Junki Takamatsu

EFFECT OF GB VIRUS C/HEPATITIS G VIRUS COINFECTION ON THE COURSE OF HIV INFECTION IN HEMOPHILIA PATIENTS IN JAPAN

OBJECTIVE A novel virus, GB virus C (GBV-C)/hepatitis G virus (HGV), has been isolated. This virus is parenterally transmissible, but its effect on various diseases remains to be disclosed. We investigated the effect of GBV-C/HGV coinfection on the course of HIV infection. METHODS GBV-C/HGV RNA was measured by nested reverse transcription polymerase chain reaction (RT-PCR) in 41 HIV-infected hemophilia patients in Japan. Patient characteristics, HIV RNA concentrations, and rates of progression to AIDS and to death were compared in patients with and without GBV-C/HGV coinfection. HIV RNA was quantified by the Amplicor HIV Monitor test (Roche Molecular Systems, Somerville, NJ, U.S.A.), and progression to AIDS and to death was analyzed using Kaplan-Meier plots. RESULTS GBV-C/HGV infection was present in 11 of 41 of patients (26.8%). Mean HIV RNA concentration was lower in patients with GBV-C/HGV coinfection (3.52+/-4.81 x 10(4) copies/ml) than in patients without coinfection (5.76+/-14.78 x 10(4) copies/ ml) and progression to AIDS and to death were slower in patients with GBV-C/HGV coinfection than patients without it, although the differences were not statistically significant. CONCLUSION In Japanese hemophilia patients, coinfection with GBV-C/HGV does not have an adverse effect on the course of HIV infection.

Effect of immunosuppression on composition of quasispecies population of hepatitis C virus in patients with chronic hepatitis C coinfected with human immunodeficiency virus

BACKGROUND/AIMS To study the effects of the immunosuppression caused by the reduction of CD4 activity on the composition of hepatitis C virus (HCV) populations, we analyzed the number of HCV quasispecies clones and the nucleotide diversity of the hypervariable region 1 (HVR1) of HCV in 37 patients with hemophilia with persistent HCV infection, with or without human immunodeficiency virus (HIV). METHODS The numbers of HCV quasispecies clones were measured by fluorescence single-strand conformation polymorphism analysis. Direct sequencing was used to analyze the degree of diversity of HVR1. We compared these values according to coinfection with HIV, and CD4 counts of patients. RESULTS There were no differences in either the number of HCV clones or the diversity between patients with and without HIV coinfection. In HIV coinfected patients the diversity decreased in association with the decrease in CD4 count while the number of HCV clones did not. The diversity of HVR1 was 3.64 +/- 5.03% in patients with a CD4 count < 50/microliters and 14.92 +/- 6.03% in patients with a CD4 count > or = 50/microliters; it was significantly lower in the former (p = 0.0002). CONCLUSIONS A severe reduction in the CD4 count, which is considered to cause a decline in the activity of helper T-lymphocytes, induced changes in the composition of HCV populations; one or a few quasispecies clones are predominant in the HCV population in the serum of individual patients.

α2-PLASMIN-INHIBITOR DEFICIENCY (MIYASATO DISEASE)

A 25-year-old man, born in Okinawa, Japan, had a haemorrhagic diathesis characterised by prolonged bleeding and ecchymoses after minor trauma and spontaneous joint haemorrhage. The frequency and severity of these episodes were reduced by an antiplasminic drug. Routine coagulation studies revealed no abnormalities except for significantly sshortened euglobulin-lysis time and whole-blood clot lysis time. Activities of all known clotting and fibrinolytic factors were within normal ranges but no circulating alpha2-plasmin inhibitor was found in the plasma. alpha2-plasmin inhibitor is a potent and fast-acting protease inhibitor. Studies of family members indicated that this abnormality was inherited as an autosomal and recessive gene.

Molecular Evaluation of Endothelial Progenitor Cells in Patients With Ischemic Limbs Therapeutic Effect by Stem Cell Transplantation

Objective—Although some patients with limb ischemia have recently undergone therapeutic angiogenesis by cell transplantation, their angiogenic potential has not been well characterized. It is also important to evaluate endothelial progenitor cell (EPC) contents in different stem cell sources to choose the best material for therapeutic angiogenesis. Methods and Results—We quantitated the mRNA expression of EPC-specific molecules (eg, Flk-1, Flt-1, CD133, VE-cadherin, etc) in bone marrow-derived or peripheral blood-derived mononuclear cells obtained from patients with ischemic limbs, using real-time reverse-transcription polymerase chain reaction technique. The mRNA expression level of EPC markers was significantly lower in the patients than in healthy controls, which was consistent with results of flow cytometric analysis. However, the implantation of autologous bone marrow mononuclear cells increased the circulating EPCs in the peripheral blood of patients. We furthermore revealed the different expression pattern of EPC markers in possible sources for stem cell transplantation, including normal bone marrow, peripheral blood obtained from recombinant granulocyte colony–stimulating factor-treated donor, and umbilical cord blood. Conclusions—Patients with peripheral obstructive arterial diseases may have lower angiogenic potential because of decreased expression of EPC specific molecules in their marrow and blood. Therapeutic angiogenesis by transplantation of autologous marrow mononuclear cells increased circulating EPCs in the patients and improved ischemic symptoms.

A Comparative Double-Blind Randomized Trial of Activated Protein C and Unfractionated Heparin in the Treatment of Disseminated Intravascular Coagulation

A randomized prospective double-blind trial was performed to compare the safety and efficacy of human activated protein C (APC) and unfractionated heparin for the treatment of disseminated intravascular coagulation (DIC). One hundred thirty-two patients with DIC were enrolled in this study: 63 patients received APC (12.5 U [2.5 μg]/kg body wt per hour) and 69 patients received heparin (8 U/kg body wt per hour) by intravenous infusion for 6 days. Forty-nine APC-treated patients and 55 heparintreated patients were evaluated for efficacy, and 52 APC-treated patients and 55 heparin-treated patients were evaluated for safety. The 2 groups were similar with respect to sex, age, body weight, underlying diseases, and coagulation/fibrinolysis parameters before treatment. Aggravation of bleeding was seen after treatment in 8 patients receiving heparin, but in none of the patients receiving APC. The number of patients who showed alleviation of bleeding was significantly higher in the APC group than the heparin group (P = .009). The effects on DIC-related organ dysfunction were not significantly different between the 2 groups. Fibrinogen-fibrin degradation products, D-dimer, thrombin-antithrombin complex (TAT), and plasmin-plasmin inhibitor complex (PIC) were all significantly decreased by treatment in both groups. Fibrinogen, protein C, and antithrombin were significantly increased in the APC group, whereas only protein C was significantly increased in the heparin group. Platelet count in the nonleukemic group was significantly increased in those patients receiving APC but not increased in those patients receiving heparin. Improvement of coagulation/ fibrinolysis was assessed by scoring 4 parameters (soluble fibrin monomers, D-dimer, TAT, and PIC), and the results indicated that the APC group showed significantly greater improvement than the heparin group (P = .046). There was, however, no significant difference in the rate of complete recovery from DIC between the 2 groups. The rate of death from any cause within 28 days after treatment was 20.4% in the APC group, significantly lower than the 40% death rate observed in the heparin group (P < .05). There were no severe adverse events in either group. These results suggest that APC in a relatively small dosage can improve DIC more efficiently than can heparin, without increasing bleeding, and may be a better alternative.

Characteristics of Helicobacter pylori-Induced Gastritis and the Effect of H. pylori Eradication in Patients with Chronic Idiopathic Thrombocytopenic Purpura

Background.  The association between Helicobacter pylori infection and idiopathic thrombocytopenic purpura (ITP) has been reported widely. We investigated the prevalence of H. pylori infection, its virulence profile and the effectiveness of its eradication in patients with ITP.

A phenotypically neutral dimorphism of protein S: The substitution of Lys155 by Glu in the second EGF domain predicted by an A to G base exchange in the gene

During the course of structural gene analysis of a family with type III protein S deficiency, we found a novel DNA polymorphism: an A or G variation at nucleotide 732 in exon 6 of the PS-alpha gene. This A to G mutation would lead to a substitution of Lys155 by Glu in the second EGF domain. Linkage study with restriction enzyme analysis using mutagenic PCR strategy showed that the same mutation was also present in three other members of the patients family and two individuals from an unrelated kindred, while they all had normal amounts of both immunological and functional PS levels. Restriction enzyme analysis of 182 normal Japanese genomic samples showed that 1.65% of normal population were heterozygotes for this variant allele. These findings suggest that this substitution in exon 6 is not responsible for the type III protein S deficiency but a phenotypically neutral polymorphism. Hereby we designate this polymorphism as PS-732.

A two-amino acid insertion in the Cys146- Cys167 loop of the alphaIIb subunit is associated with a variant of Glanzmann thrombasthenia. Critical role of Asp163 in ligand binding.

The ligand binding site(s) of the alpha subunit of integrin alphaIIb beta3 (GPIIb-IIIa), a prototypic non-I domain integrin, remains elusive. In this study, we have characterized a Japanese variant of Glanzmann thrombasthenia, KO, whose platelets express normal amounts of alphaIIb beta3. KO platelets failed to bind the activation-independent ligand-mimetic mAb OP-G2 and did not bind fibrinogen or the activation-dependent ligand-mimetic mAb PAC-1 following activation of alphaIIb beta3 under any condition examined. Sequence analysis of PCR fragments derived from KO platelet mRNA revealed a 6-bp insertion leading to a 2-amino-acid insertion (Arg-Thr) between residues 160 and 161 of the alphaIIb subunit. Introduction of the insertion into wild-type recombinant alphaIIb beta3 expressed in 293 cells led to the normal expression of alphaIIb beta3 having the defect in ligand binding function. The insertion is located within the small loop (Cys146-Cys167) in the third NH2-terminal repeat of the alphaIIb subunit. Alanine substitution of each of the oxygenated residues within the loop (Thr150, Ser152, Glu157, Asp159, Ser161, and Asp163) did not significantly affect expression of alphaIIbbeta3, and only Asp163AlaalphaIIb beta3 abolished the ligand binding function. In addition, Asp163AlaalphaIIb beta3 as well as KO mutant alphaIIb beta3 constitutively expressed the PMI-1 epitope. Our present data suggest that Asp163 of the alphaIIb subunit is one of the critical residues for ligand binding.

Arg260-Cys mutation in severe factor XIII deficiency : conformational change of the A subunit is predicted by molecular modelling and mechanics

To explore the implications of the structure/function relationships in factor XIII, a patient with severe A subunit deficiency was examined at the DNA and RNA levels. Nucleotide sequence analysis of the patients DNA amplified by PCR revealed that the patient had a replacement of C by T in the codon for Arg260. RT‐PCR analysis demonstrated that only one kind of mRNA coding for the Arg260‐Cys mutation was expressed in the patient at a normal level. Another possible defective allele of the A subunit gene with a G‐A polymorphism was not expressed (null allele). The substitution of Arg260 by Cys located on the interface of two A subunits would preclude the reciprocal ionic interaction (salt bridge) between Arg260 and Asp404. Molecular modelling and, for the first time, molecular mechanics calculated that Cys260 changed the local conformation of the A subunit and reduced the electrostatic interaction between two monomers, suggesting destabilization of the molecules dimer.

Impaired secretion of the elongated mutant of protein C (protein C-Nagoya). Molecular and cellular basis for hereditary protein C deficiency.

Genetic analysis of a heterozygous protein C-deficient patient revealed a novel deletion of a single guanine residue (8857G) among four consecutive guanine nucleotides [380Trp(TGG)-381Gly(GGT)] in exon IX, which encodes the carboxyl-terminal region of protein C. This deletion results in a frameshift mutation and substitution of the last 39 amino acids (381Gly-419Pro) with 81 abnormal amino acid residues, and we have designated this elongated variant as Protein C-Nagoya. A mutagenic primer was designed which replaced the third guanine residue upstream from the deletion with cytosine, thereby creating a new AvaI site in an otherwise normal allele. Analysis of the polymerase chain reaction products derived from this mutagenic primer showed that the abnormal allele has been inherited in this family. To elucidate how this molecular abnormality leads to protein C deficiency, an expression plasmid containing this mutation was transfected into COS 7, BHK, and psi-2 cells, and the secretory process of the expressed Protein C-Nagoya was analyzed. ELISA and immunoprecipitation analysis with [35S]methionine labeling indicated that the mutant protein C, which was larger in size than normal, was mostly retained within the cells, and only a small portion of it was secreted into the medium. These results suggest that most of Protein C-Nagoya undergoes degradation within the producing cells, and this frameshift mutation apparently leads to protein C deficiency by impairment of secretion of the elongated protein C into plasma.