Detection of hepatitis C virus ribonucleic acid in the serum by amplification with polymerase chain reaction.

Hepatitis C virus (HCV) RNA was detected in the sera of patients with non-A, non-B chronic liver disease by polymerase chain reaction (PCR). RNA was extracted from the serum, reverse transcribed to cDNA, and amplified by PCR. With this method, 30 patients with non-A, non-B chronic liver disease and 10 healthy subjects were tested. HCV RNA was detected in 13 of 16 (81%) anti-HCV-positive patients and also in 7 of 14 (50%) anti-HCV-negative patients, but in none of 10 anti-HCV-negative healthy subjects. Specificity of this method was confirmed by direct sequencing of amplified cDNA segment. The nucleotide sequences (37 nucleotides) obtained from 15 patients showed only 68-78% homology compared with the prototype HCV nucleotide sequence. In addition, of 15 nucleotide sequences, there were 12 different types. But the translated amino acid sequences (12 amino acids) showed 83-100% homology compared with the prototype HCV amino acid sequence. These data suggest the majority of anti-HCV-positive patients are carriers of HCV. But to detect all the viremic patients, the anti-HCV antibody testing may be insufficient. Direct detection of HCV RNA may be useful in the study of virus replication and its association with various liver diseases.

Variations in codons 84-101 in the core nucleotide sequence correlate with hepatocellular injury in chronic hepatitis B virus infection.

Individuals with chronic hepatitis B virus (HBV) infection are generally divided into asymptomatic healthy carriers and patients with chronic liver disease. Several studies have suggested that the hepatitis B core antigen could be an immunological target of cytotoxic T lymphocytes (CTL). To investigate the possible pressure site from CTL, the entire core region of HBV DNA was sequenced in 30 subjects (10 asymptomatic healthy carriers and 20 patients with chronic liver disease). No significant changes in the nucleotide sequence and deduced amino acid residue were noted in the 10 healthy carriers. In contrast, a cluster of changes in a small segment of 18 amino acids (codons 84-101 from the start of the core gene) was found in 15 of the 20 chronic liver disease patients. All these 15 patients had advanced liver diseases (chronic active hepatitis and cirrhosis), whereas only mild liver disease (chronic persistent hepatitis) was found in the five patients without mutations. These data suggest that the region with mutation clustering is the major target of CTL, and that the mutations evolve under the pressure of immune selection.

Mutations in core nucleotide sequence of hepatitis B virus correlate with fulminant and severe hepatitis.

Infection with hepatitis B virus leads to a wide spectrum of liver injury, including self-limited acute hepatitis, fulminant hepatitis, and chronic hepatitis with progression to cirrhosis or acute exacerbation to liver failure, as well as an asymptomatic chronic carrier state. Several studies have suggested that the hepatitis B core antigen could be an immunological target of cytotoxic T lymphocytes. To investigate the reason why the extreme immunological attack occurred in fulminant hepatitis and severe exacerbation patients, the entire precore and core region of hepatitis B virus DNA was sequenced in 24 subjects (5 fulminant, 10 severe fatal exacerbation, and 9 self-limited acute hepatitis patients). No significant change in the nucleotide sequence and deduced amino acid residue was noted in the nine self-limited acute hepatitis patients. In contrast, clustering changes in a small segment of 16 amino acids (codon 84-99 from the start of the core gene) in all seven adr subtype infected fulminant and severe exacerbation patients was found. A different segment with clustering substitutions (codon 48-60) was also found in seven of eight adw subtype infected fulminant and severe exacerbation patients. Of the 15 patients, 2 lacked precore stop mutation which was previously reported to be associated with fulminant hepatitis. These data suggest that these core regions with mutations may play an important role in the pathogenesis of hepatitis B viral disease, and such mutations are related to severe liver damage.

Analysis of ras gene mutations in human hepatic malignant tumors by polymerase chain reaction and direct sequencing.

The ras gene is one of the oncogenes most commonly detected in human cancers, and it consists of three families (H-ras, K-ras, N-ras). These genes are converted to active oncogenes by point mutations occurring in either codon 12, 13, or 61. We analyzed mutations of these codons in 23 primary hepatic malignant tumors (12 hepatocellular carcinomas, nine cholangiocarcinomas, and two hepatoblastomas) by a method to directly sequence nucleotides, using polymerase chain reaction and a direct sequencing method. Of 23 hepatic malignant tumors, point mutations at K-ras codon 12 or K-ras codon 61 were found in six of nine cholangiocarcinomas. In contrast, there were no point mutations in any of 12 hepatocellular carcinomas or two hepatoblastomas around codon 12, 13, or 61 of the ras genes. These observations suggest that ras gene mutations are not related to pathogenesis of hepatocellular carcinoma, but play an important role in pathogenesis of cholangiocarcinoma.

Detection of ras gene mutations in pancreatic juice and peripheral blood of patients with pancreatic adenocarcinoma.

Pancreatic adenocarcinomas are known to have a high incidence of K-ras gene mutations. Differential diagnosis of pancreatic cancer and chronic pancreatitis sometimes presents a clinical dilemma. We recently developed a highly sensitive and specific polymerase chain reaction capable of detecting 3-30 copies of mutant K-ras genes harboring codon 12 single base changes in the presence of 300,000 normal copies. Mutant ras genes were detected in DNA purified from pancreatic juice from all 6 cases of pancreatic adenocarcinoma and 1 case of intraductal papillary neoplasms of the pancreas. In 2 of 6 other cases with pancreatic adenocarcinoma, circulating metastatic cells were detected in DNA purified from peripheral blood. Activated ras genes were not found in pancreatic juice of three control cases (chronic pancreatitis and choledocholithiasis) or in the peripheral blood of two patients with insulinomas. Notable conclusions of this study are that there can be significant levels of shed tumor cells in peripheral blood and an even higher number in pancreatic juice. In addition, two different K-ras mutations were found in some patients.

Noninvasive quantitation of liver iron in dogs with hemochromatosis using dual-energy CT scanning.

The concentration of iron deposited in the livers of two dogs with experimentally induced iron overload was determined by use of dual energy computerized tomographic (CT) scanning. A phantom was constructed, containing known amounts of iron-dextran solutions. CT scans of the phantoms, at 80 and 120 kVp, corrected for the response of water, showed a linear relationship between known iron concentrations and difference in CT number at the two scanning energies, with a change of 24 H units per 1000 mg% iron. Using the graph of this linear relationship, the amount of iron in dog liver was predicted, compared with the amount of iron measured from biopsy specimens, and analyzed by neutron activation analysis. A close correlation existed between predicted liver iron and measured iron concentration (r = 0.99). Dual-energy CT scanning appears to provide an accurate, noninvasive method of quantitating liver iron.

Effects of cobalamin, cobalamin analogues and cobalamin binding proteins on P388D1 mouse leukemic cells in culture.

Cobalamin-deficient P388D1 mouse leukemic cells were created by propagation in a cyanocobalamin-free medium in which the original fetal bovine serum was replaced by bovine serum albumin. These cobalamin-deficient cells gradually ceased to multiply when the medium contained 5-methyltetrahydrofolate. The growth of cells that had been cultured with this coenzyme was recovered following the addition of cyanocobalamin (CNCbl), at concentrations above 37 pM. In contrast to the effect of CNCbl, cobinamide, and cobalamin analogues prepared from hydroxy cobalamin by reaction with ascorbic acid, did not have a growth-inducing effect on these cells, nor did these analogues inhibit CNCbl-dependent growth. Transcobalamin II-cobalamin complex had a remarkably stimulating effect on cell growth. The growth inducing effect became apparent with a cobalamin concentration of only 0.37 pM. This was about 1/100th the level of free cobalamin required for cell growth. However, no growth-inducing effect was seen at an R protein-bound cobalamin concentration of 37 pM, indicating that once cobalamin has been bound to R protein, it loses its growth-promoting effect on these cells in culture.

Danazol therapy in idiopathic thrombocytopenic purpura: the efficacy of low-medium dose therapy.

To examine the effects and optimal dose of danazol on idiopathic thrombocytopenic purpura (ITP), we administered a low-medium dose to 14 patients with this disease. A low-medium dose of danazol was effective in maintaining the platelet count at a high level, even after the dose of prednisolone was reduced. A low-medium dose of danazol without other therapy was effective in 3 of 6 patients even after they had been refractory to other treatment. A low dose of danazol was also effective in some patients for whom the other regimes were not indicated. It is concluded that a low-medium dose of danazol instead of a high dose is worth trying in ITP when the patient has become refractory to other therapeutic approaches or when these are not indicated.

Study of repeated arterial infusion chemotherapy with a subcutaneously implanted reservoir for advanced hepatocellular carcinoma.

We performed repeated arterial infusion chemotherapy (RAIC) in 114 advanced hepatocellular carcinoma (HCC) patients, using a subcutaneous reservoir implanted under ultrasonic guidance. In 60 patients, this was the initial therapy for the primary tumor and the other 54 patients being treated for recurrent tumor. One hundred and seventy-one patients with advanced HCC who had been treated by transcatheter arterial embolization (TAE) or single bolus arterial infusion chemotherapy before RAIC was available served as historical controls. In 97 patients, anticancer agents (4'-epidoxorubicin or acurarubicin) and Lipiodol emulsion were used, and in 17, anticancer agents alone were given. The response rates were 39.2% in the Lipiodol group and 17.6% in the non-Lipiodol group. The dose of Lipiodol and the degree of liver invasion were the most important factors influencing the response rate. The 1-, 2-, and 3-year survival rates were 55.0%, 30.9%, and 21.2%, respectively. The long-termsurvival was compared in relation to Child's classification and the presence or absence of portal vein tumor thrombosis (PVTT). In non-PVTT patients, the results of initial therapy and therapy for recurrence were similar, but recurrent Child's C patients showed a poorer prognosis. In PVTT patients, initial therapy had a better prognosis than treatment for recurrence, but initial Child's C patients had a poor long-termprognosis. During the observation period, no severe complications were encountered, but in Child's C patients, hepatic function sometimes deteriorated. Compared with the results in the 171 controls, RAIC was more useful for advanced HCC as initial therapy, and it was also beneficial for the treatment of recurrence after TAE.

Pulsed Doppler in the diagnosis of small liver tumours.

Doppler ultrasound detection of the blood flow associated with liver tumours was studied in primary hepatocellular carcinoma as well as in metastatic liver cancer and haemangioma. Doppler signals were detected from 48 of 55 hepatocellular carcinomas (87.3%), seven of 25 metastatic liver cancers (28.0%) and four of 30 haemangiomas (13.3%). The waveforms of Doppler signals were divided into two types: the pulsatile wave, which was detected from hepatocellular carcinoma (in 35 of the 48 with Doppler signals) and metastatic liver cancer (in all seven with positive signals), and the continuous wave, which was seen from hepatocellular carcinoma (41 out of 48) and haemangioma (in all four with signals). In six patients with hepatocellular carcinoma who underwent transcatheter arterial embolization, the pulsatile wave detected before therapy disappeared immediately thereafter and it is possible that this type of wave originates from tumour vessels. In the study of small, hypoechoic, mass lesions appearing in liver cirrhosis, such signals were also demonstrated, even in eight of 10 small hepatocellular carcinomas less than 2 cm in diameter, whilst they were not detected from nine regenerative nodules related to cirrhotic change. In conclusion, the Doppler ultrasound method may be a useful technique in detecting blood flow within liver tumours and may offer the possibility of a differential diagnosis of small tumours.

Major degradation pathway of thiuram in tap water processed by oxidation with sodium hypochlorite.

Thiuram (3 microM), a fungicide, was incubated in deionized water by adding 0-100 mg/L free chlorine at 30 degrees C for 30 min, and the solution was analyzed by HPLC and IC. The byproducts were identified by LC/MS, EI-MS, infrared, and (13)C NMR spectra and a reduction technique using 2-mercaptoethanol. On the basis of these results, it was found that the oxidation of thiuram with sodium hypochlorite initially produced an intermediate dimethylthiocarbamoyl dimethylcarbamoyl disulfide, which was finally degraded to bis(dimethylcarbamoyl) disulfide, its trisulfide, and dimethylamine. Subsequently, it was suggested that monitoring of bis(dimethylcarbamoyl) disulfide, its trisulfide, and dimethylamine should be included for the management and control of thiuram in tap water processed by oxidation with sodium hypochlorite.

Strategy for early diagnosis of hepatocellular carcinoma (HCC).

Two hundred and eighty-two patients with HCC including 89 with tumors 2 cm or smaller in size and 193 of 2-5 cm were studied on clinical and pathological findings and correlated with the value of various diagnostic imaging modalities. There were apparent differences in histological findings of HCC between 2 cm or smaller in size and larger ones; the former had much less invasion of malignant cells to the extracapsule and portal vein neighbouring the HCC than the latter. In 83 patients with HCCs 2 cm or less in diameter AFP level was normal (less than 20 ng/ml) in 39.8%, 20-200 ng/ml in 44.6% and more than 200 ng/ml in only 15.6%. Detectability of HCC measuring 2 cm or less by various imaging modalities was as follows: 97.8% by US, 54% by X-ray computed tomography (CT), 61.6% by magnetic resonance imaging (MR) and 75.4% by angiography. There was considerable limitation of imaging modalities in making a definitive diagnosis of the smaller HCCs, particularly in the differentiation from regenerative nodules of liver cirrhosis. Tissue biopsy under ultrasound control provided a correct diagnosis in 36 out of 41 patients (87.8%) with HCCs 2 cm or less in size, a better ratio than for aspiration biopsy.

[Laboratory and clinical studies CS-1170 (author's transl)].

CS-1170 is a new antibiotic, a derivative of cephamycin C. In vitro, 50 strains of E. coli and Klebsiella consisting of gentamicin-sensitive isolated from the blood and gentamicin-resistant strains isolated from the urine were inhibited at concentrations from 0.4 to 12.5 mcg/ml of CS-1170, whereas only 2 strain of Klebsiella isolated from the blood had MIC more than 50 mcg/ml of the antibiotic. Moreover, CS-1170 was significantly more effective than cefazolin and cephalothin against these strains. Ten strains of gentamicin-sensitive Serratia isolated from the blood and the 2 gentamicin-resistant strains were inhibited at concentrations from 3.2 to 50 mcg/ml of CS-1170 and only one strain was resistant to this agent. All tested Serratia were resistant to cefazolin and cephalothin. CS-1170 was not effective against Enterobacter. Three cases of biliary tract infections consisting of 2 cases of cholelithiasis and a case of carcinoma of bile duct were treated with 4 g/day dosage of CS-1170. The remarkable effects were obtained in the two cases with cholelithiasis, whereas a case with the carcinoma was treated not so effectively by administration of CS-1170.

[Theophylline pharmacokinetics in patients with liver diseases with reference to estimated hepatic blood flow].

Pharmacokinetics of theophylline were determined in patients with liver cirrhosis and idiopathic portal hypertension with reference to estimated hepatic blood flow assessed by indocyanine green (ICG). Decreased plasma clearance of theophylline was noted in patients with liver cirrhosis and the clearance was significantly lower in Child C group than in Child A, B groups (17.5 +/- 3.4 ml/Kg/hr vs 27.6 +/- 8.7, p less than 0.05). Theophylline has been classified as a drug with a low hepatic extraction ratio and it has been believed that changes in hepatic blood flow have little effect on its clearance. The results of present study indicate that theophylline clearance is basically not related to ICG clearance but to theophylline extraction ratio, supporting the common belief. However, it is noteworthy that the clearance was related to decreased hepatic blood flow rather than extraction ratio in a cirrhotic patient with huge extrahepatic shunt, suggesting that hepatic clearance of this drug could be affected by hepatic blood flow under some circumstances.

[Assessment of laboratory findings in megaloblastic anemia--measurement of serum vitamin B12 and methylmalonic acid].

The laboratory findings of 20 patients with untreated megaloblastic anemia due to vitamin B12 deficiency were analysed. The material consists of 13 patients with pernicious anemia, 6 with postgastrectomy B12 deficiency and one with malabsorption syndrome. Hematological data (RBC, Hgb, Ht, WBC, Plt) were correlated with each other and serum LDH levels. Megaloblastic changes of bone marrow were apparent in cases of which Hgb values were below 9 g/dl, although its change were not clear in cases with mild anemia (above 9 g/dl). However, giant metamyelocytic changes of bone marrow were seen even in cases with mild anemia. Serum B12 levels in 6 out of 19 cases (31.6%) measured by clinical laboratory center were within normal range. In contrast, its level in all cases measured by radiodilution assay using R-protein or intrinsic factor were lower than normal values. Serum B12 levels measured by the latter method were correlated with various hematological data and also related with hematological severity, although its level measured by clinical laboratory did not have any correlation with hematological data. Schilling test seemed to be unreliable, because sample volume which was suggested by kit manual was too small (2 ml) to catch enough radioactivity for accurate measurement. Serum methylmalonic acid levels measured by gas capillary mass spectrophotometry were higher than normal values in all cases and were well correlated with hematological data.

[Molecular evidence for a single clonal origin in a patient with multiple myeloma and non-Hodgkin's lymphoma].

A 49-year old man was admitted in November 1989, because of anemia, abnormal shadowing on chest X ray and hyperproteinemia. Biclonal gammopathy (IgG kappa + IgA kappa) was shown in serum, and Bence Jones protein in urine. The bone marrow examination showed an increased number of abnormal plasma cells (15.7%) and no evidence of lymphoma, A diagnosis of multiple myeloma (MM) was made. In April 1990, while the patient was treated with the modified M2 regiman, swelling of the right cervical lymph node was observed. Lymph node biopsy revealed that he had non-Hodgkin's Lymphoma (:NHL, diffuse, mixed, B cell type). He was retreated with the CHOP regimen for both disease, but died of respiratory failure in October. 1991. To establish the clonal origin of this case of concominant MM and B-cell NHL, the immunoglobulin gene rearrangements in his lymph node and bone marrow were analyzed. Southern blot analysis with the JH probe and Ck probe showed one common band and one different band in the two samples. Our data suggest that two B-cell malignancies may have arisen from a single B-cell progenitor.

[Primary culture of proximal tubular cells (PTC) from normal mouse kidney as an in vitro model to study mechanisms of development of tubulointerstitial nephritis. Induction of ICAM-1 in PTC by antigen-primed lymphocytes].

Cultured PTC clearly expressed intercellular adhesion molecule-1 (ICAM-1) under the influence of tubular basement membrane antigen (TBM)-primed lymphocytes. These TBM-primed lymphocytes also demonstrated a high cytotoxic activity against cultured PTC. A pure preparation of isolated PTC from BALB/c mouse kidney was brought into primary culture. PTC was prepared by the method of Boogaard PJ et al, and our modification. Briefly, kidney was perfused with buffer containing 0.08% (w/v) collagenase. The cortical tissue was then filtered through nylon-gauze. Viable PTC were separated from other materials by isopycnic centrifugation on a discontinuous Nycodenz gradient. The confluent monolayer of PTC showed a typical epithelial morphology with cobblestone-like cells in the center of the cell-islands. Typical dome formation was observed in PTC cultures. These cells also strongly expressed gamma-glutamyl transpeptitase activity. Coculture of PTC with syngeneic lymphocytes primed with TBM antigen induced ICAM-1 expression in PTC. The TBM-primed lymphocytes had a cytotoxic activity without complement. However, neither virgin lymphocytes nor liver antigen-primed lymphocytes had cytotoxic activity. This simple syngeneic experimental model may allow us further molecular biological examination of renal tubulointerstitial diseases.

[Intrarenal arterial Doppler sonography in patients with various renal disease: correlation of resistive index with biopsy findings].

Duplex Doppler sonography (ultrasonic equipment: Toshiba SSA-270A) were performed in the patients with various renal disease (male 44, female 32) admitted to our hospital between June 1990 and August 1991. Interlobar arterial blood flow velocity was measured at the side of renal pyramid through a longitudinal scan. Both the maximum blood flow velocity (Vmax) and the minimum blood flow velocity (Vmin) were measured quantitatively and resistive index (RI: defined as (Vmax-Vmin)/Vmax) were calculated. Vmax and Vmin correlated well with the creatinine clearance (Ccr) (r = 0.56, r = 0.66 respectively), whereas RI, by which we can detect Doppler waveform changes, correlated weak with Ccr (r = 0.39). We found twenty-nine patients (40%) with an elevated RI (greater than or equal to 0.70). They had severe renal dysfunction and active pathologic findings in the tubulo-interstitial or vascular compartment of the kidney. Doppler examination of renal blood flow velocity was valuable not only to estimate renal function but also to assess pathophysiology of renal disease.

[The diagnostic process in carcinoma of the pancreas].

Early diagnosis of carcinoma of the pancreas is considerably difficult because the disease is often in the advanced stage at the time of clinical manifestation. We have, however, no choice for improving the prognosis of the disease but to make an early diagnosis by detecting small tumors. Ultrasonography (US) is the most useful diagnostic technique in detecting the initial sign of carcinoma of the pancreas. The patient suspected of having this disease should first undergo US combined with measurement of serum pancreatic enzymes (amylase or elastase-1) and the tumor marker (CA 19-9). Those patients with positive results on the first examinations should undergo dynamic CT, ERCP or endoscopic US (EUS) to corroborate suspicions of the disease. Percutaneous fine needle biopsy of the pancreas may be recommended as a reliable method for making a definite diagnosis, when these diagnostic modalities fail to elucidate the disease. In conclusion, appropriate combined use of these diagnostic techniques can facilitate the early diagnosis of carcinoma of the pancreas.

[Non-vascular interventional radiology--percutaneous ethanol injection (PEI) in hepatocellular carcinoma smaller than 3 cm in diameter].

Percutaneous ethanol injection (PEI) was carried out for 109 lesions in 85 patients with hepatocellular carcinoma (HCC) smaller than 3 cm in diameter during the period from Dec. 1982 to Mar. 1989. Therapeutic effect of PEI on HCC was evaluated by ultrasound showing that 15 of 68 HCCs disappeared and the remaining 53 HCCs decreased in size with regression rates ranging from 8% to 88% at 6 months after PEI. Survival rates from PEI calculated by the Kaplan-Meier method showed a 1-yr survival of 92%, a 2-yr survival of 81%, a 3-yr survival of 61%, a 4-yr survival of 55%, and a 5-yr survival of 41%. These survival rates were better than those of patients with HCCs smaller than 3 cm in size who had not received anti-cancer treatment (p less than 0.05).