cathepsin G, that is Gla-domainless-FVII, showed a rapid loss in the binding ability of FVII to TF (FVII-TF binding). After adsorption with Al(OH)3 of plasma from 8 out of 44 warfarin-treated patients, no FVII clotting activity (FVII:c) was detected in the supernatant, whereas FVII antigen (FVII:ag) and FVII-TF binding remained 19.2% and 17%, respectively, as compared with those before adsorption. In the plasma from 44 warfarin-treated patients the FVII:c (mean +/- SD, 54.26 +/- 12.50%) was significantly lower than the FVII:ag (77.15 +/- 18.24%) (p0.001), although the FVII:c was significantly correlated with FVII:ag (r = 0.628). FVII-TF binding (68.27 +/- 21.16%) was significantly higher than FVII:c (p0.001), but not than FVII:ag (p0.05). The FVII-TF binding was significantly correlated with FVII:ag (r = 0.738), but somewhat poorly with FVII:c (r = 0.415). These results show that artificial Gla-domainless-FVII digested with cathepsin G loses both the clotting activity and the binding ability to TF. However, PIVKA-VII has little or no clotting activity but the binding ability to TF. This suggested that the low specific activity of FVII (FVII:c/FVII:ag) in the plasma of warfarin-treated patients would not entirely depend on the decreased FVII-TF binding.

We previously reported a new analytical immunoblotting method to detect reduced plasma von Willebrand factor (vWF) fragments using polyclonal anti-reduced vWF antibody and demonstrated that the reduced normal plasma vWF consists of at least five or six fragments, 240kDa for the main fragment and 216 (220, 210), 194, 164 and 144kDa for minor fragments. In this study, we describe the reduced fragments in patients with various types of von Willebrand disease (vWD) (type I: 7 patients, type IIA: 4 patients and type IIB: 3 patients) using this method. Of seven patients with type I vWD, one patient with an equally low level of ristocetin cofactor activity (vWF: RCo) and vWF antigen (vWF: Ag) showed normal relative proportions of each fragment. However, three of four patients with a low level of vWF: RCo compared to vWF: Ag showed a relatively increased proportion of the 164kDa fragment. The remaining two patients demonstrated a new 200kDa reduced fragment in addition to those of normal plasma vWF. In type II A vWD, all patients showed a relatively increased proportion of the 164kDa fragment. In type IIB vWD, two of three patients also showed a relatively increased proportion of the 164kDa fragment. These results suggest that plasma vWF with a low vWF: RCo compared with the vWF: Ag ratio tends to show an increased proportion of the 164kDa reduced fragment, even in type I vWD with a normal multimeric structure. Fragment analysis of reduced vWF in patients with vWD is useful to understand the abnormality at the molecular level, especially in type I vWD.

The sixth edition of the haemophilia B database lists in easily accessible form all known factor IX mutations due to small changes (base substitutions and short additions and/or deletions of

SummaryA multicentre retrospective survey was conducted to re-assess the use of porcine factor VIII (HYATE:C), its side effects and the selection of patients for regular or home-therapy. 15,152,000 units of HYATE:C were used by 154 patients. The median inhibitor cross-reactivity to porcine VIIIC of 137 patients was 15%, 27% of patients lacking cross-reactivity. An absent, intermediate or brisk specific antiporcine anamnestic response was observed in 29, 40 and 31% of patients respectively. Seven patients were treated on-demand as home-therapy for a median 6.2, range 1.5-13 years. 23 further patients were treated regularly in hospital for a median of 3, range 2-7 years. This group used 8,319,000 U of porcine VIIIC for 2,000 bleeding episodes.The incidence of transfusion reactions was 0.001%, 0.64% and 2.3%, for domiciliary infusions, infusions in multiply treated inpatients, and unselected in-patient infusions, respectively. The risk of reactions was dose-related. A post-infusion fall in platelet count was common, but usually transient and clinically insignificant. This was also dose-related (r = -0.64, p = 0.002). Marked reductions in platelet count were occasionally seen, usually with intensive replacement therapy. The relative lack of side effects observed amongst patients treated at home is attributable to the low, median 33 U/kg, dose used by this group.A subgroup of inhibitor patients, identifiable by their absent or modest anamnestic response to porcine factor VIII may be treated regularly and safely with this product in small doses, over a period of years.

Thrombelastgram (TEG) is an old but automated instrument that demonstrates changes occurring during blood coagulation and fibrinolysis. TEG was evaluated to be better than activated partial thromboplastin time (APTT) as a monitor of hemostatic effects when using recombinant factor VIla (65-80 μg/kg) in 3 hemophilia A patients with a high titer of factor VIII inhibitors. TEG was more suitable than APTT, because r, r+k and ma values of TEG were normalized at least for 4 h after the infusion, whereas APTT was variably shortened and was not always maintained at a normal level for 4 h.

Advances in molecular biology have improved the screening for carriers and the prenatal diagnosis of many genetic disorders, including hemophilia B. For instance, based on the eight described dimorphic restriction fragment length polymorphisms within the factor IX gene, a Caucasian female has a 94% chance of being heterozygous (informative) for at least one of these markers. However, ethnic limitations of molecular genetic techniques have been found in diagnosing hemophilia B families in the Japanese and other populations. Previously, we heve demonstrated that three dinucleotide polymorphisms, which locate in the 5' flanking region at nucleotide-793 (FIX-793), in intron A at nucleotide 192 (FIX192), and in the 3' flanking region (FIXHhaI), of the factor IX gene, are present in normal Japanese. Each of these polymorphisms was able to be rapidly ascertained by the polymerase chain reaction technique.In this study, we analyzed 23 Japanese families with hemophilia B, and found that 19 families (82.6%) were heterozygous for at least one of these polymorphisms. Using these polymorphisms together with the extragenic DXS99/Sac I PFLP, which was previously reported as a useful gene marker for Japanese hemophilia B, the informative rate impproved to 87.0%. Carrier detection and the prenatal diagnosis of hemophilia B can be achieved effectively and rapidly in Japanese with these polymorphisms. In fact, we have successfully performed the prenatal diagnosis in two Japanese families with hemophilia B by using FIXHhaI or FIX192 polymorphism analysis. These polymorphisms may also be present and useful for hemophilia B carrier detection in other Oriental population.

A 38-year-old hemophiliac, who had been infected with HIV by the administration of blood products and had been diagnosed as AIDS by the onset of Pneumocystis carinii pneumonia, was admitted to our hospital with the complaints of headache and vomiting. After he was diagnosed as cryptococcal meningitis using the microscopy, cryptococcal antigen detection and culture of cerebrospinal fluid, treatment with amphotericin-B and fluconazole was started. As there was no clinical improvement, spinal drainage was performed and acetazolamide administered in order to reduce the intracranial pressure. Treatment was changed from AMPH-B and FLCZ to a combined therapy of AMPH-B and itraconazole. As his clinical features showed improvement, he was discharged home on a maintenance dose of ITCZ and acetazolamide after having been hospitalized for three months. This case-report may be of use in the management of cryptococcal meningitis in patients with AIDS.

SummaryWe studied the molecular basis of protein C deficiency in 28 Japanese families including 4 asymptomatic families. Two showed a decreased level of function with a normal antigen concentration consistent with type II protein C deficiency and the remaining 26 showed type I deficiency with decreases in both function and antigen level. All the exons and intron/exon junctions of the protein C gene were studied using a strategy combining polymerase chain reaction (PCR) amplification and rapid nonradioactive single-strand conformational polymorphism (SSCP) analysis. The PCR-amplified fragments with aberrant migration on SSCP analysis were sequenced. We identified 11 missense mutations, 1 nonsense mutation, 2 neutral polymorphisms, 1 frameshift deletion, 1 inframe deletion, and 1 splice site mutation. We also identified two different rare mutations in the 5-untranslated region in the protein C gene that may be responsible for the phenotype. Of these molecular defects, ten were novel. From the results of genetic analysis of 47 Japanese families with protein C deficiency reported in this and previous studies, Phel39Val and Met364Ile substitutions and a G8857 deletion were only found in Japanese subjects and seem to be a founder effect. In contrast, Argl69Trp and Val297Met substitutions, both occurring at CG dinucleotides, were commonly observed in not only Japanese but also Western populations, indicating that these are hot spots for mutation in the protein C gene. These molecular defects were found in 22 families in total, accounting for 47% of Japanese families with protein C deficiency. The structural models of the second EGF and protease domains of activated wild-type and mutant human protein C suggest a possible substrate binding exosite on two loops; one from amino acid position 349 to 357 and the other from position 385 to 388, both of which are close to each other in the three-dimensional model.

Factor VIII (FVIII) inhibitor alloantibodies obtained from seven severe haemophilia A patients were examined for their binding regions and their effects on FVIII binding to von Willebrand factor (vWF). Immunoblotting analysis with a panel of recombinant fragments demonstrated that the binding regions of antibodies in cases 1-5 were contained in the C2 domain of the light chain. Antibodies from cases 1 and 2, which recognized an epitope within residues 2248-2312, completely inhibited FVIII/vWF binding in an ELISA (IC50: 5.0 and 9.0 micrograms/ml, respectively). Antibodies from case 3 recognizing 2170-2312 and case 5 recognizing 2170-2327 also inhibited FVIII/vWF binding (IC50: 110 and 400 micrograms/ml, respectively). Case 4 antibodies recognizing 2218-2307 showed barely detectable inhibition and cases 6 and 7 antibodies recognizing the 44 kD heavy chain, did not inhibit. Our results demonstrate that all anti-C2 alloantibodies with epitopes that extend to the residue 2312 inhibit vWF binding and that an overlap of the inhibitor epitope with residues 2308-2312 is critical for maximal inhibition of vWF binding. Prevention of FVIII/vWF binding appears to be a common property of anti-C2 domain inhibitor alloantibodies.

By screening a human fetal brain cDNA expression library using a monoclonal anti-phosphotyrosine antibody, we have isolated a cDNA clone encoding a receptor type protein-tyrosine kinase belonging to the EPH family, NET (neuronally expressed EPH-related tyrosine kinase). NET shows 87% homology in nucleotide sequence and 99% homology in the deduced amino acid sequence to rat elk, suggesting that NET is the human homologue of elk. The NET gene is mapped to human chromosome 3q21-q23 by PCR screening of a human-rodent somatic cell hybrid panel and by fluorescence in situ hybridization. Examination of NET mRNA expression in several human tissues has shown that the NET gene is expressed preferentially in brain as a 5-kb transcript. Steady-state levels of NET mRNA in human brain are greater in the midterm fetus than in the adult. Lower levels of NET mRNA are found in fetal kidney and adult skeletal muscle. The expression pattern of NET mRNA thus differs from that of elk, suggesting that these two gene products may perform distinct roles in human and rat. NET transcripts are detected in human NTera-2 teratocarcinoma cells after retinoic acid-induced neuronal differentiation. Several human tumor cell lines derived from neuroectoderm including primitive neuroectodermal tumor, small cell lung carcinoma, and neuroblastoma also express NET transcripts. Since the NET mRNA expression in human brain is developmentally regulated and is induced during neuronal differentiation, NET potentially plays important roles in human neurogenesis.

von Willebrand factor (vWF) is a multimeric plasma protein with ABO (H) blood group sugar chains. We investigated a total of 330 plasmas from normal individuals having various ABO genotypes, with special reference to vWF antigen and its platelet glycoprotein-Ib-related biological activities, termed ristocetin cofactor (RCof) and botrocetin cofactor (BCof). RCof reflects the biological activity of higher vWF multimers, while BCof reflects that of vWF of multimers of all sizes. Plasmas from normal individuals carrying one O gene (genotypes AO and BO) had slightly, but proportionally lower levels of vWF antigen, RCof, and BCof than those carrying no O gene (genotypes AA, AB, and BB). Normal plasmas from individuals carrying two O genes (genotype OO) showed much lower values for these parameters than the other plasmas, as previously reported. However, multimeric analysis of plasma vWF antigen revealed no differences among the different genotypes.

10025Background: There are few reports about patient’s prognosis depending on primary sites of cancer in palliative care settings. Methods: This study was conducted as a secondary analysis for a la...

Zimmerman et al reported that normal plasma vWF contains proteolytic fragments of 225, 189, 176, and 140 kDa with anti-vWF monoclonal antibodies in the presence of protease inhibitors and partial cleavage of the plasma vWF subunit is a normal event. Now we report here the analysis of normal plasma vWF with home-made polyclonal anti-reduced vWF antibody by using a small amount of normal plasma itself. With this reference, the vWF fragments with mobility corresponding to 240, 216 (220, 210), 194, 164, and 144 kDa were detected. Also by means of employing anti-reduced SP III and SP II antibody, we suggested that the 164 kDa fragment is located close to the carboxy terminal and the 194 and 144 kDa fragments are located close to the amino terminal. Furthermore, the vWF fragments showed no difference in mobility with or without protease inhibitor. In vivo proteolytic degradation of vWF occurring normally was also shown by our technique.

We report a factor VII (FVII) variant, FVIIShinjo, characterized by normal FVII antigen levels and variable procoagulant activity using tissue thromboplastin from different sources. Normal FVII activity is obtained using human placenta thromboplastin but low activity using rabbit or bovine brain thromboplastin. Exons 2-8 and the intron-exon junctions of the FVII genes of the propositus were amplified by PCR from DNA extracted from peripheral white blood cells, and screened by single-strand conformational polymorphism (SSCP) analysis. DNA fragments showing aberrant mobility were cloned and sequenced. We detected a single-point mutation, a homozygous G to A transition at nucleotide position 6,055 in exon 4, which results in the substitution of Arg 79 by Gln in the first EGF-like domain. This mutation results in a loss of a site for the restriction endonuclease Msp I. The Msp I digestion pattern of the PCR-amplified exon 3+4 fragments from each member of the family was determined. The Msp I haplotypes were consistent with this G to A transition being associated with reduced FVII activity as detected using thromboplastins from various species. We conclude that the Arg 79 to Gln substitution in the first EGF-like domain of FVII identified in the propositus is responsible for the inherited FVII abnormality in this Japanese family. We postulate that one of the sites of interaction between FVII and tissue thromboplastin includes Arg 79 in the first EGF-like domain of factor VII.

Cogenital deficiency of α 2-plasmin inhibitor (α 2-PI) is a very rare hereditary disease associated with poor hemostasis. We successfully performed hemostatic management for intraoral bleeding on 11 occasions in three sisters with this condition in the manner described below.Hemostatic laboratory tests in these patients showed an accelerated whole blood clot lysis time (3-4 h) and a short euglobulin lysis time (1.5-2 h). A very low activity and concentration of a 2-PI were also characteristically noted.The following policy is recommended for the management of hemostasis:1) For surgery, hemostasis can be obtained by oral administration of 10mg/kg of tranexsamic acid (t-AMCHA) every 6 hours (40mg/kg/day) from 3 hours preoperatively to the 7 th postoperative day.2) When urgent hemostasis is necessitated by continuous bleeding, intravenous injection of 50mg/kg of t-AMCHA followed by oral administration of 25mg/kg/day of t-AMCHA for several days is effective for the maintainance of a high level of serum α 2-PI activity and proper hemostasis.3) Hemostasis can be achieved by oral administration of 25-35mg/kg/day of t-AMCHA for several days in the case of minor superficial bleeding and hematoma.4) Proper local hemostatic management is necessary for intraoral bleeding, in addition to improving the abnormal fibrinolytic state by t-AMCHA.

The hemostatic effect of 1-deamino-8-D-arginine vasopressin (DDAVP) on tooth extraction was investigated in 7 patients with mild and moderate hemophilia A and von Willebrand disease. Tooth extraction was performed uneventfully on 10 occasions under DDAVP treatment. Primary hemostasis was completely achieved, and postoperative bleeding was not noted in any case. Four activities (VIII: C, VIEW: Ag, VIII R: Ag, VIII R: RC) of F VIII/vWF increased several times 30-60 minutes after DDAVP injection and remained high for 1-3 hours and returned to the initial levels after 24 hours. The increase was high enough to control the hemostasis in most cases, although the cases of hemophilia A showing low VIII: C and von Willebrand Type II A showing low VIII R: RC had a poorer response. These results indicate that DDAVP is useful for the control of hemostasis after tooth extraction in patients with hemophilia A and von Willebrand disease.

Summary. Allergic reactions to concentrates containing factor IX (FIX) are serious complications in the treatment of haemophilia B patients with inhibitor. We have established a therapeutic protocol for such cases using an initial skin test followed by step-wise infusions of FIX concentrates under hydrocortisone cover. We have successfully treated three patients whose treatment with FIX had been suspended.

Vehicle Safety Communications (VSC) have been proposed as part of ITS development to reduce the traffic accidents by providing critical road traffic information beyond driver's view. The system is planned to be implemented on 700MHz band in Japan, and 5.9GHz band in Europe and America. Some applications require the communication in Non-line-of-Sight (NLOS) conditions. However, the feasibility study of the communication performance in NLOS conditions is still an open issue, especially in the urban environment with the traffic flow. In this paper, we explain the differences of radio specifications among EU, USA and Japan based on the ITS standardization work carried out in each region respectively to clear the implementation method for VSC. Then we show the VSC performance results in NLOS conditions through the computer simulation, which duplicates the urban road traffic environment in Tokyo. We compare the simulation results between 5.9GHz and 700MHz bands, and conclude there are some improvements needed for 5.9GHz band to realize the NLOS VSC in the urban environment.

A 41-year-old man was admitted to our hospital because of multiple liver tumors. Colonoscopy showed a mass lesion in the cecum. He was given a diagnosis of endocrine cell carcinoma by immunostaining technique, and received chemotherapy of CAPOX regimen for 3 courses. After that, he underwent second-line chemotherapy of EP(CDDP/VP-16)regimen due to deterioration of his performance status(PS), and his tumor marker NSE. He then showed dramatically improved PS, and improvement in the size of liver mets and NSE(4. 3mg/mL).

We previously reported that cell-based therapies using isolated hepatocytes including hepatocyte transplantation and liver tissue engineering approaches provide therapeutic benefits to hemophilia. For clinical application of these approaches, it is important to establish an active hepatocyte proliferation system that enables providing a sufficient number of hepatocytes. We also reported that human hepatocytes, which were transplanted into the liver of urokinase-type plasminogen activator transgenic severe combined immunodeficiency (uPA/SCID) mice, were able to proliferate while retaining their ability to produce coagulation factor IX. The objective of this study was to explore the functionalities of other coagulation and anticoagulation factors of the propagated human hepatocytes in uPA/SCID mice. Human hepatocytes were transplanted into the liver of uPA/SCID mice, and the propagation status of human hepatocytes in the mice was monitored by the increase in serum human albumin levels and immunohistochemical evaluation on the liver sections. Using uPA/SCID livers with various stages of human hepatocyte propagation, we analyzed the gene expression levels of coagulation factors (prothrombin, factor VII, factor X, and factor VIII) and anticoagulation factors (protein C and protein S) by real-time polymerase chain reaction (PCR) using human-specific primers. As a result, the total amount of raw messenger RNA expression levels increased in all genes analyzed according to the progress of hepatocyte propagation and proliferation. Except for factor VIII, the gene expression levels of the highly repopulated uPA/SCID mouse livers with human hepatocyte showed higher levels than those of normal human livers, indicating that propagated human hepatocytes in the uPA/SCID system possess full functions to produce most of the coagulation-related factors. The current work demonstrated that human hepatocytes can be propagated in experimental animals while maintaining normal gene expression levels of coagulation-related factors. It could be speculated that the propagated cells serve as a cell source for the treatment of various types of coagulation factor deficiencies.

This paper reports that the switching controllability of high-voltage GaN-HEMTs and the cascode connection depends on the feedback capacitance design. The switching behavior of the GaN-HEMT can be controlled by the external gate resistance as the same manner as the conventional Si-MOSFETs. The switching controllability was improved by the substrate connection due to the parasitic capacitance change. The controllability of the cascode connection was slightly worse compared with the Si-MOSFET, because the effective feedback capacitance became small by the step by step switching operation.

Hemophilia A is a bleeding disorder resulting from coagulation factor VIII (FVIII) deficiency. Exogenously provided FVIII effectively reduces bleeding complications in patients with severe hemophilia A. In approximately 30% of such patients, however, the 'foreignness' of the FVIII molecule causes them to develop inhibitory antibodies against FVIII (inhibitors), precluding FVIII treatment in this set of patients. Moreover, the poor pharmacokinetics of FVIII, attributed to low subcutaneous bioavailability and a short half-life of 0.5 d, necessitates frequent intravenous injections. To overcome these drawbacks, we generated a humanized bispecific antibody to factor IXa (FIXa) and factor X (FX), termed hBS23, that places these two factors into spatially appropriate positions and mimics the cofactor function of FVIII. hBS23 exerted coagulation activity in FVIII-deficient plasma, even in the presence of inhibitors, and showed in vivo hemostatic activity in a nonhuman primate model of acquired hemophilia A. Notably, hBS23 had high subcutaneous bioavailability and a 2-week half-life and would not be expected to elicit the development of FVIII-specific inhibitory antibodies, as its molecular structure, and hence antigenicity, differs from that of FVIII. A long-acting, subcutaneously injectable agent that is unaffected by the presence of inhibitors could markedly reduce the burden of care for the treatment of hemophilia A.

Background/Aim. Hemophilia A patients with inhibitors are treated effectively with immune tolerance induction (ITI) therapy. Although anti-idiotypic antibodies may play a certain role in the underlying mechanism, the detailed mechanism by which ITI produces a curative effect remains unknown. The aim of this study was to clarify the immunological aspect of ITI. Methods. Longitudinal T-cell receptor (TCR) analysis was performed during ITI. TCR variable region α-chain and β-chain repertoires were serially analyzed for peripheral blood mononuclear cells (PBMCs), CD4 T cells, and CD8 T cells from 2 hemophilia inhibitor patients treated with ITI (Patients 1 and 2). Furthermore, to see whether skewing observed in TCR analysis resulted from clonality alterations, T-cell clonality was investigated using complementarity-determining region 3 (CDR3) size spectratyping. Results. In the patient 1, inhibitor titer remained to be 19.6 BU/mL for 596 days after ITI commencement, and ITI was unsuccessful. In the patient 2, inhibitor titer disappeared 434 days after ITI commencement, and ITI was successful. In both cases, skewing of TCR variable region α/β-chain repertoires was observed in CD8 T cell subset, whereas not in CD4 T cell subset. Conclusion. Alteration of TCR repertoires, especially TCR variable region β-chain repertoire of CD8 T cells, was distinct between successful and unsuccessful cases, suggesting that immunological response in the early phase affected the ITI outcomes.

Source populations of polyphagous pests often occur on host plants other than the economically damaged crop. We evaluated the contribution of patches of a non-native meadow grass, Lolium multiflorum Lam. (Poaceae), and other weeds growing in fallow fields or meadows as source hosts of an important native pest of rice, Stenotus rubrovittatus (Matsumura) (Hemiptera: Miridae), in an agricultural landscape of northern Japan. Periodical censuses of this mirid bug by using the sweeping method, vegetation surveys, and statistical analysis revealed that L. multiflorum was the only plant species that was positively correlated with the density of adult S. rubrovittatus through two generations and thus may be the most stable and important host of the mirid bug early in the season before the colonization of rice paddies. The risk and cost of such an indirect negative effect on a crop plant through facilitation of a native pest by a non-native plant in the agricultural landscape should not be overlooked.

We show that the MIC-Kepler probrem is simply solved via the phase-space formulation of non-relativistic quantum mechanics. The MIC-Kepler problem is the Hamiltonian system behind the hydrogen atom subjected to the influence of the Dirac’s magnetic monopole field and the square inverse centrifugal potential force besides the Coulomb’s potential force. We get the energy spectrum of the bound states explicitly and construct the Green’s functions for $E \lt 0$ by means of the Moyal product, which is one of the “star” products denoted by “$\ast$”.

Star products parametrized by complex matrices are defined. Especially commutative associative star products are treated, and star exponentials with respect to these star products are considered. Jacobi's theta functions are given as infinite sums of star exponentials. As application, several concrete identities are obtained by properties of the star exponentials.

We investigated the structural elements in human von Willebrand factor (vWF) that influence binding affinity for platelet glycoprotein (GP) Ib using a dispase-digested vWF fragment as a prototype (residues Leu480/Val481-Gly718 of the vWF subunit; Andrews et al, Biochemistry 28:8326, 1989). The major structural features of this fragment are a large A1-loop formed by an intrachain disulfide bond between Cys509 and Cys695 and six O-linked sugar chains. The fragment was chemically modified by (1) reduction and S-carboxyamido-methylation (R/A), (2) desialylation (DS), or (3) a combination of both (R/A-DS). The GPIb binding affinity of these fragments was basically evaluated by competitive binding assay with anti-GPIb monoclonal antibody (LJ-Ib1), a receptor blocker for vWF (Sugimoto et al, Biochemistry 30:5202, 1991). Both the prototype and the R/A fragments were also assessed for their function in shear-induced platelet aggregation. Results unambiguously demonstrated that the presence of a disulfide bridge (Cys509-Cys695) within this domain downregulates the affinity of vWF to GPIb. In addition, it was also demonstrated that the terminal sialic acids attached to six o-linked sugar chains within this domain contribute to optimal functional modulation by the antibiotic ristocetin, but not by snake venom botrocetin.