Quantitative analysis of carcinoembryonic antigen messenger RNA in peripheral venous blood and portal blood of patients with pancreatic ductal adenocarcinoma.

One major therapeutic failure of pancreatic adenocarcinoma treatment is metastasis to the liver. To screen patients with high risk for such hematogenous dissemination, we previously developed a very sensitive system to detect carcinoembryonic antigen (CEA) in blood. For a more practical application, we improved this system by making it quantitative and capable of analyzing both preoperative peripheral blood and intraoperative portal blood for the presence of CEA mRNA. CEA mRNA was not detected in the peripheral venous blood of any of the three patients examined, but it was identified in the portal blood without fail. In addition, the quantities of CEA mRNA identified in the portal blood before and after pancreatectomy were different. This study suggests that analysis of the portal blood seems to be important for the precise evaluation of hematogenous dissemination and of the pathophysiology of pancreatic ductal adenocarcinoma.

Continuous hepatocyte growth factor supply prevents lipopolysaccharide-induced liver injury in rats.

We present a rat model in which continuous supply of hepatocyte growth factor (HGF) prevents liver injury induced by carbon tetrachloride (CCl4) and E. coli 011:B4 lipopolysaccharide (LPS). Rat fibroblasts genetically modified to secrete rat HGF were implanted in syngenic rat spleen 7 days before administration of the hepatotoxins. Rats with HGF-secreting fibroblasts in the spleen showed a dramatic resistance to CCl4- and LPS-induced liver injury. In the LPS-induced liver injury model, blood chemical analysis revealed that the increase in serum glutamic oxalacetic transaminase level and the decrease in blood sugar level were remarkably suppressed in rats with HGF-secreting cells in the spleen. Most importantly, their survival rate was greatly improved compared to other control groups of rats. Thus our results indicate a new role of HGF in liver protection during endotoxemia and convey important clinical implications for developing new therapeutic modalities in the treatment of liver failure caused by endotoxemia.

Histologic features of thyroid gland in a patient with bromide-induced hypothyroidism.

A case of a 22-year-old woman with bromide-induced hypothyroidism is presented. There have been no reports dealing with changes in thyroid function and thyroid morphologic characteristics associated with bromide intoxication. The characteristic histologic features of the affected thyroid gland included a marked hyperplasia of follicles lined by tall columnar follicular cells with foci of papillary proliferation and a loss of colloid materials in the follicular lumen. Ultrastructurally, thyroid follicular cells showed a marked dilatation of rough endoplasmic reticulum containing no electron-dense materials. X-ray microanalysis of the thyroid follicular cells revealed the presence of significant amounts of bromide and an absence of iodine. These indicated that severe hypothyroidism in this patient might be related to the accumulation of bromide in the thyroid follicular cells.

Perioperative quantitative analysis of cytokeratin 20 mRNA in peripheral venous blood of patients with colorectal adenocarcinoma.

Hematogenous dissemination is a significant short-coming of colorectal carcinoma treatment. To screen patients with high risk for such blood-borne metastasis, we previously developed a highly sensitive system for the detection of cytokeratin 20 (CK-20) mRNA in blood. For a more practical application, we improved this system by making it quantitative and capable of analyzing peripheral venous blood for the detection of perioperative changes in CK-20 mRNA. CK-20 mRNA was not always detected in the preoperative blood, even in patients in an advanced stage, but it was identified without fail in intra- and post-operative blood. In addition, more copies of CK-20 mRNA were observed in the intra-operative blood than in pre- and post-operative blood. This study suggests that analysis of perioperative changes may provide important information for the precise evaluation of hematogenous dissemination and of the effect of surgical maneuvers on recurrence.

Membrane fluidity correlates with liver cancer cell proliferation and infiltration potential.

We developed a method to measure membrane fluidity of living cancer cells in two- and three-dimensional cultures, and found that there was a close relationship between the membrane fluidity of cancer cells and their proliferative and infiltrative ability. Membrane fluidity is thus a promising indicator of the probability of cancer recurrence.

Quantitative analysis of alpha-fetoprotein mRNA in circulating peripheral blood of patients with hepatocellular and alpha-fetoprotein-producing gastric carcinomas.

In conjunction with strategies introduced in recent years to identify cancer micrometastasis through amplification of cancer-associated mRNA, we developed a highly sensitive system to detect alpha-fetoprotein mRNA in circulating peripheral blood of hepatocellular carcinoma patients. The aim of the present study was to make our original system quantitative. Peripheral venous blood from patients with hepatocellular carcinoma and alpha-fetoprotein-producing gastric carcinoma was subjected to reverse transcription followed by our original three-step polymerase chain reaction co-amplifying both the original sequence and our synthetic competitor. We succeeded in modifying our system for quantitative analysis, and investigated the perioperative change, the postoperative change and the change after chemotherapy in order to illustrate the possible application of this method. The quantitative analysis of alpha-fetoprotein mRNA present in the peripheral blood represents a useful tool for analyzing the relationship of surgery to recurrence, the effect of chemotherapy, and to predict impending recurrence in patients with hepatocellular and alpha-fetoprotein-producing gastric carcinomas.

HLA-DR antigen and interleukin-2 receptor expression on primary-cultured human hepatic macrophages in relation to liver cirrhosis and hepatitis virus infection.

We analyzed primary-cultured human hepatic macrophages (HHMphi) from 12 patients with non-cirrhotic and cirrhotic livers for cell surface expression of HLA-DR antigen and interleukin-2 receptor (IL-2R). Compared to the relatively abundant HLA-DR antigen, IL-2R expression was generally low. No significant difference was observed between HLA-DR antigen expression nor IL-2 receptor expression. HHMphi from patients with serum hepatitis viral markers, however, expressed significantly more HLA-DR antigen than did HHMphi of patients without viral markers, which suggest a possible role of HHMphi as antigen-presenting cells (APC) in viral hepatitis. This direct, quantitative measurement of cell surface molecule expression on hepatic macrophages of human may provide an important clue to the pathophysiology of human liver disorders.

Chemical mediator release and surface marker expression of hepatic macrophages in rats with CCl4-induced liver cirrhosis.

The present study was performed to analyze possible functional alterations of hepatic macrophages (HM phi) in rats with carbon tetrachloride (CCl4)-induced liver cirrhosis. HM phi from rats injected with CCl4 for 13 weeks and cultured for 24 hours released less than normal amounts of prostaglandin E2 (PGE2) and tumor necrosis factor (TNF) and very large amounts of interleukin-1 (IL-1). In rats injected with CCl4 for 9 weeks, only PGE2 production was reduced. Interleukin-2 receptor (IL-2R), Ia antigen and asialo GM1 antigen expressions on HM phi from both the 9- and 13-week groups were significantly decreased. IL-2R and Ia antigen expressions showed larger decreases in the 13-week group. Thus, it is concluded that HM phi derived from CCl4-induced cirrhotic livers show a functional alteration in the release of cytokines (except for IL-1) and a decrease in surface marker expression, as cirrhosis advances. These results should provide a basis for further investigation into the host-compromised status in the presence of liver cirrhosis.

Highly-sensitive identification of alpha-fetoprotein mRNA in circulating peripheral blood of hepatocellular carcinoma patients.

In order to capture hepatocellular carcinoma (HCC) cells in circulating peripheral blood, we made analysis to see if alpha-fetoprotein (AFP) mRNA exists in the peripheral blood obtained from patients with HCC and also, as a control, from hepatitis-viral-marker-positive patients without HCC and a healthy volunteer. As the number of HCC cells in peripheral blood and the quantity of AFP mRNA are expected to be very small, the analysis was performed by the reverse transcription followed by an original three-step polymerase chain reaction. By this highly-sensitive method, 5 of 7 HCC patients were positive for AFP mRNA. These 5 positive patients consisted of three with clinically apparent recurrence, one preoperative patient with tumor thrombus in the portal vein and one recurrence-free patient who developed clinically detectable recurrence three months after this analysis. Neither 4 patients with positive viral markers nor a healthy volunteer was positive. The results suggest that detection of AFP mRNA from HCC patients' peripheral blood by our highly-sensitive RT-PCR may be a practical and powerful tool to diagnose the preoperative spreading of HCC and to monitor its recurrence.

Identification of carcinoembryonic antigen mRNA in circulating peripheral blood of pancreatic carcinoma and gastric carcinoma patients.

To detect adenocarcinoma cells in the circulating peripheral blood, we "analyzed the presence of carcinoembryonic antigen (CEA) mRNA in the peripheral blood obtained from patients with pancreatic carcinoma (PC) or with gastric carcinoma (GC) and also, as controls, from pancreatitis or gastritis patients without carcinomas, a gastric lymphoma patient and four healthy volunteers. Because of the small number of carcinoma cells expected in the peripheral blood, the analysis was performed by the reverse transcription followed by an original two-step polymerase chain reaction. By this sensitive method, 3 of 9 PC patients and 2 of 9 GC patients were positive for CEA mRNA. Except for 1 highly advanced PC patient, 3 of 4 CEA mRNA-positive patients developed recurrence after curative resection or liver metastasis after palliative operation within 9 months after the analysis. None of the control patients was positive for CEA mRNA in the peripheral blood. The results suggest that our sensitive RT-PCR method for detecting CEA mRNA in the peripheral blood is practically useful to find the hematogenous spreading of adenocarcinoma cells.

Prostaglandin E2 production by hepatic macrophages and peripheral monocytes in liver cirrhosis patients.

We analyzed PGE2 production in primary-cultured human hepatic macrophages (HHM phi) and peripheral monocytes (MO) from patients with and without liver cirrhosis, and correlated PGE2 production with the patients' liver function. Serum choline esterase (ChE) levels were used as an indicator of liver function. PGE2 production in both HHM phi and MO from cirrhotic patients was significantly lower than in HHM phi and MO from non-cirrhotic patients. PGE2 production in cirrhotic HHM phi was inversely correlated with ChE levels, whereas PGE2 production in cirrhotic MO showed no obvious correlation. In conclusion, both HHM phi and MO might contribute to the pathophysiology of liver cirrhosis via attenuated PGE2 production. Furthermore, HHM phi activity appears to be more strongly affected by the chronic pathological changes observed in the cirrhotic liver.

Detection of colorectal carcinoma cells in circulating peripheral blood by reverse transcription-polymerase chain reaction targeting cytokeratin-20 mRNA.

For the detection of circulating colorectal carcinoma cells, we investigated the presence of cytokeratin 20 (CK 20) mRNA in the peripheral blood of colorectal carcinoma patients. Application of our published technique resulted in analysis by reverse transcription followed by three-step nested polymerase chain reaction. This analysis could detect a single Colo 205 colon cancer cell mixed with 1 ml of blood. Our system also successfully detected the presence of CK 20 mRNA in actual patients' peripheral blood samples. Our highly sensitive and specific system for the detection of CK-20 mRNA from patients' peripheral blood thus seems to be useful for screening for circulating colorectal carcinoma cells.

Tumoricidal activity of Kupffer cells augmented by anticancer drugs.

The effect of Mitomycin-C (MMC) and Adriamycin (ADM) on the antitumor-associated function of Kupffer cells was examined. MMC and ADM enhanced the production of superoxide by Kupffer cells in cultures at low concentrations likely to occur in clinical use. The expression of interleukin-2 receptor, Ia antigen and asialoGM1 antigen, measured by flowcytometry, was increased by contact with MMC. Growth inhibition of AH130, rat ascites hepatoma, and P815, murine mastocytoma, by Kupffer cells treated with anticancer drugs was greater than that by Kupffer cells alone or anticancer agent alone. These results show that MMC and ADM activate Kupffer cells, leading to synergistic antitumor activity. The results suggest that some anticancer agents act through immunological mechanisms as well as through direct antineoplastic activity.

Effect of PGE2 on interleukin-1 and superoxide release from primary-cultured human hepatic macrophages.

In order to learn more about how human hepatic macrophages function, we analyzed the effect of exogenous PGE2 on the amounts of interleukin-1 (IL-1) and superoxide (O2-) released from primary-cultured human hepatic macrophages (HHM phi). When endogenous PGE2 production was blocked by indomethacin, exogenous PGE2 reduced IL-1 release from HHM phi in a dose-dependent manner, whereas it tended to increase O2- release from HHM phi. These results may suggest the probable contribution of PGE2 in regulating HHM phi mediator release in vivo.

Immunohistochemistry of DNA fragmentation factor in human stomach and colon: its correlation to apoptosis.

DNA fragmentation factor (DFF) is an important factor in the pathway leading to apoptosis, which is activated by caspase-3 and is involved in the formation of nuclear DNA fragments. DFF is a heterodimic protein of 40kDa and 45kDa that becomes activated when DFF is cleaved by caspase-3. Of the two enzymatically cleaved fragments of DFF, it is the 40kDa fragment (DFF40) that is the active component of DFF and is responsible for triggering chromatin condensation when incubated with nuclei. However, the topological correlation between apoptosis and DFF expression in human tissues has not been examined. Therefore, in this study, we first immunolocalized DFF in non-neoplastic mucosa, hyperplastic polyp, adenoma and carcinoma of human stomach and colon. We then examined apoptosis in serial tissue sections. Labeling index (LI) of DFF and TUNEL positive cells in the same areas of serial tissue sections were obtained using computer-assisted image analysis. In the stomach, the DFF LI in non-neoplastic mucosa (9.8 +/- 5.0%, n = 3) and carcinoma (18.2 +/- 3.6, n = 3) were significantly lower than that of hyperplastic polyp (73.3 +/- 9.2%, n = 3) and adenoma (66.5 +/- 18.3%, n = 3) [p < 0.0001]. In colon, the DFF LI in non-neoplastic mucosa (10.2 +/- 6.4%, n = 3) was significantly lower than that of hyperplastic polyp (56.0 + 34.7%, n = 3) [p = 0.0013] and adenoma (30.1 +/- 16.3%, n = 3) [p = 0.0037]. Cells positive for DFF were much more widely distributed than TUNEL positive cells in both non-pathologic and pathologic mucosa of human stomach and colon. Notably, DFF positive cells were present beneath the TUNEL positive cells in non-pathological gastric and colonic epithelium. In addition, there was a significant positive correlation between DFF and TUNEL LIs in human stomach and colon [p < 0.0001]. These results suggest that DFF may be involved in the process of apoptosis in human gastric and colonic mucosa.

Intrahepatic mast cell population correlates with clinical outcome in biliary atresia.

PURPOSE: To determine if mast cells influence the clinical outcome in biliary atresia (BA), the authors examined the intrahepatic mast cell population in BA. METHODS: Mast cells were identified histochemically using Toludin Blue and immunohistochemically using antimast cell tryptase antibody in formalin-fixed paraffin-embedded sections from 21 cases of BA. Patients were divided into 3 groups; group I (n = 8) with good liver function, group II (n = 8) with moderate liver dysfunction, and group III (n = 5) with severe liver dysfunction. Liver biopsies from patients with choledochal cysts (CDC, n = 5), and normal liver (NL, n = 4) served as controls. The results were compared among the groups. RESULTS: Both histochemical and immunohistochemical methods showed similar data. Mast cells were seen mostly in the portal tracts. Mast cell numbers per medium power field (20 x magnification) were higher in BA than in the controls (15. 03 +/- 2.25 v 3.85 +/-.65, [mean +/- SEM], P <.05, BA v CDC; 15.03 +/- 2.25 v 1.73 +/-.06, [mean +/- SEM], P <.05, BA v NL, immunohistochemical data). Clinical correlation showed an association between higher mast cell number and liver dysfunction (32.62 +/-.80 v 8.52 +/-.87 [mean +/- SEM], group III v group I; P <. 05, immunohistochemical data). CONCLUSION: Increased mast cell population in BA adversely affects liver function and raises the possibility that type I allergic reaction may play role in the pathology of BA.

[Concentration of antibiotics (cefoperazone) in human brain, brain tumor tissue and cerebrospinal fluid].

The transfer of cefoperazone (CPZ) into cerebrospinal fluid (CSF), brain or brain tumor tissue was studied in 13 cases with brain tumor, chronic subdural hematoma and benign intracranial hypertension in 1982. The peak values of CPZ in serum came up immediately after its rapid intravenous administration and then decreased exponentially. The concentration of CPZ in CSF started to increase with a long delay of about 60 min. The average peak level in CSF remained 21.6 micrograms/ml and corresponded to 10.3% of the peak level in serum. The best transfer of chloramphenicol into CSF has been reported, while that of CPZ would be one of the next. The CPZ levels in CSF showed a slower decay than in serum. The concentration of CPZ in brain reached the peak level in less than 30 min and the average peak level was 36.5 micrograms/g cerebral tissue. The brain to blood rate of the CPZ concentration was 11.1%. The CPZ levels in the brain showed a rapid decrease like the transition of antibiotic levels in serum. The antibiotic levels in brain tumors were divided into two groups. The one showed sharp peak about one tenth of the values in serum. The other was of a slowly increasing type.

[A case of primary anterior mediastinal embryonal carcinoma: report of a case].

A 15-year-old man was admitted to our hospital because of chest pain. The chest x-ray film and CT scan revealed a large anterior superior mediastinal mass. The serum alpha-fetoprotein (AFP) value was raised. Percutaneous biopsy of the tumor suggested embryonal carcinoma. The tumor was totally removed. Postoperatively combination chemotherapy including CDDP was performed. However the patient died of tumor recurrence 8 months after operation. AFP is very useful for its diagnosis and the follow-up of the clinical course.

[Assay of serum collagen markers in chronic liver diseases and liver cancer].

In order to identify the most useful marker to diagnose hepatic fibrosis, type III procollagen peptide (P-III-P), laminin P1, and prolyl-hydroxylase (PH) in sera obtained from patients with liver diseases were simultaneously measured and compared with histological features of chronic hepatitis and with tumor size estimated on abdominal CT scan. Further more, the diagnostic accuracy of these markers was evaluated by using discriminant analysis. P-III-P and laminin P1 were closely correlated with the activity of chronic hepatitis. These two markers most accurately discriminated between the compensated stage of liver cirrhosis and the decompensated stage, and between liver cirrhosis and hepatocellular carcinoma. Laminin P1 was found to most clearly distinguish chronic hepatitis from liver cirrhosis. P-III-P was significantly correlated with the size of hepatocellular carcinoma. However, PH failed to discriminate among liver diseases, and showed no significant correlation with a liver tumor size. And none of the markers examined were correlated with a degree of hepatic fibrosis. From these results, the analysis of both serum P-III-P and laminin P1 is a useful approach to evaluate hepatic collagen metabolism.