Cytogenetic studies of Hynobiidae (Urodela)XVIII. A ZZ/ZW sex-determining mechanism in a hynobiid salamander species, Hynobius tokyoensis Tago.

The karyotype of Hynobius tokyoensis (2n = 56) was analyzed using three kinds of banding methods to determine the morphological differentiation of the sex chromosomes of this species. Salamanders and egg sacs were collected from seven localities around Tokyo, Japan. Of 28 chromosome pairs, microchromosome No. 21 was identified as a ZZ/ZW-type sex chromosome. The Z chromosome was acrocentric, whereas the W chromosome was submetacentric, with a heterochromatic, elongated short arm. Interestingly, the W chromosome is of three distinct types, W(A), W(B), and W(C), based on R-banding and Ag-NOR patterns. W(A) was detected in five populations from southern habitats, whereas W(B) and W(C) were detected in one population each from northern habitats. W(A), W(B), and W(C) were all found to carry Ag-NORs on their heterochromatic short arms. Considering the karyotypes of other species belonging to the same genus, we discuss the evolution of the sex chromosomes of H. tokyoensis.

Th1/Th2 cytokine profiles and their relationship to clinical features in patients with chronic hepatitis C virus infection.

PURPOSE: An imbalance in helper T-cell type 1 (Th1) and type 2 (Th2) cytokines is suggested to play an important role in the pathogenesis of chronic viral infections, but this issue is not resolved in patients with hepatitis C virus (HCV) infection. The aim of this study was to clarify the relationship between the balance of Th1 and Th2 cytokines and liver damage. METHODS: We investigated cytokine levels in the peripheral blood and liver tissue of patients with chronic HCV infection (n = 59) by three different methods; we used flow cytometry to detect intracellular cytokines, and we measured cytokine titers in sera and in the supernatants of lymphocyte cultures with enzyme-linked immunosorbent assays (ELISAs). RESULTS: In both CD4+ and CD8+ cells, interferon (IFN) gamma-producing cell populations increased, while there was no difference in interleukin (IL)-10 production, indicating a shift to a Th1 cytokine profile with the progression of liver disease. With respect to the ratio of IFN-gamma to IL-10, a correlation was found in CD4+ cells between peripheral blood and liver tissue (r = 0.98; P = 0.0011). Th1 cytokine was predominant in intrahepatic CD4+ cells, while it was predominant in peripheral blood CD8+ cells. CONCLUSIONS: These findings indicate a correlation between dominant Th1 response and disease activity and progression. In addition, we suggest that intrahepatic CD4+ T cells play a pathogenetic role in the hepatic injury of HCV infection.

Percutaneous radiofrequency ablation therapy using a clustered electrode in the animal liver.

BACKGROUND/AIMS: The aim of the present study was to examine the safety and effectiveness of percutaneous radiofrequency ablation therapy using a needle with cluster radiofrequency electrodes in an animal model. METHODOLOGY: A total of 10 radiofrequency applications were performed in the normal liver of 5 domestic pigs with real-time ultrasonography until roll-off occurred two times. Aspartate aminotransferase, alanine aminotransferase, lactic dehydrogenase, and total bilirubin were evaluated before the procedure and 1 h, 24 h, and 7 days following percutaneous radiofrequency ablation therapy. The animals were euthanized 1 or 2 weeks after percutaneous radiofrequency ablation therapy, and the livers were removed for gross and histopathologic analysis for coagulation necrosis. RESULTS: There were no complications in any of the experimental animals. Aspartate aminotransferase, alanine aminotransferase, and lactic dehydrogenase levels peaked 24 h following percutaneous radiofrequency ablation therapy, and decreased with time thereafter. Total bilirubin was not elevated in any of the animals at any time. Macroscopic examination revealed that the area of coagulated necrosis was 28 x 21 mm when using a 2.0-cm needle, and 41 x 35 mm when using a 3.5-cm needle. Coagulation necrosis did not occur near large vessels. Microscopic examination of the fixed tissue revealed that coagulation necrosis occurred in preserving lobular structure. CONCLUSIONS: Percutaneous radiofrequency ablation therapy using a clustered electrode is a safe and effective treatment for liver tumor. Incomplete coagulation necrosis, however, can occur when percutaneous radiofrequency ablation therapy is performed for tumors located near large vessels.

Treatment of solitary small hepatocellular carcinoma: consideration of hepatic functional reserve and mode of recurrence.

BACKGROUND/AIMS: Hepatocellular carcinoma (HCC) recurs frequently after initial treatment. The subsequent prognosis varies with the mode of recurrence. Some patients die of hepatic failure even though the HCC is controlled. We consider the clinical stage (CS), using the modified Child-Pugh classification, to be an important factor influencing the prognosis of these patients. METHODOLOGY: To determine the most effective treatment for HCC, we examined 105 patients with solitary small HCC who were followed-up for more than 1 year after initial treatment. All of them were judged to be cured according to imaging or histological studies. The initial treatments were hepatic resection (n = 43), percutaneous ethanol injection therapy (PEIT, n = 33), and percutaneous microwave coagulation therapy (PMCT, n = 29). The modes of recurrence were divided into intrahepatic metastasis (IM) and multicentric occurrence (MO). RESULTS: Prognosis of MO was superior to that of IM in CS I patients, but there was no difference in prognosis between these modes in CS II. The hepatic resection group had more MO recurrences in CS I patients and more IM recurrences in CS II patients. IM developed frequently after PEIT and PMCT, regardless of the CS. Prognosis with hepatic resection was superior to that of the other treatments in CS I patients, but there was no difference in prognosis among the 3 treatment modalities in CS II patients. CONCLUSIONS: These data indicate that hepatic resection is the first choice for treating HCC in CS I patients, and that PEIT or PMCT is preferable for CS II patients.

Coexistence of primary biliary cirrhosis and myasthenia gravis: a case study.

We present a case that suggests a relationship between primary biliary cirrhosis and myasthenia gravis. A 43-year-old Japanese woman was admitted to the Nagoya City University Medical School, First Department of Internal Medicine with abnormal liver function in August 1991. She had had ptosis of the right eye since 1990. She had not been treated for liver disease. Ptosis of the right eye and hepatomegaly were present. Serum laboratory examinations revealed elevated biliary enzymes and IgM levels; tests were positive for antimitochondrial antibody and antiacetylcholine antibody. Liver histology revealed chronic non-suppurative destructive cholangitis and led to a diagnosis of primary biliary cirrhosis. The tensilon test was positive. Electromyography with repetitive motor nerve stimulation revealed a neuromuscular junction defect; i.e., the primary characteristic of myasthenia gravis. The patient was diagnosed with myasthenia gravis. Although the development of myasthenia gravis has previously been reported in patients with primary biliary cirrhosis during D-penicillamine administration, this is a very rare case of the coexistence of both diseases before such treatment.

Diagnosis of small hepatocellular carcinoma--imaging diagnosis and significance of tumor biopsy.

BACKGROUND/AIMS: To compare the effectiveness of different imaging modalities and the significance of tumor biopsy for diagnosing small hepatocellular carcinoma. METHODOLOGY: Nodules (n = 352) with diameters of 30 mm or less newly detected by periodic ultrasonography and computed tomography in 234 patients with chronic liver disease were investigated with magnetic resonance imaging and digital subtraction angiography. These findings were compared with histologic findings. Histologic diagnoses were dysplastic nodule (n = 23), well-differentiated hepatocellular carcinoma (n = 163), moderately differentiated hepatocellular carcinoma (n = 159), and poorly differentiated hepatocellular carcinoma (n = 7). We compared three groups based on-diameters of 10, 11-20, and 21-30 mm. Nodules were diagnosed as hepatocellular carcinoma if they had hypervascular staining on digital subtraction angiography, hyperintensity on magnetic resonance T2-weighted images, arterial phase enhancement on enhanced magnetic resonance imaging, or low-high-low density on enhanced computed tomography. RESULTS: Imaging alone was sufficient to diagnose hepatocellular carcinoma in 66.3% of the well-differentiated nodules and 91.6% of the moderately and poorly differentiated nodules (P < 0.001) The size of the nodule influenced the diagnosis of hepatocellular carcinoma by imaging alone in 65.5% (< or = 10 mm), 77.2% (11-20 mm), and 92.3% (21-30 mm) (< or = 10 vs. 21-30: P < 0.0001, 11-20 vs. 21-30: P < 0.0005). It was impossible to determine the degree of differentiation of the hepatocellular carcinoma by imaging alone. CONCLUSIONS: The effectiveness of imaging for the diagnosis of hepatocellular carcinoma improved with decreasing differentiation and increasing diameter of the nodules. Tumor biopsy was required to make a histological accurate diagnosis.

Standards for selecting percutaneous ethanol injection therapy or percutaneous microwave coagulation therapy for solitary small hepatocellular carcinoma: consideration of local recurrence.

OBJECTIVE: Percutaneous ethanol injection therapy (PEIT) and percutaneous microwave coagulation therapy (PMCT) are effective treatments for small hepatocellular carcinoma (HCC). There are no clear standards, however, for the selection of PEIT or PMCT. We determined standards based on local recurrence. METHODS: The subjects were 88 patients with solitary HCC measuring < or = 30 mm in diameter, who were treated by PEIT (n = 45) or PMCT (n = 43) and judged to be cured using computerized tomography (CT) with contrast medium after treatment. Patient characteristics, including age, gender, viral markers, Child-Pugh classification, tumor size, tumor cell differentiation, and serum alpha-fetoprotein (AFP) concentration we analyzed, and the factors influencing the local recurrence in the PEIT and PMCT groups were determined, using univariate and multivariate analysis. RESULTS: Univariate analysis indicated that tumor cell differentiation and serum AFP concentration influenced local recurrence in the PEIT group, and tumor size did so in the PMCT group. Multivariate analysis revealed that tumor cell differentiation influenced local recurrence in the PEIT group, and tumor size did so in the PMCT group. PEIT was effective for treating well-differentiated HCC, and PMCT was effective for treating HCC measuring < or = 15 mm in diameter. PMCT was superior to PEIT for treating patients with HCC measuring < or = 15 mm in diameter. In such cases with well-differentiated HCC, PEIT was as effective as PMCT. CONCLUSIONS: The selection of PEIT or PMCT to treat patients with HCC should be based on tumor size and cell differentiation.

Limitations of imaging diagnosis for small hepatocellular carcinoma: comparison with histological findings.

BACKGROUND AND AIMS: The purpose of this study was to clarify the value and limitation of imaging modalities for diagnosing small hepatocellular carcinoma (HCC). METHODS: Nodules (n = 207) with diameters of 20 mm or less detected by periodic ultrasonography and computed tomography in 139 patients with chronic liver disease were investigated with digital subtraction angiography (DSA) and magnetic resonance imaging (MRI). These findings were compared with histological findings. RESULTS: Histological diagnoses were adenomatous hyperplasia (AH, n = 27), well-differentiated HCC (n = 99), moderately differentiated HCC (n = 79) and poorly differentiated HCC (n = 2). We compared two groups: group A (n = 62), nodules of 10 mm diameters or less; and group B (n = 145), nodules 11-20 mm. Adenomatous hyperplasia accounted for approximately 30% of group A, but was difficult to diagnose with imaging modalities alone. We diagnosed those nodules showing hypervascular staining on DSA or hyperintensity on MRI T2-weighted images as HCC. Imaging alone was sufficient to diagnose HCC in 58% of the well-differentiated nodules and 87% of the moderately and poorly differentiated nodules (P < 0.01). It was possible to diagnose HCC by imaging alone in 60% of all nodules or 45% of group A and 68% of group B (A vs B, P < 0.005). CONCLUSIONS: With decreasing differentiation and increasing diameter of nodules, the use of imaging modalities to diagnose HCC improved. Tumour biopsy was required to diagnose 55% of the cases in group A and 32% of the cases in group B.

Endoscopic injection sclerotherapy for esophagogastric variceal bleeding in children with biliary atresia.

BACKGROUND/AIMS: To determine the safety and effectiveness of endoscopic injection sclerotherapy (EIS) for children with biliary atresia. METHODOLOGY: Subjects were 7 patients with biliary atresia with esophagogastric varices and variceal bleeding. Intravariceal injection using 5% ethanolamine oleate was performed under fluoroscopy until varices were eradicated. RESULTS: Endoscopic examination revealed that bleeding occurred in the junctional gastric varices in most of the cases. The mean number of EIS sessions required for obliteration of the varices was 2.3. In the observation period (mean: 21 months), recurrent esophagogastric varices occurred in 2 patients. One had variceal bleeding that was treated successfully by additional EIS. There were no severe complications associated with EIS. CONCLUSIONS: EIS under fluoroscopy was safe and effective for variceal bleeding in children with biliary atresia.

Differential inhibition of epidermal growth factor signaling pathways in rat hepatocytes by long-term ethanol treatment.

BACKGROUND & AIMS: Long-term ethanol intake suppresses liver regeneration in vivo and ethanol interferes with epidermal growth factor (EGF)-induced DNA synthesis in vitro. Therefore, the effects of long-term ethanol treatment on EGF-activated signaling reactions in rat hepatocytes were investigated. METHODS: Hepatocytes from long-term ethanol-fed rats and pair-fed controls were stimulated with EGF (0.5-20 nmol/L) for 15-120 seconds. Tyrosine phosphorylation of EGF receptor (EGFR), Shc, and phospholipase-C gamma1 (PLC gamma), and growth factor receptor binding protein 2 (Grb2) coprecipitation with EGFR and Shc were analyzed by Western blotting. RESULTS: EGFR autophosphorylation was suppressed at all EGF concentrations in ethanol-fed cells compared with pair-fed cells, without significant differences in total EGFR protein or EGFR tyrosine kinase activity detected in cell lysates, suggesting that intracellular factors suppressed EGFR function. EGF-induced PLC gamma tyrosine phosphorylation and inositol 1,4,5-trisphosphate (InsP3) formation were suppressed, but cytosolic [Ca2+]c elevation was little affected, indicating enhanced InsP3-mediated intracellular Ca2+ release in ethanol-fed cells. Grb2 binding to EGFR was suppressed, but EGF-induced Shc tyrosine phosphorylation and Grb2 association with Shc were not significantly decreased. CONCLUSIONS: Long-term ethanol feeding suppressed EGF-induced receptor autophosphorylation in rat hepatocytes with differential inhibition of downstream signaling processes mediated by PLC gamma, Shc, and Grb2. Altered patterns of downstream signals emanating from EGFR may contribute to deficient liver regeneration in chronic alcoholism.

Inhibitory effect of ethanol on hepatocyte growth factor-induced DNA synthesis and Ca2+ mobilization in rat hepatocytes.

Hepatocyte growth factor (HGF) is the most potent mitogen identified for hepatocytes and is thought to be an important growth factor in the regulation of liver regeneration. Its effects are mediated through a tyrosine kinase receptor, the product of c-met proto-oncogene. One of the downstream signaling processes activated by HGF is phospholipase C-gamma. HGF stimulation of liver cells causes formation of inositol 1,4,5-triphosphate, which releases Ca2+ from intracellular Ca2+ ([Ca2+]i) stores, and causes elevation of cytosolic Ca2+ levels. It is known that liver regeneration is inhibited by both acute and chronic ethanol (EtOH) treatment. We investigated the effect of EtOH on HGF-induced DNA synthesis and mobilization of [Ca2+]i in rat hepatocytes in primary culture. DNA synthesis was monitored by [3H]thymidine incorporation in primary cultures of hepatocytes 42 hr after stimulation with HGF. HGF concentration required for maximum DNA synthesis was 0.3 to 1 ng/ml, and DNA synthesis was inhibited by 100 mM EtOH at HGF concentrations in the range of 0.1 to 5 ng/ml. This inhibition was strongest (45 to 47% inhibition) at a low concentration of HGF (0.1 to 0.3 ng/ml) and decreased at an HGF concentration > 1 ng/ml. HGF-induced changes in [Ca2+]i were measured in single fura 2-loaded hepatocytes by fluorescence imaging techniques. The Ca2+ response induced by HGF (0.3 to 5 ng/ml) was inhibited by EtOH, with an EC50 of approximately 50 mM. Analysis of Ca2+ response patterns in individual cells indicated that EtOH suppressed the number of responsive cells and made Ca2+ responses more transient, but did not affect peak [Ca2+]i elevation; thus suggesting an inhibition at the level of phospholipase C-gamma-activation. These data indicate that inhibition by EtOH of the response of liver cells to HGF may contribute to the inhibitory effect of EtOH on liver regeneration.

Interaction of protein phosphatases and ethanol on phospholipase C-mediated intracellular signal transduction processes in rat hepatocytes: role of protein kinase A.

Phospholipase C (PLC)-mediated signal transduction processes in rat hepatocytes are subject to modulation by protein phosphatases (PPases) and protein kinases, including protein kinase A (PKA) and protein kinase C. Ethanol (EtOH) stimulates PLC activity in liver cells in the absence of hormones, and EtOH pretreatment inhibits the subsequent stimulation of PLC by hormonal stimuli. There is evidence that protein kinase activities are involved in these actions of EtOH. We investigated the effects of okadaic acid (OKA), a PPase inhibitor, and 8-(4-chlorophenylthio)adenosine 3':5'-cyclic monophosphate (cpt-cAMP), a cell permeant cAMP analog that activates PKA, on EtOH-induced PLC activation. In addition, we studied the combined effects of cpt-cAMP and EtOH/OKA on vasopressin-induced PLC activation. PLC activation (cytosolic Ca2+ mobilization and inositol trisphosphate accumulation) induced by EtOH and vasopressin was inhibited by treatment with OKA, and was potentiated by cpt-cAMP. OKA treatment prevented the effect of cpt-cAMP. Pretreatment with EtOH caused inhibition of vasopressin-induced PLC activation. EtOH also decreased the enhancing effect of cpt-cAMP on the responses to vasopressin. The susceptibility to enhancement by cpt-cAMP plotted as a function of the initial rate of vasopressin-induced Ca2+ mobilization in EtOH-treated cells was similar to the pattern observed in OKA-treated cells. These data suggest that interactions of OKA and PKA on EtOH-induced PLC activation occurred at the level of G-protein, and indicate that EtOH may act as an inhibitory agent of PPase.

Effect of glutathione on inositol 1,4,5-triphosphate-induced Ca2+ release in permeabilized hepatocytes from control and chronic ethanol-fed rats.

The effect of oxidized and reduced glutathione on inositol 1,4,5-trisphosphate (InsP3)-induced Ca2+ release from endoplasmic reticular Ca2+ stores was studied in digitonin-permeabilized hepatocytes from chronically ethanol-fed rats and pair-fed control animals. The fractional Ca2+ release induced by a subsaturating concentration of InsP3 was significantly enhanced in cells from ethanol-fed rats in the absence of a change in maximal InsP3-releasable Ca2+ pool size, and this difference was not affected by preincubation with reduced glutathione. Incubation with oxidized glutathione (1 mM) increased the efficacy of Ca2+ release by subsaturating concentrations of InsP3 in both control preparations and in cells from ethanol-fed rats. The shift in the InsP3 dose-response curve was not significantly different between the two preparations. These findings suggest that the enhanced efficacy of InsP3-induced Ca2+ release in hepatocytes from ethanol-fed rats is not caused by the oxidation of protein-bound thiol groups on the InsP3 receptor.

Transfer index of MR relaxation enhancer: a quantitative evaluation of MR contrast enhancement.

To clarify the effects of Gd-DTPA on biological water, we examined the effects of the compound on spin-lattice relaxation rate with various concentrations of gelatin solutions. The results indicate that the effects of relaxation rate of Gd-DTPA in biological water fundamentally correspond to those in aqueous solution. To evaluate the distribution of Gd-DTPA in tissues, we introduced a transfer index that represents the product of tissue-blood ratio of Gd-DTPA and the ratio of extracellular volume of a tissue based on the above findings. The index depends neither on dose of the compound nor on Larmor frequency. The clinical significance of the index was studied in patients with brain tumors. The indexes varied from 0.038 to 0.51, depending on the biological characteristics of the tumors. The transfer index may be used in the quantitative evaluation of MR relaxation enhancement, which may be applied to monitoring therapeutic efficacy and to estimating tissue perfusion.

Assessment of T1 time course changes and tissue-blood ratios after Gd-DTPA administration in brain tumors.

Sequential T1 changes in brain tumor tissue after Gd-DTPA administration were investigated in 10 patients, including 4 meningiomas, 2 gliomas, 3 metastatic cerebral tumors and 1 brain abscess. T1 values were measured serially for 60 minutes following Gd-DTPA injection using a magnetic focusing technique. In vitro T1 of the whole blood samples was also comparatively examined. Time processes in the tissue-blood ratio (TBR) were calculated from two-point relaxation rates at 5 and 30 minutes. The obtained ratios of TBR were ranged from 1.0 to 3.0, probably depending on histological types of brain tumor (the value of 1.0 to 1.5 for meningioma and 1.5 to 3.0 for glioma and metastatic tumor). No significant changes in the T1 value were observed in the examined normal tissue and peritumoral edema. These results indicate that Gd-DTPA plays an important role not only as an image enhancer for tumor tissue but also as an indicator for estimating the blood-brain barrier function.

[Evaluation of peritumoral edema by proton T1 values with special remarks on time courses following intracranial surgery].

Proton NMR is said to be sensitive to change in tissue water content. Much more attention has been paid to clinical NMR technique for evaluating pathophysiological states including intracranial edema dynamics. This paper describes the follow up data in NMR imaging and spin-lattice relaxation time (T1) measurement in peritumoral brain tissues. Special attention was directed to the time course of focal T1 values before and after surgery. Twenty-one patients with brain tumor, including 6 meningiomas, 7 gliomas and 8 metastatic tumors, were subjected to the present studies in the term from March 1983 to September 1985. Age distribution of this series ranged from 39 to 74 years (average: 58.2). Fonar QED 80-alpha system was applied for NMR examinations of dual modes (static magnetic field: 433 gauss), i.e. image display by steady state free precession (SSFP) and in vivo T1 measurement by field focussing technique. Standard T1 values for healthy brain were 290 +/- 41 ms and 230 +/- 34 ms (mean +/- SD) in cerebral gray and white matter, respectively. Both T1 values in tumors and in peritumoral tissues before craniotomy were obviously prolonged as compared to standard T1 of corresponding site. However, it remained difficult to discriminate statistically these histological differences by preoperative T1 values. Time course of peritumoral T1 was characteristic in accordance with the sorts of brain tumor. T1 values in the brain parenchyma around meningioma were shortened after operation in the earlier stage up to 1 month, showing relatively rapid normalization in T1.(ABSTRACT TRUNCATED AT 250 WORDS)

Spin-lattice relaxation times of bound water--its determination and implications for tissue discrimination.

Measurements were made of T1 of bound water (T1b) and bound water fraction (alpha) for gelatin solutions and human tissues (sera, brain tumor, cerebral white matter). Bound water fraction in each sample was measured by means of thermal analysis (differential scanning calorimetry: DSC). T1 values were measured by FONAR QED 80-alpha. T1b values were calculated by an equation derived from the fast-exchange two-state model. In the study of gelatin solutions, the relationship between T1 and water content differed depending on the sort of solutions. This was considered to be due to differences in T1b values. In each biological tissue the values of T1b and alpha had different distributions. These results indicate that values of T1b and alpha for biological tissues may be altered in correspondence to the changes in pathophysiological states in those tissues.

[Time course of NMR images and T1 values associated with hypertensive intracerebral hematoma].

The present study describes time courses in tissue T1 values, as well as in NMR imagings, associated with hypertensive intracerebral hematoma (ICH). Non-operative 21 cases of ICH were examined by FONAR QED 80-alpha NMR system, which possessed dual modes of image display and focal T1 measurement (static magnetic field: 433 gauss). As the first step of examination SSFP images are displayed and then, at the regions of interest, absolute values of T1 are measured by field focusing technique. The extent of ICH was revealed as high density zone in NMR imaging, occasionally represented much wider extent of high density area than the finding on X-ray CT. Prolonged T1 values were obtained from such high density zone. This wide-spread high density area was regarded to reflect the spread of perifocal brain edema. T1 value of the hematoma itself was rather shortened in its initial phase within 2 weeks, thereafter followed by prolongation in the time lapse. This seemed to reflect the alterations in the properties of hematoma such as clot formation in earlier phase and resolution in later phase. On the contrary, T1 in the brain tissue surrounded to hematoma was apparently prolonged in the early phase within 2 weeks, representing the maximal values of 312 msec around 2 to 4 weeks after the onset, and then gradually normalized in the period over 1 month. This alteration in tissue T1 likely represents the processes of edema formation and its regression in perifocal zone. T1 values measured in perifocal region might be available for the evaluation of edema state in association with cerebrovascular accident.