Administration of FGF-2 to embryonic mouse brain induces hydrocephalic brain morphology and aberrant differentiation of neurons in the postnatal cerebral cortex.

Fibroblast growth factor-2 (FGF-2) was injected into mouse cerebral ventricles at embryonic day (E) 14 in utero and its effects on developing brain morphology and expression of various cell- or differentiation-associated protein markers in the cerebral cortex were examined. High doses of FGF-2 (200 or 300 ng) caused encephalic alternations such as deformation of the calvarium, enlargement of the ventricular spaces, and thinning of the cerebral cortex. There was no gross abnormality in the alignment of the cerebral neuronal layers, however, both cell number and cell density of the upper layers (II/III) and the lower layers (IV-VI) of the cerebral cortex were increased. Brain-derived neurotrophic factor (BDNF), tyrosine hydroxylase, nestin, and microtubule-associated protein 2 were aberrantly or ectopically expressed in the deep areas of the cerebral cortex. A substantial number of these cells coexpressed these antigens. These observations demonstrate that a subpopulation of neurons in the cortical deep layer abnormally differentiated or partly sustained their immature state following a single administration of FGF-2 at E14. Developmental analysis of localization of BDNF-positive cells suggested that the abnormality started around P5. Furthermore, cell migration was not affected by FGF-2 administration. FGF-2 seems to play predominant roles in the proliferation of neuronal precursors and in neuronal differentiation in the developing mouse cerebral cortex even at relatively late stages of brain neurogenesis.

Effectiveness of interferon treatment for patients with chronic hepatitis C virus infection and normal aminotransferase levels.

To determine the effects of interferon treatment, we studied 77 Japanese patients with hepatitis C virus (HCV) infection and normal alanine aminotransferase (ALT). Of 77 patients, 37 were given natural interferon-alpha for 24 weeks, and 40 not given interferon acted as controls. Serum samples were tested for HCV RNA and genotypes by polymerase chain reaction (PCR). HCV RNA levels were measured by competitive PCR. Of 37 treated patients, 11 (29.7%) had sustained elimination throughout a six-month follow-up, while HCV RNA was not eliminated in any untreated patients. At 24 months, the number of patients with elevated ALT was not significantly different between treated (13.5%) and untreated patients (15%). Interferon eliminates HCV RNA in patients with normal ALT without severe side effects. The natural history of HCV infection should be clarified so that the interferon treatment regimen can be tailored to the needs of each patient.

Aberrant expression of neurotrophic factors in the ventricular progenitor cells of infant congenitally hydrocephalic rats.

OBJECTS: This study was conducted to investigate the roles of neurotrophic factors in the development of hydrocephalus in HTX rats. METHODS: Expressions of brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and fibroblast growth factor (FGF)-1 were examined immunohistochemically in the cerebral cortex and ventricular zone of 6-day-old rats with congenital hydrocephalus (HTX rats). In the ventricular zone of hydrocephalic rats, potent BDNF-like immunoreactivity (-LI) and weak but significant signals for NT-3- and FGF-1-LIs were observed. However, no significant signals were detected in non-HTX rats. A small subpopulation of ventricular cells was positive for microtubule-associated protein 2 in HTX and non-HTX rats. The positive cells in the HTX rats had neurites much longer than those in the non-HTX animals, suggesting that some ventricular cells of the hydrocephalics had ectopically differentiated into mature neurons. CONCLUSIONS: This abnormal differentiation may have been responsible for the aberrant expressions of neurotrophic factors. In contrast, the cerebral neuronal layers did not show such prominent alterations in neurotrophic factor expression.

Induction of a physiologically active brain-derived neurotrophic factor in the infant rat brain by peripheral administration of 4-methylcatechol.

Effects of 4-methylcatechol (4MC), a known potent stimulator of nerve growth factor (NGF) synthesis, on expression of brain-derived neurotrophic factor (BDNF) mRNA and BDNF-like immunoreactivity (BDNF-LI) was investigated in infant rat brains. A single intraperitoneal administration of 4MC caused transient increases in the levels of BDNF mRNA and BDNF-LI in neurons of the cerebral cortex from 1 to 3 h and 3 to 12 h, respectively, after the injection. Repetitive injections of 4MC to newborn rats (12-h intervals for 10 days) caused a marked and dose-dependent elevation of the level of BDNF mRNA in the whole brain besides elevating the number of cells containing calbindin D-28 and enhancing its immunoreactive intensity in the pyriform cortex and hippocampus. These findings demonstrate that 4MC stimulates de novo synthesis of BDNF in the infant rat brain, resulting in acceleration of the developmental expression of calbindin D-28.

4-methylcatechol increases brain-derived neurotrophic factor content and mRNA expression in cultured brain cells and in rat brain in vivo.

Practical use of brain-derived neurotrophic factor (BDNF) as therapy is limited by two serious problems, i.e., its inability to cross the blood-brain barrier and its instability in the bloodstream. In the present study, we investigated the effects of 4-methylcatechol (4-MC), which stimulates nerve growth factor synthesis and protects against peripheral neuropathies in rats, on BDNF content and mRNA expression in cultured brain cells and in vivo in the rat brain. 4-MC elevated BDNF content in culture media of both rat astrocytes and neurons with different dose-response relations. The increase in BDNF mRNA level was correlated with the increase in BDNF content, demonstrating that 4-MC can stimulate BDNF synthesis of both neurons and astrocytes. Then we examined the in vivo effects of 4-MC. First, we found that ventricularly administered 4-MC facilitated an increase in the BDNF content in the cerebral cortex and hippocampus in association with its diffusion into the brain parenchyma. Second, i.p. administration of 4-MC enhanced BDNF mRNA expression in the infant rat brain, in which the blood-brain has not yet fully been established. These results demonstrate that 4-MC, once delivered into the brain, can stimulate BDNF synthesis.

Brain-derived neurotrophic factor prevents neuronal cell death induced by corticosterone.

Corticosterone (CORT), one of the glucocorticoids, causes neuronal damage in the hippocampus, but the mechanism(s) of action underlying its effects remains unknown. Brain-derived neurotrophic factor (BDNF) is a neurotrophic factor that belongs to the neurotrophin family, affects the survival and/or differentiation of various types of neurons in vitro, and is able to antagonize neuronal death induced by various brain insults or neurotoxins in vivo. In this study, the effects of CORT on BDNF protein contents and mRNA expression were investigated in relation to neuronal survival/death of cultured rat hippocampal neurons, because the colocalization of BDNF with its receptor, TrkB, suggests that BDNF may exert its putative protective and trophic effects through an autocrine mechanism in the hippocampus. Administration of CORT accelerated the neuronal death that proceeds after serum deprivation, and simultaneously reduced the levels of BDNF mRNA and intracellular BDNF content. Exogenously added BDNF actually attenuated CORT-induced neuronal death, but not in the presence of K252a, an inhibitor of the tyrosine kinase activity of Trk family receptors. These observations suggest that CORT induces damage to hippocampal neurons, at least partly, via reducing their BDNF synthesis.

Endogenous neurotrophin-3 is retrogradely transported in the rat sciatic nerve.

To address the active transport of neurotrophins, nerve growth factor, brain-derived neurotrophic factor, and neurotrophin-3 in the peripheral nerves, we examined the levels of proteins and messenger RNAs in the sciatic nerve of adult rats following transection, using enzyme immunoassays and reverse transcription polymerase chain reaction method, respectively. Neurotrophin-3 protein increased one day after transection only in the distal segment next to the transection site and returned to the original level two days later. This was considered to reflect accumulation of neurotrophin-3 transported from the periphery toward the neuronal cell bodies, because the neurotrophin-3 messenger RNA level was not changed in any sciatic segments during this experimental period. An increase in brain-derived neurotrophic factor protein was observed simultaneously in both the distal and proximal stumps three days after transection. Brain-derived neurotrophic factor messenger RNA was elevated in the same stumps two days after transection, suggesting that brain-derived neurotrophic factor was produced within the transected stumps. These observations demonstrate that neurotrophin-3, like nerve growth factor, is retrogradely transported in the sciatic nerve but that brain-derived neurotrophic factor is not. This suggests that neurotrophin-3 plays a role in the conveyance of trophic signals from target organs to neurons.

Differences between interferon-alpha and -beta treatment for patients with chronic hepatitis C virus infection.

To compare virological, biochemical, and immune responses to human lymphoblastoid interferon (IFN-alpha) and human fibroblast interferon (IFN-beta) in patients with chronic hepatitis C virus (HCV) infection, 120 patients were randomly assigned to three groups (group A, 60 patients receiving IFN-alpha, 6 million units (MU) once a day, daily for one month and thrice weekly for five months; group B, 40 patients receiving 6 MU IFN-beta once a day daily for two months; and group C, 20 patients receiving 3 MU IFN-beta twice a day (6 MU/day) daily for two months). Serum soluble interleukin-2 receptor (sIL-2R) and interleukin-6 (IL-6) levels were measured by enzyme-linked immunosorbent assay. Patients with sustained clearance of serum HCV RNA detected by polymerase chain reaction (PCR) at six months after IFN treatment were defined as having complete response to IFN treatment. A low level of HCV RNA (< or = 10(4) copies/50 microl, measured by competitive PCR) and HCV RNA of genotype 2a were favorable factors for a complete response to both IFNs. Complete response in group A treatment was strongly associated with early HCV RNA clearance, in contrast with group B. A significantly higher HCV RNA negativity at the second week from start of treatment was noted in group C (80.0%), compared with groups A (41.6%) and B (27.5%). sIL-2R levels rose in each group during IFN administration. In group C, alanine aminotransferase (ALT) and IL-6 levels were remarkably elevated. These findings indicate that timing of serum HCV RNA negativity in sustained response differs between IFN-alpha and IFN-beta administrations and that early HCV RNA clearance was induced by twice-a-day IFN-beta treatment.

Elevation of serum soluble interleukin-2 receptor levels in patients with hepatitis C virus infection.

To determine serum soluble interleukin-2 receptor (sIL-2 R) levels in hepatitis C virus (HCV) infection, serum sIL-2 R was measured in 260 subjects with chronic HCV infection, including 100 patients who had previously been treated with natural interferon (IFN) alpha, and in 51 HCV RNA-negative controls. Serum sIL-2 R levels in asymptomatic HCV carriers, patients with chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (HCC) were significantly higher than those of healthy controls and subjects who were positive for anti-HCV and negative for HCV RNA (P < 0.01, respectively). Moreover, serum sIL-2 R levels were also significantly higher in patients with HCC than in other HCV RNA-positive groups. There was some correlation between serum sIL-2 R levels and histological activity index scores (r = 0.287, P < 0.01) and serum alanine aminotransferase levels (r = 0.272, P < 0.01). In patients in whom HCV RNA was eliminated following IFN treatment, serum sIL-2 R levels decreased to those seen in healthy controls by one year post treatment. Serum sIL-2 R levels increase due to HCV infection, and the amount of increase corresponds to the degree of inflammation.

Large dose natural interferon alpha therapy for patients with chronic hepatitis C.

To evaluate the efficacy of large dose interferon treatment for patients with chronic hepatitis C virus (HCV) infection, we studied 99 Japanese patients treated with either 6 million units (MU) or 9 MU natural interferon alpha. Serum samples were tested for HCV RNA by polymerase chain reaction (PCR). HCV RNA genotypes were determined by PCR with type-specific preimers, and the HCV RNA level was measured by competitive PCR. HCV RNA was detected in all patients, prior to the initiation of treatment. We examined interleukin-1 receptor antagonist (IL-1 Ra) by enzyme-linked immunosorbent assay. Forty-four patients were treated with 9 MU natural interferon alpha for 24 weeks (group A), and fifty-five patients were treated with 6 MU natural interferon alpha for 24 weeks (group B). There were no significant differences in HCV RNA levels, HCV RNA genotype or histological activity index (HAI) score between the two groups. Of the 94 patients who completed this treatment, nine (23.1%) in group A and 14 (25.5%) in group B sustained elimination of HCV RNA throughout a 6-month follow-up. There were no differences in the rate of complete response when comparing HCV RNA genotype, levels and HAI score and no significant differences in elevation of IL-1 Ra levels between the two groups. Five of group A patients refused further treatment because of severe side effects such as retinal hemorrhage, while no patient in group B had severe side effects. Thus, large dose natural interferon alpha treatment confers no additional benefit to the patient, compared with the current use of a lower dose.

Long term survey of hepatitis C virus infection in hemodialysis units in Fukuoka, Japan.

To examine prevalence of hepatitis C virus (HCV) infection and liver dysfunction in hemodialysis units, we surveyed markers for HCV infection and serum alanine aminotransferase (ALT) in hemodialysis patients. 204 hemodialysis patients (111 men and 93 women; mean age, 53 +/- 12 years) in four hemodialysis units in Fukuoka, Japan were investigated. All serum samples were tested for antibody to HCV (anti-HCV) by second-generation enzyme-linked immunosorbant assay (ELISA). HCV RNA was detected to identify present HCV infection in the anti-HCV-positive patients by polymerase chain reaction (PCR) using primers deduced from the 5'-noncoding region. Liver dysfunction was defined as an elevated concentration of serum ALT (above 36 IU/liter) tested by a multiple autoanalyser. 105 patients (51.5 percent) were initially positive for anti-HCV, 95 (90.5 percent) of whom were also positive for HCV RNA. Ten became positive for anti-HCV in hemodialysis units during the observation, eight (80 percent) of whom had sustained HCV viremia. The route of transmission of HCV was not clear, but two of these patients had received blood transfusions. Of 95 patients with HCV viremia, 43 (45.3 percent) had had liver dysfunction at least once. In conclusion, HCV infection continues to occur in hemodialysis units not through blood transfusion and many of them become HCV carriers. Liver dysfunction was found in about a half of HCV-infected hemodialysis patients during the observation.

Serum levels of soluble interleukin-2 receptors and effects of interferon-alpha for patients with chronic hepatitis C virus.

To characterize the role of serum soluble interleukin-2 receptor (sIL-2R) in hepatitis C virus (HCV) infection, the level of sIL-2R was measured by ELISA in 117 subjects with chronic HCV infection and in 23 healthy controls. HCV RNA was detected by polymerase chain reaction in all subjects with HCV infection. Forty-seven patients with chronic hepatitis and 10 with liver cirrhosis were treated for six months with natural interferon-alpha. The sIL-2R levels of 40 asymptomatic HCV carriers (632 +/- 340 units/ml), 47 patients with chronic hepatitis (547 +/- 204 units/ml), 10 with cirrhosis (679 +/- 239 units/ml, and 20 with hepatocellular carcinoma (1145 +/- 487 units/ml) were significantly higher than those of healthy controls (380 +/- 191 units/ml) (P < 0.05, respectively). The levels of sIL-2R increased, as did the histological activity index scores (r = 0.348, P < 0.01). The level of sIL-2R rose after the initial administration of interferon in all 57 patients. In patients whom HCV RNA was eliminated from the sera within a six-month follow-up after cessation of treatment, the level of sIL-2R reverted to basal values, but in patients in whom HCV RNA was not eliminated the value was significantly higher than that before treatment. These results suggest that monitoring serum sIL-2R in patients with chronic HCV infection treated with interferon may provide information concerning the possibility of the elimination of HCV RNA.

A preliminary study of retreatment of chronic hepatitis C with interferon.

Nine patients with chronic hepatitis C virus (HCV) infection and no complete response to the first treatment with natural interferon (IFN)-alpha, were prescribed a second treatment with IFN. Five patients (Group A) with unsustained levels serum alanine aminotransferase (ALT) after the first treatment were administrated the same species of IFN but in a higher dose. The remaining four patients (Group B), with no normalization of ALT throughout the observation period of the first treatment, were administrated IFN-beta. HCV RNA was eliminated in three patients of group A and in two of group B patients during 6 months follow up and ALT reverted to normal levels. These results suggest that retreatment with a higher dose of the same species of IFN-alpha can be effective in case of a relapse and that IFN-beta can be effective for those not responding to IFN-alpha.

A statistical analysis of predictive factors of response to human lymphoblastoid interferon in patients with chronic hepatitis C.

OBJECTIVE: To determine markers predictive of effective interferon treatment for patients with chronic hepatitis C virus (HCV) infection, we studied 80 Japanese patients treated for 6 months with natural interferon-alpha. METHODS: Serum samples were tested for HCV RNA by two-stage polymerase chain reaction (PCR). HCV RNA were grouped into four genotypes by amplification of core-gene sequences by PCR with type-specific primers, and the level of HCV RNA was measured by competitive PCR. HCV RNA was detected in all patients before the interferon treatment. For the purposes of this study, a complete response was defined as the elimination of HCV RNA for at least 6 months after termination of the treatment. RESULTS: HCV RNA was eliminated from the sera of 27 patients (33.8%) at the termination of the interferon treatment, and the elimination was sustained throughout a 6-month follow-up (complete response). Four of eleven variables proved to be associated with a complete response when assessed by univariate analysis. With multiple logistic regression analysis assessment, however, only two variables (HCV RNA level (p < 0.001) and genotype (p = 0.048)) were significant. CONCLUSION: These results suggest that factors associated with the HCV infection are more important than patient characteristics for effective interferon treatment of patients with chronic HCV infection and that a low level of HCV RNA and HCV of genotype III are useful predictors of a complete response.

Inverse correlation between the titre of antibody to hepatitis C virus and the degree of hepatitis C viraemia.

We titrated 277 hepatitis C virus (HCV) antibody-positive serum samples from 235 volunteer blood donors as well as from 42 outpatients of a hospital for elderly people and studied the relation of the titre of HCV antibody to the presence of HCV RNA, of antibody to C100 protein (anti-c100) and of antibody to GOR epitope (anti-GOR). Liver dysfunction was measured also. Of 177 HCV RNA-positive serum samples, 87 were tested for the degree of HCV viraemia by means of a competitive assay. Among the 277 samples, prevalences of HCV RNA, anti-c100, anti-GOR and liver dysfunction were 63.9%, 71.8%, 75.7% and 17.5%, respectively. The prevalence of HCV RNA became higher as the titre of HCV antibody increased. The titre tended to increase with age but the tendency was not statistically significant. The mean titre was higher in females (2(10.4 +/- 1.8)) than in males (2(9.4 +/- 2.2)) (P < 0.01). In the HCV RNA-positive serum samples, the HCV antibody titre was significantly higher in the anti-c100-positive samples than in the negative ones. This difference between the positive and negative samples, however, was not statistically significant for anti-GOR and liver dysfunction. Low degrees of HCV viraemia were accompanied by high titres of HCV antibody while high degrees of HCV viraemia went with low titres of HCV antibody. The study revealed that titres of HCV antibody were higher in females and the degree of HCV viraemia correlated inversely with the titre of HCV antibody.

Prevalence and role of hepatitis C viraemia in haemodialysis patients in Japan.

A second generation assay for antibody to hepatitis C virus (anti-HCV) was used in order to establish the exact prevalence of HCV infection in haemodialysis patients. HCV RNA was sought by the polymerase chain reaction in order to determine whether haemodialysis patients with anti-HCV had been infected with HCV in the past or were presently infected. Of 357 patients, 198 (55.5%) were positive for anti-HCV, compared to 113 (31.7%) positive for original antibody to c100-3 protein (P < 0.001). HCV RNA was detected in 171 (86.4%) of the 198 patients with anti-HCV. Liver dysfunction was found in 63 (17.6%) of all haemodialysis patients. Of these, 55 (87.3%) had HCV infection, one (1.6%) hepatitis B virus infection while, in the remaining seven, the origin was unknown. Thus, in almost all anti-HCV-positive patients, HCV viraemia was present. We conclude that HCV is an important cause of liver disease in haemodialysis patients.

Improved detection of antibodies to hepatitis C virus by the second-generation assay in patients with chronic non-A, non-B liver disease.

Serum samples from 337 Japanese patients with chronic non-A, non-B liver disease were tested for antibodies to hepatitis C virus (HCV) by means of first-generation (c100-3; anti-c100) and second-generation (pHCV-34, pHCV-31, c100-3; anti-HCV II) enzyme linked immunosorbent assays (ELISA) and for antibody to the GOR epitope (anti-GOR) also by ELISA. Anti-HCV II was detected in 314 (93.2%), anti-c100 in 247 (81.3%) and anti-GOR in 211 (62.6%) samples. Thus, anti-HCV II was more sensitive in detecting HCV infection than either anti-c100 or anti-GOR (P < 0.001). All serum samples reactive with anti-c100 or anti-GOR reacted with anti-HCV II. Among 314 anti-HCV II-positive patients, we found that 185 (58.9%) were positive for both anti-c100 and anti-GOR while 14 (4.5%) were positive for anti-HCV II alone. Nine (64.3%) of the 14 are presently infected with HCV, as revealed by detection of HCV RNA in their serum; the remaining five may have been infected in the past with HCV. These findings indicate that HCV is a major causative agent of chronic non-A, non-B liver disease in Japan and that detection of anti-HCV II is a specific and more sensitive diagnostic test for HCV infection.

Amino acid sequence of the major component of Nostoc muscorum ferredoxin.

The amino acid sequence of the major component of ferredoxin isolated from a blue-green alga, Nostoc muscorum, grown under N2 as the sole nitrogen source has been studied. The use of a combination of sequence analyzer, carboxypeptidases, and manual Edman degradations on tryptic and chymotryptic peptides of carboxymethylferredoxin has established the amino acid seuqence, which consists of 98 amino acid residues. Only four cysteine residues were present, located at positions 41, 46, 49, and 79. These residues must fulfil the minimum requirement in this ferredoxin for the chelation of two iron atoms, as postulated previously. The sequence is similar to those of Spirulina ferredoxins in having two extra residues at positions 10 and 14 compared with other chloroplast-type ferredoxins. Sequence comparison among blue-green algal ferredoxins suggests that Nostoc muscorum ferredoxin is more closely related to Spirulina ferredoxins than to Aphanothece major ferredoxin.