Enzyme-linked immunoassay for midkine, and its application to evaluation of midkine levels in developing mouse brain and sera from patients with hepatocellular carcinomas.

Midkine (MK) is a growth factor that promotes neurite outgrowth and survival of neurons, and enhances the plasminogen activator in endothelial cells. A highly sensitive enzyme-linked immunoassay for MK was developed, involving affinity-purified anti-MK antibodies, their biotinylated form, and avidin-beta-galactosidase. The amount of bound avidin-beta-galactosidase was determined using a fluorogenic substrate, 4-methylumbelliferyl-beta-D-galactoside. This method allowed the detection of human and mouse MK in the range of 50 pg-10 ng. Pleiotrophin, which is related to MK in its amino acid sequence, did not show any cross reactivity. Employing this method, the MK levels in the developing mouse brain were determined. The MK level was 2 micrograms/g of wet tissue on the 12th day of gestation, and then steadily decreased during embryogenesis and postnatal development to 30 ng/g two months after birth. The assay method can also be applied to serum samples. Although the MK levels in the sera of normal human subjects were low or undetectable, 0.6-8 ng/ml of MK was detected in samples in the majority of cases of hepatocellular carcinomas.

Treatment of hepatocellular carcinoma by transcatheter hepatic arterial injection of radioactive iodized oil solution.

After 12 days of culture, VX2 carcinoma cells were inoculated into the liver of 16 rabbits; 14 days later, 131I-labeled iodized oil ([131I]-Lp) suspended in lipiodol was injected into the hepatic artery. Selective accumulation of the contrast material in the tumor for an extended time was evident on X-rays and hepatic scintiphotographs. The antitumor effect was remarkable. [131I]-Lp agents warrant further examination for their clinical usefulness. Internal radiation therapy by transcatheter hepatic arterial injection of [131I]-Lp (group A) was evaluated in 9 patients with hepatocellular carcinoma (HCC, tumor stage III or IV) associated with liver cirrhosis (LC) and compared with combination therapy of Lp-TAE (group B) in 18 patients with HCC (tumor stage III or IV) associated with LC. In group A, serum AFP levels dropped rapidly in eight of the nine patients who had an elevated initial level of more than 500 ng/ml. The average reduction in tumor size was 50% in eight cases as determined by computed tomography. Histological examination of one resected liver specimen at 3 months after the third injection of [131I]-Lp revealed microscopic features highly suggestive of a radiation effect in the [131I]-Lp-containing area. The 1-year survival value for patients with HCC was estimated at 49.0% using the Kaplan-Meier method. The survival of patients treated with internal radiation therapy tended to be better than that of those treated with Lp-TAE (P = 0.119).

Unruptured intracranial aneurysms in elderly patients.

A total of 556 patients with 769 intracranial aneurysms, of which 256 were unruptured and 513 were ruptured, were included in the present study. The patients were divided into three age groups: those aged 59 years or younger, those aged 60 to 69 years, and those aged 70 years or older. Small aneurysms of 4 mm or less in diameter were more common in the series of unruptured aneurysms than in the ruptured aneurysms. The rupture rate in anterior communicating artery aneurysms was the highest, and it increased with age. A follow-up study was performed on 47 patients with 55 unruptured aneurysms, and only one giant basilar artery aneurysm ruptured during the average follow-up period of 5.2 years. Direct operation was performed on 52 patients with unruptured aneurysms. While the surgical mortality rate was 0%, the morbidity rate was 6% (three of 52 cases), which was not directly related to the patients' age. When considering surgery for unruptured aneurysms, rupture rate of aneurysms at each site is one of the most important factors, especially in elderly patients.

Rapid spontaneous diminution of cisternal blood on computed tomography in patients with subarachnoid hemorrhage.

BACKGROUND: The purpose of this study is to investigate whether rapid spontaneous diminution of cisternal subarachnoid hemorrhage (SAH) alleviates vasospasm in the corresponding arterial territory. METHODS: The subjects were 103 patients in whom initial computed tomography scans were performed within 24 hours after SAH and repeated within 72 hours. We analyzed the effect of diminution of cisternal SAH on vasospasm in 16 sites in each patient. Of the total 1642 cisterns. SAH was found in 1362 (83%), of which 539 (40%) had a decrease in SAH. The highest diminution rate was 64% in quadrigeminal cistern, and the lowest rate was 27% in frontal interhemispheric fissure (IHF). In basal frontal IHF, both the incidence of diminution of SAH and its degree were significantly lower in patients with ruptured anterior cerebral artery aneurysms than in those with other site aneurysms, while in suprasellar cisterns, sylvian stems, and sylvian fissures, diminution of SAH was not affected by the side of ruptured aneurysms. The diminution of SAH in basal frontal IHF and sylvian stems was associated with less vasospasm in adjacent arteries. CONCLUSIONS: We concluded that in patients with SAH, rapid spontaneous diminution of cisternal blood, which is affected by several factors, makes vasospasm in the corresponding arterial territory less likely.

Complication of silastic dural substitute 20 years after dural plasty.

A 63-year-old woman was admitted because of right hemiparesis. Both computed tomography scan and magnetic resonance imaging revealed a mass lesion in the left parieto-occipital lobe. Twenty years previously, meningioma of the left parieto-occipital region was removed at another hospital, and a dural plasty was performed using a silastic dural substitute. The patient was operated on under the diagnosis of recurrent meningioma. However, most of the mass lesion was composed of an old hematoma, which seemed to be a complication of the silastic dural substitute.

Detection of hepatitis C virus RNA in circulating immune complexes by RT-PCR.

BACKGROUND/AIMS: We investigate whether hepatitis C virus (HCV) forms a circulating immune complex (CIC) in patients with chronic HCV infection. MATERIALS AND METHODS: We examined HCV-RNA immunoprecipitated with anti-human IgG, A and M antibodies by reverse transcription-polymerase chain reaction. RESULTS: In thirty-nine (91%) of 43 patients, composed of 35 chronic hepatitis (CH) and 8 liver cirrhosis (LC), HCV-RNA was detected in the CIC. All 43 patients analyzed were classified into the following three categories; HCV-RNA was detected only in the supernatant (S pattern, 4 patients), both in the supernatant and the precipitate (SP pattern, 27 patients), and only in the precipitate (P pattern, 12 patients). SP pattern was most common in chronic HCV infection, and the frequency of SP pattern decreased with the progression of liver disease. P pattern was significantly more frequent in patients with higher gamma-globulin levels, histologically indicated LC, and antibody to HCV envelope protein. CONCLUSION: We found that HCV formed a CIC in most patients with chronic HCV infection, and that the formation of CIC might be related to the stage of chronic HCV infection.

Point mutations in the S and pre-S2 genes observed in two hepatitis B virus carriers positive for antibody to hepatitis B surface antigen.

Two hepatitis B virus (HBV) carriers who had antibodies to HBV surface antigen (anti-HBs) were studied. Case 1 was a 47 year old woman positive for hepatitis B e antigen (HBeAg), and case 2 was a 61 year old man positive for antibody to HBeAg (anti-HBe) and DNA-polymerase (DNA-p). Neither case had received the HBV vaccine. The nucleotide sequences of the HBV-DNA extracted from the patients' sera were determined within the pre-S2 and S genes. Seven out of nine S gene clones from case 1 and six out of nine S gene clones from case 2 had an amino acid replacement from Thr or Ile to Ser at codon 126 in the alpha-determinant of the S gene. Amino acid substitution of codon 145 of the S gene previously reported was not observed. Although two previous reports on HBV escape mutant carriers with both anti-HBs and HBeAg described some deletions in the pre-S2 gene, our cases did not show these deletions. Our analysis indicated that carriers with the HBV escape mutant did not always have pre-S2 gene deletions. We found two HBV escape mutant carriers who had amino acid substitutions at codon 126 in the S gene due to point mutation without any deletions in the pre-S2 gene.

Expression of the midkine gene in human hepatocellular carcinomas.

BACKGROUND/AIMS: Aberrant expression of Midkine (MK) has been found in various human carcinomas including hepatocellular carcinoma (HCC). The aim of study is to identify the incidence of MK expression in tumor and surrounding non-tumor tissues of the liver, and to find the correlation of MK expression with other tumor markers. METHODOLOGY: Liver tissues were obtained from 16 patients with HCC and 4 with metastatic liver cancer. Background diseases of the HCC patients include liver cirrhosis and chronic hepatitis of type B or C. RNA was prepared from both cancerous and surrounding non-cancerous tissues, and analyzed for the presence of MK mRNA by RT-PCR, PCR-Southern blot, and Northern blot analysis. RESULTS: MK expression was detected in 12 (75%) of 16 HCCs by PCR-Southern blot analysis, the most sensitive of the 3 methods. Three of 9 surrounding cirrhotic tissues were weakly positive for MK expression, and none of chronic hepatitis and 4 normal tissues were negative. No significant difference was found in clinical and pathological parameters between MK negative and positive cases. Among metastatic cancers, 1 of gastric origin was positive for MK expression, but 1 each of chorangiocellular, gall bladder, and gastrinoma origin was negative. CONCLUSIONS: These results suggest that MK is expressed in the majority of HCC tissues and rarely in surrounding tissues in chronic liver diseases.

Reciprocal gene expression of rat fibroglycan and beta-actin during the course of regeneration after D-galactosamine liver injury.

BACKGROUND/AIMS: Fibroglycan (FG) is a major heparan sulfate proteoglycan (HSPG) in the rat liver that is mainly distributed on the surface of hepatocytes. HSPG may play some important roles in the regeneration of liver by interacting with various growth factors such as bFGF and HB-EGF. However, little is known about the function of FG. We reported that after injury caused by D-galactosamine, regeneration started on the following day and peaked on day 2. To clarify the function of FG in liver regeneration, we investigated the gene expression of FG during regeneration after D-galactosamine injury. MATERIALS AND METHODS: Rats were given D-galactosamine on day 0. Liver RNA was collected from day 0 to day 7. The gene expression of FG and beta-actin (as a representative cytoskeleton) was examined by Northern and/or Slot blotting. RESULTS: FG gene expression was markedly decreased on day 2, but totally recovered on day 3. In contrast, beta-actin gene expression was markedly increased on day 2 and returned to the normal level on day 3. Expression of the FG and beta-actin genes was reciprocal. CONCLUSION: FG expression is transiently suppressed when cytoskeleton gene expression is enhanced at the early phase of liver regeneration.

Promoter-independent loss of mRNA and protein of the Rb gene in a human hepatocellular carcinoma.

BACKGROUND: Inactivation of the retinoblastoma (Rb) gene is considered to play a fundamental role in the genesis and progression of several human cancers. In retinoblastoma, the inactivation of Rb promoter by mutations or hypermethylation has been reported. Although genetic changes of Rb gene have been described in hepatocellular carcinoma (HCC), an epigenetic change such as hypermethylation of the Rb promoter as reported in retinoblastoma has not been described. MATERIALS AND METHODS: We examined the hypermethylation in the promoter region of Rb gene by restriction fragment length polymorphism in 19 HCCs, as well as the expression of Rb mRNA and protein by RT-PCR and by immunoblotting, respectively. RESULTS: We found no evidence of hypermethylation in the promoter region of the Rb gene in all HCCs analyzed. However, the expression of Rb mRNA and protein was lost in one HCC, and no mutation was detected in the Rb promoter region of this patient. The inactivation of Rb promoter by hypermethylation or by inhibition of binding of transcription factors due to point mutations did not contribute to the loss of mRNA and protein in the patient. CONCLUSIONS: Hypermethylation in the Rb promoter region appeared to have little causal effect on HCC.

Gene expression of liver-specific proteins in hepatocyte spheroids in primary culture.

Adult rat hepatocytes assemble to form multicellular spheroids under non-adherent environments such as immobilized chondroitin sulfate-proteoglycan in primary culture. Previously, we demonstrated that hepatocyte spheroids exhibited various differentiated structures as observed in the liver tissue. It was also shown that hepatocyte growth was highly suppressed and several differentiated functions, including albumin production and gluconeogenesis, were well preserved in spheroids. To investigate the differentiated functions of cultured hepatocytes in relation to cell morphology, we compared the expression of the albumin and transferrin genes in spheroids with those in monolayers by Northern blot analysis. Production of these proteins in the culture medium was simultaneously examined by ELISA. Gene expression and protein production of both albumin and transferrin were better preserved in spheroids. We also examined changes in the expression of liver-specific genes in response to IL-6. Reduced mRNA levels of both albumin and transferrin was only found in spheroids and no change was observed in monolayers. These results suggest that the regulation of tissue-specific gene expression is better preserved in spheroids, in which hepatocytes are in close contact with each other.

Sequence variations in the envelope protein of the hepatitis C virus: comparison with partial cDNA sequence of a new variant virus obtained by the polymerase chain reaction.

It has been reported that the envelope region located at the 3' portion of the structural protein coding region is one of the most variable regions at both nucleotide and amino acid sequence levels in the hepatitis C virus (HCV) genome. We cloned HCV cDNA fragments of an envelope protein coding region (HCVNK), which were derived from serum of a Japanese patient with hepatocellular carcinoma and were amplified by polymerase chain reaction. After determining the nucleotide sequence, deduced amino acid sequence of the envelope protein region was compared with those of six HCV strains already published (HCJ1, HCVUS, HCJ4, HCVJH, HCVJ and HCVBK). Homology analysis among the strains revealed that the seven strains were classified into two subtypes; a US subtype (HCJ1 and HCVUS) and a Japanese subtype (HCJ4, HCVJH, HCVJ, HCVBK and HCVNK), since percentage homologies between two subtypes (70.3-77.3%) were significantly lower than those within each subtype (83.9-93.5%). Detailed analysis of the amino acid sequences also indicates that the region at aa246-aa258, tentatively named intersubtype variable region-1, may distinguish the US subtype from the Japanese subtype.

Detection by western blotting of an antibody to the hepatitis C virus E1 envelope protein in sera of patients with chronic liver disease.

We detected an antibody to HCV envelope protein (E1) in sera of patients with HCV-related chronic liver diseases (20 patients with chronic hepatitis and 5 patients with liver cirrhosis) by Western blotting using the fusion protein of E1 envelope protein and beta-galactosidase as an antigen. The antibody to HCV E1 (anti-HCV E1) was detected in 8 (42%) of 19 patients positive for HCV-RNA (16 were positive and 3 were negative for antibody to C100-3) and in 1 (17%) of 6 patients negative for HCV-RNA but positive for antibody to C100-3. HCV-RNA was detected in 8 (89%) of 9 anti-HCV E1 positive sera. The value of alanine aminotransferase was significantly higher in patients positive for anti-HCV E1 than in patients negative for the antibody. Although an antibody to the envelope protein of HCV is suspected to be one of the candidates of virus-neutralizing antibodies, our results suggest this hypothesis appears to be unlikely.

Relationship between response to interferon therapy and detection of hepatitis C virus RNA by differential flotation centrifugation.

Circulating hepatitis C virus (HCV) particles can be fractionated by means of differential flotation centrifugation. It is reported that in the bottom fraction HCV is in the form immune complexes, whereas in the top, it is free of antibodies. We evaluated the significance of circulating complex and free HCV in chronic hepatitis C, and assessed the relationship in terms of the response to interferon (IFN) therapy. We examined sera before, just after, and 1 year after administering IFN to 18 patients with chronic hepatitis C, 10 of whom responded (group CR), and 8 did not (group NR). The amounts of virus were similar between both groups before therapy. After differential flotation centrifugation with 1.063 g/ml of NaCl, the top and bottom fractions were assayed for HCV RNA. Before therapy, HCV RNA was detected in the top fraction in 1 of 10 in group CR, and in 6 of 8 in group NR (P < 0.05, chi-square test). HCV RNA was positive in the bottom fraction of all samples. In a follow-up study of group NR, HCV RNA was detected in the top fraction in 3 of 8 just after IFN therapy, and in 7 of 8 after 1 year. This study suggests that the presence of HCV in the top fraction can predict a poor response to IFN therapy.

[A case of visual perseveration attack caused by transverse-sigmoid sinus dural arteriovenous fistula].

A case of visual perseveration attack in the right superior quadrantic visual field caused by left transverse-sigmoid sinus dural arteriovenous fistula (AVF) was reported. A 75-year old right-handed man noticed that when he saw an object, the image sometimes persisted even after he looked away or the object was removed. The palinoptic images always appeared in the right superior quadrant visual field, consisted of a row of multiple objects all identical to the real one in shape, and were accompanied by photopsia. The palinoptic images disappeared within a few minutes. He was neurologically normal and showed no hemianopia or quadrantanopia on admission. EEG showed no epileptic discharge. CT scanning of the brain revealed a small high density area in the left occipital lobe. MRI demonstrated hyperintensity in the left inferior occipital lobe corresponding to the lower part of Brodmann's areas 18 and 19 on T2 weighted image and flow-voids caused by the dilated left occipital artery. 99mTc-ECD SPECT disclosed a decrease of regional cerebral blood flow in the left occipital lobe. Cerebral angiography revealed dural AVF fed by three branches of the left occipital artery and another branch of the left ascending pharyngeal artery with retrograde drainage into the left transverse sinus, sigmoid sinus, and dilated cortical veins. The left transverse and sigmoid sinuses were occluded. Visual perseveration disappeared following the treatment of the dural AVF by transarterial embolization. Because the lesions on MRI and SPECT improved after the treatment, these lesions were considered to represent not infarction but vasogenic edema due to venous congestion. We emphasized the role of the left inferior occipital lesion including the secondary visual cortex (Brodmann's areas 18 and 19) as the cause of visual perseveration in the right superior quadrantic visual field.

[A case of neurofibromatosis type I associated with basal meningocele and abnormal vessels].

A 21-year-old man with neurofibromatosis type 1 (NF 1) had many widespread cutaneous neurofibroma on his right face. Magnetic resonance imaging (MRI) revealed basal meningocele due to dysplasia of the skull base. Carotid and vertebral angiograms revealed occlusion of the right internal carotid artery, persistent primitive trigeminal artery. We have reviewed the clinical and radiographic features of this case of neurofibromatosis, meningocele and cerebral arterial abnormalities. NF associated with both intracranial vascular malformation and meningocele is very rare, and in our case both were thought to arise congenitally as a manifestation of mesodermal dysplasia. Careful follow up using MRI and MR angiography should be performed for such patients.

Enhancing effects of sound on methamphetamine-induced behavioral aberrations in the rat: a model of relapse of schizophrenia-like symptoms.

Conditioning of methamphetamine (MAP)-induced behavioral aberrations, hyperactivity (HA), stereotyped (SB) and bizarre behavior (BB) in rats were examined using Pavlovian conditioning methods. A speaker-sound (SS) was used as the conditioning stimulus (CS) and MAP-induced behavior as the unconditioned stimulus (UCS). The rats belonging to the Sound Group were repeatedly administered with MAP or saline paired with SS in an observation room and those in the Soundless Group with MAP only in the housing room. After chronic administration with MAP, conditioned behavioral responses (CBR) such as HA, SB, and BB were observed more intensely in the Sound Group than in the Soundless Group while being subjected to SS. CBR were greatest on the 30th day after the last MAP administration and they did not disappear for 120 days. CBR were considered as analogous to the spontaneous relapse of schizophrenia-like symptoms. Furthermore, as a certain behavior which had been conditioned could easily be observed as CBR with both specific (SS) and nonspecific environmental cues (handling), it is difficult to distinguish the withdrawal behavioral effects of MAP that lacks those of CBR.