Nobuhiko Yoshida

Very high prevalence of TT virus (TTV) infection in general population of Japan revealed by a new set of PCR primers

Clear evidence for TT virus (TTV) to be a hepatitis virus remains unseen despite its origination from a hepatitis patient. Epidemiological studies on TTV have been limited more or less by the prematurity of the diagnostic systems. We sought after PCR systems that could detect TTV DNA more efficiently than those reported previously, and found that a PCR with new primers designated T801 and T935 was 10–100 times more sensitive than that with published primers NG061 and NG063. To our surprise, TTV DNA was identified by the new PCR system with T801/T935 in 92% of 100 individuals who visited our hospital for health screening, compared to 23% with NG061/NG063. Liver function test values were comparable between TTV DNA-negative and positive individuals, but mean age was younger in the former than in the latter: 29.7±6.9 versus 41.4±14.7 years (P<0.05). Our results suggest that asymptomatic carriers of TTV are very common in general population of adults in Japan.

Abnormal Plasminogen: A HEREDITARY MOLECULAR ABNORMALITY FOUND IN A PATIENT WITH RECURRENT THROMBOSIS

A patient who suffered a recurring thrombosis over the last 15 yr has been investigated. The only abnormality found in this patient was a significantly depressed level of plasminogen activity in plasma. In spite of the depressed plasminogen activity, the patient was found to have a normal level of plasminogen antigen concentration. It was calculated that the activity per milligram of plasminogen of the patient was approximately one-half the values of normal subjects. The same discrepancy between biological activity and antigen concentration was found in the other members of the kindred. A niece was found to have practically no plasminogen activity but possessed a normal concentration of plasminogen antigen. Both her parents were found to have approximately half the normal plasminogen activity and normal antigen levels. These studies suggested that the molecular abnormality was inherited as an autosomal characteristic, and the family members who had half the normal levels of activity with normal plasminogen antigen were heterozygotes whereas the one with practically no plasminogen activity was homozygote. Subsequent studies showed that the pattern of gel electrofocusing of purified plasminogen of the heterozygotes consisted of 10 normal bands and 10 additional abnormal bands, each of which had a slightly higher isoelectric point than each corresponding normal component. This indicates that plasminogen of the heterozygote is a mixture of normal and abnormal molecules in an approximately equal amount, which was substantiated by active site titration of purified plasminogen preparations obtained from the propositus and a normal individual. The gel electrofocusing pattern of the homozygote consisted of abnormal bands only. The defect is a hereditary abnormality of plasminogen.

Hepatitis C virus infection and mutations of mannose-binding lectin gene MBL

SummaryWe assessed the genetic polymorphism of mannose-binding lectin (MBL) in 93 patients with chronic hepatitis C (45 responders and 48 nonresponders to interferon) and 218 healthy controls. Mutant allele was identified only at codon 54 (Gly→Asp), leading to three genotypes (54 m/m, 54 W/m, and 54 W/W). Frequency of 54 m/m was significantly lower in interferon-responders (2.2%) compared to those in nonresponders (14.6%) and controls (10.6%): p<0.05. Our results suggest that homozygous carriage of the variant allele of codon 54 of MBL may predict poor response to interferon in chronic hepatitis C patients.

Distribution of cold-insoluble globulin in plasma and tissues.

Cold-insoluble globulin is normally present in plasma and serum at concentrations of 27.52 +/- 4.60 and 23.46 +/- 5.18 mg/dl, respectively (means +/- SD). The concentration of CIg in blood samples was significantly decreased in DIC syndromes (14.69 +/- 6.55 mg/dl; p less than 0.001). A strong, positive correlation was found with AT-III (r = 0.68) and a less striking one with Plg. Although alpha 2-PI was shown to be significantly decreased in DIC syndromes (p less than 0.001), a weak, inverse correlation was found between CIg and alpha 2-PI (r = -0.29). Immunologically cross-reactive substances were found to be widely distributed in association with the cells and tissues of mesenchymal origin, including fibroblasts, adipose cells, smooth muscle cells, and basement membranes. The glomerular basement membrane was an exception and is currently believed to be of different origin. In the kidney, fluorescence was found in the mesangium. Cold-insoluble globulin is also present as a component of cryofibrinogen that forms a solid gel at low temperatures. Sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed that CIg in this fraction was rather homogeneous. Although closely migrating doublets were occasionally seen in the 440,000-dalton region on gels of unreduced samples, monomeric derivatives with a molecular weight of 220,000 or less, which have been claimed to occur in circulating plasma, were not observed. Thus, intact dimeric CIg appears to be the form of the molecule that complexes with fibrinogen. Cold-insoluble globulin is the fraction that was shown to exist as an independent entity from fibrinogen at an ambient temperature by immunoelectrophoresis and ultracentrifugation. However, very rapid formation of highly polymerized complexes in the sol phase at low temperatures was manifested by the finding of a sharp increase in light-scattering intensity using the technique of quasielastic light scattering. A control study on a mixture of normal CIg and fibrinogen disclosed no appreciable change in the temperature range between 37 and 8.5 degrees C. A comparative study on a mixture of cryofibrinogen-derived CIg and normal fibrinogen revealed an intermediate light-scattering pattern. After 2 hr at 8 degrees C, this mixture reached a state of equilibrium, where no further polymerization occurred. The secondary structures of normal and cryofibrinogen-derived CIg, determined by circular dichroism, showed no appreciable difference. A noteworthy finding was the almost complete absence of alpha-helices and a relatively high proportion of beta-structure in both forms of CIg. Amino termini of the fibrinogen moiety of cryofibrinogen were found to consist of alanine, tyrosine, and a small quantity of aspartic acid, consistent with the NH2 terminal moiety composition of normal fibrinogen but not of soluble fibrin monomer complex.

Treatment of DIC with Antithrombin III Concentrates

We administered AT III concentrates to 21 patients with DIC who failed to respond initially to heparin therapy. About 60% of these 21 patients were effectively treated with AT III concentrates by enhancing the effect of heparin and alleviating the burden of excessive plasma transfusions on the cardiovascular system.

Preparation and characterization of monolconal antibodies against the human thrombin-antithrombin III complex

Monoclonal antibodies were raised against human thrombin-antithrombin III complex by a hybridoma technique. Among them, five monoclonal antibodies, designated as JITAT-4, -14, -16, -17 and -19, were found to react with thrombin-antithrombin III, but not with its nascent components, alpha-thrombin or antithrombin III. Their respective immunoglobulin classes are IgG1 for JITAT-16 and -19, and IgG2a for JITAT-4, -14 and -17. Besides the thrombin-antithrombin III complex, they all bound to the Factor Xa-antithrombin III complex and the active-site-cleaved two-chain antithrombin III as well. Moreover, the reactivity of these two antibodies to the neoantigens was not affected by heparin, suggesting that their epitopes are independent of heparin-induced conformational changes of antithrombin III. Two of them, JITAT-16 and -17, were categorized as high-affinity antibodies to thrombin-antithrombin III complex, the dissociation constants being 6.7 nM and 4.8 nM, respectively. However, they do not share antigenic determinants. These monoclonal antibodies may allow us to explore more precisely the reaction between antithrombin III and thrombin or its related enzymes.

Endoscopic injection sclerotherapy for esophageal variceal hemorrhage in myeloproliferative disorder: Case report

A 7-year-old woman with myeloproliferative disorder and massive splenomegaly presented with hematemesis. Emergency endoscopy demonstrated bleeding from esophageal varices. Management of variceal hemorrhage by endoscopic injection sclerotherapy, using 5% ethanolamine oleate, was successful. Following the control of variceal bleeding, she was treated with hydroxyurea, a myelosuppressive agent. The spleen size markedly decreased and she was discharged 3 months later. Variceal hemorrhage in myeloproliferative disorder has been reported to be fatal on many occasions, despite different therapeutic approaches, including surgery. In this report, we demonstrated that endoscopic injection sclerotherapy followed by treatment with a myelosuppressive agent was effective in a patient with myeloproliferative disorder and variceal hemorrhage.

Abnormal Fibrinogens with Two Structural Defects

Among the abnormal fibrinogens which we recently analyzed, two types of abnormality were found to have two structural defects: a genetically determined amino acid substitution and an additional secondary modification of the molecule due to the mutation (1, 2). Here we briefly describe these abnormal molecules.

Potentiation by collagen or epinephrine of platelet responsiveness to aggregation. The possible role of arachidonic acid released from platelet membranes

When human platelets in plasma were exposed to a small amount (nonaggregating concentration) of collagen, epinephrine or arachidonic acid, their responsiveness to aggregating agents were potentiated and they were aggregated by a subsequent addition of nonaggregating concentration of the stimulants. Otherwise the nonaggregating concentrations of the stimulants were uncapable to induce platelet aggregation. The potentiation of platelet responsiveness to aggregating agents was also caused by collagen- or epinephrine-treated platelet menbranes, but not by arachidonic acid-treated membranes. Furthermore, the soluble fraction of collagen- or epinephrine-treated membranes contained some material responsible for platelet potentiation, indicating that the responsible material was released from platelet membranes by collagen or epinephrine. It is suggested that the material may be arachidonic acid since the soluble fraction of collage- or epinephrine-treated membranes contained a larger amount of the precursor of prostaglandin F2α than the untreated membranes and arachidonic acid is the precursor of Prostaglandin F2α. The potentiation of platelet aggregability by the small amounts of the stimulants was increased in nephrotic syndrome whose albumin concentration in plasma is low, and the increased potentiation was abolished by the addition of albumin. The abolishment of the potentiation of platelets by albumin could be explained by postulating that some part of arachidonic acid released from membranes immediately binds to albumin and becomes unavailable to platelets.