A monoclonal antibody against gamma-glutamyltransferase from human primary hepatoma: its use in enzyme-linked immunosorbent assay of sera of cancer patients.

A monoclonal antibody, gamma-120, was raised against a highly purified gamma-glutamyltransferase (gamma GT) from human primary hepatoma. The antibody preferentially bound to the small subunit of gamma GT from human hepatoma and kidney as evidenced by immunoblot analysis. Weak binding to the normal liver enzyme could be detected by solid-phase enzyme-linked immunosorbent assay (ELISA). With the use of this antibody, an ELISA was developed for the quantitation of immunoreactive gamma GT in human serum. Sera of 188 normal control subjects displayed a low level (9.4 micrograms/ml) of immunoreactive gamma GT. Highly elevated levels of immunoreactive gamma GT were detected in the sera of patients with primary hepatoma, metastatic liver cancer, pancreatic cancer, and certain types of lung cancer. Slightly elevated levels of immunoreactive gamma GT were seen in the sera of patients with liver cirrhosis. The levels of gamma GT were within a normal range in the sera of patients with gastrointestinal cancers and patients with nonmalignant diseases such as hepatitis and gallstones. The antibody has been shown to be useful for the diagnosis of some of the neoplastic diseases.

A collaborative study for the evaluation of lectin-reactive alpha-fetoproteins in early detection of hepatocellular carcinoma.

Lectin-affinity electrophoretic separation of serum alpha-fetoprotein (AFP) was carried out using AFP Differentiation Kits, which used Lens culinaris agglutinin-A (Kit L) and erythroagglutinating phytohemagglutinin (Kit P). Separated AFP bands were detected with a sensitive antibody-affinity blotting technique and determined quantitatively by densitometry, and the results were expressed as percentages of the intensity of total AFP bands. Sera from 424 patients with acute hepatitis, chronic hepatitis, liver cirrhosis, hepatocellular carcinoma, and extrahepatic tumors were assayed for proportion of AFP present as Lens culinaris agglutinin-A-reactive AFP (AFP-L3) and erythroagglutinating phytohemagglutinin-reactive AFPs (AFP-P4+P5). From the maximum Youden indices determined, cutoff levels were set at 15% for both AFP-L3 and AFP-P4+P5 to discriminate between patients with chronic hepatitis and liver cirrhosis and patients with hepatocellular carcinoma. AFP-L3 and AFP-P4+P5 showed sensitivities of 55.3 and 61.0% at specificities of 93.9% and 82.3%, respectively. Thirty-eight % of tumors that measured less than 20 mm in diameter were positive for AFP-L3 and AFP-P4+P5. AFP-L3 exceeded the cutoff level of 15% 4.0 +/- 4.9 months before detection of hepatocellular carcinomas by imaging techniques with a sensitivity of 48% and a specificity of 81%. Thus, these tests are useful for the early detection of hepatocellular carcinomas in patients with hepatitis or liver cirrhosis.

Measurement of human serum immunoreactive gamma-glutamyl transpeptidase in patients with malignant tumors using enzyme-linked immunosorbent assay.

An enzyme-linked immunosorbent assay for human immunoreactive gamma-glutamyl transpeptidase (gamma-GTP) was developed. This assay was found to be simple, reproducible, and sensitive to 10 ng of the enzyme. Serum immunoreactive gamma-GTP content was significantly elevated in patients with various malignant tumors including liver cell cancer, lung cancer, gastric cancer, esophageal cancer, and colorectal cancer. On the other hand, in sera of patients with nonneoplastic diseases, the immunoreactive gamma-GTP content was not significantly elevated. No correlation was found between the serum levels of gamma-GTP determined by enzymatic assay and enzyme-linked immunosorbent assay, which indicates that, due to the presence of endogenous inhibitors and/or activators in sera, the enzyme activity may not reflect the true amount of enzyme protein. The measurement of immunoreactive gamma-GTP protein in sera appears to be useful for the detection and monitoring of certain malignant tumors.

Serum-manganese-superoxide dismutase: normal values and increased levels in patients with acute myocardial infarction and several malignant diseases determined by an enzyme-linked immunosorbent assay using a monoclonal antibody.

An enzyme-linked immunosorbent assay (ELISA) has been developed for human manganese-superoxide dismutase (Mn-SOD), using a specific monoclonal antibody raised against the purified enzyme. The Mn-SOD molecule comprises four identical sub-units and this permitted the development of a symmetrical assay, using the same monoclonal antibody as both capture and detector. The assay offers a specific, sensitive and convenient means of measuring immunoreactive Mn-SOD in human sera. Under optimum conditions, the sensitivity of the assay permits the detection of 2-200 ng of purified Mn-SOD from human liver. The mean serum Mn-SOD levels of normal healthy males and females were 99.8 +/- 24.8 (mean +/- SD) and 88.8 +/- 20.8 (mean +/- SD), respectively. A high level of the enzyme was found in the sera of patients with acute myocardial infarction as well as malignant diseases such as acute myeloid leukemia, primary hepatoma and gastric cancer. This is the first report of an ELISA using a monoclonal antibody specific for a distinct epitope of Mn-SOD.

Changes in the plasma abnormal prothrombin level following treatment of hepatocellular carcinoma.

The Hokkaido Liver Cancer Study Group focused on the changes in PIVKA-II levels observed in 61 HCC patients after several regimens of treatment in comparison with the AFP levels and the pathophysiological characteristics of HCC. The overall positivity rate for PIVKA-II was 47%, and there was no correlation between the PIVKA-II values and the AFP levels. Accordingly, the HCC detection rate was increased by about 20% by the measurement of both markers. In all, 13 patients underwent hepatic resection, and nonsurgical therapy was carried out in the other 48 subjects. Of the 6 surgically treated patients, 5 (83%) showed a fall in PIVKA-II levels to the normal range immediately after surgery, whereas 14/29 (48%) subjects receiving nonsurgical treatment showed a decrease in PIVKA-II values. Although inconsistency between these tumor markers was detected in four treated cases, we concluded that assay for both of these two parameters may expand their clinical utility for the diagnosis of HCC and monitoring of patients after treatment.

Cooperative study on arterial regional chemotherapy for primary liver cancer in Hokkaido.

The Liver Study Group of Hokkaido analyzed a total of 57 patients with non-resectable primary liver cancers, which were treated by intra-arterial adriamycin infusion chemotherapy combined with lipiodol and/or the Gelform embolization of the hepatic arteries. Of the ten patients considered clinical responders, three complete response patients and seven partial response cases were obtained. The overall response rate was 17.5%. The median survival period at each clinical stage was as follows: stage I: 13.0 months, stage II: 16.0 months, stage III: 11.5 months and stage IV: 4.7 months. The common side-effects of this treatment were nausea, vomiting and anorexia. Hematological toxicities were also found, but there was no patient who suffered from severe complications.

Indomethacin blocks the anorexic action of interleukin-1.

It has been reported recently that the central nervous system actions of interleukin-1 are mediated by the prostaglandin system in the brain. The present study was therefore performed in order to examine the hypothesis that indomethacin, an inhibitor of prostaglandin biosynthesis, might alleviate the interleukin-1-induced suppression of food intake in rats. The i.p. injection of interleukin-1 (2 micrograms/rat) resulted in a significant decrease in food intake. The pre-injection of indomethacin (0.5 mg/rat), however, completely blocked the anorexic action of the monokine, while indomethacin on its own did not affect food intake. These results suggested that indomethacin might be clinically useful for improvement of the anorexic state of patients with acute infectious diseases.

Changes in free radical-metabolizing enzymes and lipid peroxides in the liver of Long-Evans with cinnamon-like coat color rats.

We report changes in free radical-metabolizing enzymes and the increased generation of lipid peroxides associated with extreme metal accumulation in the liver of the Long-Evans with cinnamon-like coat color (LEC) rat, a new mutant strain displaying hereditary hepatitis and subsequent hepatocellular carcinoma. The activity of free radical-metabolizing enzymes and lipid peroxides, and the concentration of metal in the liver were determined sequentially after birth. Mn-superoxide dismutase activity significantly increased immediately after the onset of hepatitis in LEC rats, whereas no remarkable change was observed in control rats. Cu, Zn-superoxide dismutase activity in LEC rats was similar to that in control rats. Glutathione reductase activity increased, while glutathione peroxidase activity was lower in LEC rats than in control rats throughout the observation periods. Lipid peroxides, estimated by thiobarbituric acid reaction, also increased 4- to 5-fold immediately after the onset of hepatitis in LEC rats. Copper concentration was 30- to 50-fold higher in the liver of LEC rats than in control rats, and the iron content also increased significantly before and after the onset of hepatitis. These findings suggested that an oxidant injury generated by toxic metals could be one of the factors responsible for hepatocellular damage in this unique hereditary hepatitis.

Importance of cytotoxic T lymphocytes in the rejection of transplanted hepatocellular carcinoma.

Fischer rats became resistant to syngeneic hepatocellular carcinoma (FAA-HTC1) cells on repeated sensitization with mitomycin C-treated FAA-HTC1 cells. In contrast, FAA-HTC1 cells injected into the liver killed normal control Fischer rats within 2 months. Histopathological studies revealed massive accumulation of mononuclear cells in the tumor tissues of sensitized rats that rejected syngeneic FAA-HTC1 cells, whereas very few mononuclear cells were found in the tumor tissues of control rats. Cell populations infiltrating the tumor tissues were identified by flow cytometric analysis. Mononuclear cells found within the regressing tumors of the sensitized rats were identified as mostly T cells, and two-thirds of these T cells were CD8-positive. Compared with the activity in control rats, the killer activity of mononuclear cells infiltrating tumors was significantly increased in the sensitized rats 7 days after tumor inoculation. Depletion of CD8(+) T cells significantly reduced the cytotoxicity of mononuclear cells infiltrating tumors obtained from sensitized rats. In contrast, depletion of CD16(+) cells reduced the cytotoxicity of mononuclear cells infiltrating tumors obtained from both control and sensitized rats. Furthermore, the CD16(+) cell-depleted fraction of mononuclear cells infiltrating tumors showed significant cytotoxicity against FAA-HTC1 cells, but failed to show cytotoxicity against other syngeneic tumor cells or allogeneic hepatoma cells.

Serum immunoreactive beta-glucuronidase determined by an enzyme-linked immunosorbent assay in patients with hepatic diseases.

An enzyme-linked immunosorbent assay (ELISA) was developed for human beta-glucuronidase, using a specific polyclonal antibody raised against the purified enzyme. beta-Glucuronidase from human liver consisted of three subunits with molecular mass of 76, 64 and 18 kDa. The assay offered a specific, sensitive and convenient means of measuring immunoreactive beta-glucuronidase in human sera. beta-Glucuronidase activity determined by the conventional method appeared to be extremely low, indicating that in human sera beta-glucuronidase exists in an enzymatically inactive form. The sensitivity of the assay permitted the detection of 1-100 ng of purified beta-glucuronidase. A mean serum level in normal subjects was 108 +/- 25 ng/ml (mean +/- S.D.). A high level of beta-glucuronidase was found in sera of patients with severe hepatocellular necrosis, including liver cirrhosis (152 +/- 130 ng/ml) and chronic active hepatitis (220 +/- 99 ng/ml), whereas no significant increase of the enzyme protein was observed in chronic persistent hepatitis (102 +/- 42 ng/ml). beta-Glucuronidase was also increased in sera of patients with primary hepatoma (156 +/- 125 ng/ml). The immunoreactive beta-glucuronidase determined in this assay was thought to be a supplementary serological indicator for hepatocellular necrosis.

Detection of PIVKA II produced by human hepatoma cells in nude mice.

A novel experimental nude mouse model, which is useful for investigation of the mechanisms of PIVKA II synthesis, was established by inoculation with PIVKA II-producing human hepatoma cells (huH-1). We have found markedly elevated levels of PIVKA II in the plasma of nude mice transplanted with huH-1 cells and increased PIVKA II content in huH-1 tumor tissues. Whereas we have not found detectable level of PIVKA II neither in the plasma nor in tumor tissues of nude mice transplanted different human hepatoma cells (HLF) which is not producing PIVKA II. Histology of the tumor tissues produced by huH-1 cells revealed a thick trabecular pattern with blood spaces.

Effects of exercise, cold, and immobilization stresses on gamma-glutamyltransferase activity in rat kidney.

Effects of acute and chronic stresses (exercise, cold, and immobilization) on gamma-glutamyltransferase (gamma GT) activity in the rat kidney were investigated. In the extramicrosomal fraction there were significant decreases in acute-exercised rats but significant increases in cold-adapted and immobilized rats. In the microsomal fraction, on the other hand, the gamma GT activity of acute-exercised rats increased definitely. The results suggest that different types of stresses have different effects on gamma GT activity in the rat kidney.

[Diagnosis of type C chronic hepatitis using PCR technology].

Many studies have suggested that examination of a patient's serum for viral RNA by reverse transcriptase-polymerase chain reaction (RT-PCR) is the most sensitive and specific serological means of determining chronic HCV infection. Moreover, technique to quantify HCV RNA in serum using PCR technology has been developed, and diagnosis of HCV genotype by PCR with mixed primers has been used for diagnosis of type C hepatitis. Herein, we discuss the laboratory technique and significance of various PCR methods. HCV RNA can be detected by amplification technique of RT-PCR. Primers in the 5'-noncoding region are frequently used to detect HCV RNA, because the RNA sequence in this region is the most conserved. We usually perform 30 cycles of amplification with outer primers and another 30 cycles with inner primers (nested RT-PCR). Using this technique, HCV RNA was detected in 96% of anti-HCV-positive chronic hepatitis patients. This PCR technique gives us important information for the diagnosis of type C chronic hepatitis indicating HCV infection. The competitive PCR technique is usually used to quantify HCV RNA. This method is based on complification of the target RNA with known amounts of synthetic mutant RNA. The mutant RNA should be amplified by the same primers for amplifying target RNA, and should be distinguished from target RNA by the size of the amplified DNA product by making a deletion or restriction site artificially. Quantifying HCV RNA before interferon therapy is useful to predict the effectiveness of the therapy; patients with a large amount of HCV RNA tend to have a poor outcome.(ABSTRACT TRUNCATED AT 250 WORDS)

[Parasitic cirrhosis of the liver].

The shistosomiasis (caused by S. japonicum) has markedly decreased in Japan, as the most of other parasitic diseases. S. japonicum inhabits the portal venous system and its egg attacks the host's immunologic defences, resulting in inflammation and fibrosis of the liver. Laparoscopy reveals the characteristic, tortoise-shell-like liver, and liver biopsy is able to make a correct diagnosis. Portal-hypaertention is one of the most popular clinical features. Multilocular echinococcosis is a less frequent disease. However, this disease has gradually increased these several years in Hokkaido. This disease progresses to cirrhotic stage after long terms of latent and asymptomatic stages. This slowly enlarging lesion gradually occupies a large part of the liver and metastases to another intrahepatic parts or organs as well, just like a malignant tumor. Laparoscopically, the most of lesions appear ash-colored tumor with granular surface. Biopsy reveals the characteristic alveolar cysts with thick chitin membrane. Clinical features looks like liver cirrhosis or hepatoma.

[Survey on intestinal Behçet's disease in Hokkaido (author's transl)].

The occurrence of intestinal Behçet's disease is not common so that misdiagnosis with inappropriate treatment often results in death. Due to this circumstance intestinal Behçet's disease is noted as a very serious complication of Behçet's disease. The identity of intestinal Behçet's disease and its patient manifestation remain obceure. As members of the national project team for intestinal Behçet's disease, we surveyed the state of intestinal Behçet's disease in Hokkaido. The results are summarized as follows: 1) The occurrence in Hokkaido of intestinal Behçet's disease is higher than the national average in concordance with a high rate of Behçet's disease itself. 2) Intestinal Behçet's disease, incomplete type, was observed to be more frequent in the female which differs from the other complications of Behçet's disease. 3) Ulcers which develop in the ileus are apt to perforate (85.7%) giving rise to frequent emergency operations. On the other hand, ulcers in the ascending colon are not as liable to the perforate (25.0%). 4) Ulcers which develop in the ileum are often round with the part under the surface being quite deep and large. Ulcers in the ascending colon are usually of various shapes and are not very deep. 5) Through a retrospective review of the cases of Behçet's disease, the probability of an ulcer occurring or not is difficult to predict at this time.

[Studies on development of viral hepatitis with HBs antigenemia (author's transl)].

47 cases of viral hepatitis with HBs antigenemia, who were diagnosed by peritoneoscopy and biopsy, were studied. 41 cases were males, and 6 cases were females. The onset ages were mostly in 25--29 years old. The histories of blood transfusion were found in only 4.3%. On the other hand, the families clustered with HBs antigen were found in 68%. Recidivums were often seen (76%), and yet the intervals between recidivum were short (the neighborhood of 8 months). The transaminases at the recidivums were not so high (200 units or thereabout). 20 cases whom biopsies were performed more than twice, were studied histologically. 14 cases were prognessive. 5 were stationary. 1 was improved. In all of progressive cases, sublobular hepatic necrosis was seen. But in other cases, sublobular necrosis was not seen. After this sublobular necrosis, various distortion of the lobule architecture was appeared, and some of them progressed to liver cirrhosis. As to GOT and GPT, GPT was dominant before sublobular neerosis, but GOT was liable to be dominant after sublobular necrosis. Peritoneoscopically, peculiar red spots were found on liver surfaces at the time of sublobular necrosis.

[A case of subacute thyroiditis associated with acute hepatitis].

We report one case of subacute thyroiditis associated with acute hepatitis, which is histopathologically diagnosed. A 43-year-old woman visited our hospital with chief complaints of fever, sore throat and anterior neck pain. Thyroid gland was found to be swollen and tender. Laboratory findings gave high ESR and positive test for CRP. High values of T3, T4 and RT3U indicated that the patient had hyperthyroidism. However no autoantibodies against thyroglobulin and microsome were found. High activities of serum AIP, LAP and gamma-GTP were observed. Serum GOT and GPT activities increased moderately. AIP type 2 was dominant in zymograms. Histopathological findings of liver specimen obtained by needle biopsy showed ballooning degeneration of hepatocytes with a slight focal necrosis and hyaline bodies. In addition bile plugs were observed in some biliary tubules. These findings were consistent with those of acute hepatitis. After three months all laboratory data were found to be within normal ranges and no recurrence has been observed. Subacute hepatitis associated with liver dysfunction is considered to be relatively frequent. However very few reports have been published on the case in which histopathological evidence for acute hepatitis was presented.

Mitochondrial glutamic oxaloacetic transaminase and its clinical significance.

The m-GOT were studied in 57 patients with liver diseases, diagnosed by peritoneoscopy and biopsy. In acute hepatitis, the m-GOT rose to 100 units or there-about. But in chronic hepatitis, even in hepatitis B with sublobular necrosis, the m-GOT were low and 20--30 units. On the other hand, in alcoholic hepatitis, the m-GOT were 50--60 units which may be considered to be higher than expected for comparable their histological necrotic findings. These values were higher than OCT, which were measured at the same time. This results showed that there might be m-GOT induction by alcohol. In liver cirrhosis lower values were obtained. Generally m-GOT was liable to be lower as the lobular distortion was getting severer.

[Hepatic GOT and GPT activities in patients with various liver diseases--especially alcoholic liver disease].

GOT and GPT activities were measured in percutaneous needle biopsy specimens of human liver tissue from 98 cases including normal subjects and patients with various liver diseases. Hepatic GOT activity was markedly decreased in liver tissue of patients with nonalcoholic liver cirrhosis. Hepatic GPT activity was markedly decreased in liver tissue of patients with alcoholic liver cirrhosis. The GOT/GPT ratio in liver tissue was increased in patients with alcoholic liver cirrhosis (5.32 +/- 2.03) and alcoholic liver disease (4.78 +/- 2.43). The increased SGOT/SGPT ratio in patients with alcoholic liver disease is due to primarily to the increased LGOT/LGPT ratio.

[Clinical significance of HBeAg and anti-HBe in HBsAg positive liver diseases].

Variation of incidence of HBe antigen (HBeAg) and HBe antibody (anti-HBe) was examined by use of RIA in 72 patients with HBsAg positive liver diseases. 1) Percentage of positive HBeAg was highest (71.5%) in chronic active hepatitis with lobular distortion, followed by chronic active hapatitis without lobular distortion (70.0%) and acute hepatitis in asymptomatic HBsAg carriers (66.7%). In contrast, it was low in chronic inactive hepatitis (35.7%) and liver cirrhosis (38.5%). None of liver cancers showed HBeAg positive reaction. 2) Percentage of positive HBe antibody (anti-HBe) was highest in liver cancer (100%), followed by liver cirrhosis (61.5%) and chronic inactive hepatitis (50.0%). In acute hepatitis from asymptomatic HBsAg carriers no anti-HBe was found. In chronic active hepatitis the percentage of positive anti-HBe was low, 21.4 and 30.0% with and without lobular distortion, respectively. 3) In 45 patients with persistently positive HBsAg liver diseases, fluctuations of HBeAg and anti-HBe were followed over a period of one year in relation to serum GPT values, an indicator of clinical conditions. Serum GPT tended to fluctuate or to remain high in patients with persistently positive HBeAg or with sporadically positive HBeAg or anti-HBe, whereas it tended to become low or normal with persistently positive anti-HBe or with seroconversion from HBeAg to anti-HBe. However, there were some exceptions to this tendency. From these results we concluded that it is clinically of significant value to determine HBeAg and anti-HBe levels for the effective assessment of the activity and time course of HBsAg positive liver diseases.