Immunohistochemical assessment and prognostic value of hepatitis B virus X protein in chronic hepatitis and primary hepatocellular carcinomas using anti-HBxAg monoclonal antibody.

Hepatitis B virus (HBV) is the most meaningful risk factor in chronic hepatitis, cirrhosis and primary hepatocellular carcinoma (PHC). The hepatitis B virus X protein (HBxAg) is a multifunctional protein with many important functions in hepatocellular carcinogenesis. A monoclonal anti-HBxAg antibody was developed in our laboratory and characterized by different methods. Using this antibody HBxAg was detected in formaldehyde fixed paraffin embedded tissue sections of 72 liver biopsies from patients with acute hepatitis, chronic hepatitis, cirrhosis and primary hepatocellular carcinoma. The co-expression of hepatitis B surface antigen (HBsAg), hepatitis B core antigen (HBcAg) and HBxAg was compared. The histological and cytological localization of the detected HBxAg showed a characteristic distribution in different stages of HBV infection. Strong and diffuse nuclear reaction was detected in PHC cases in contrast to the focal, cytoplasmic and nuclear labeling in the acute and chronic B hepatitis cases. Our antibody seems to be a suitable prognostic marker for routine pathohistological diagnosis and for comparative pathological and epidemiological research on the development of PHC.

Metabolism of carnitine in phenylacetic acid-treated rats and in patients with phenylketonuria.

The effect of metabolites accumulating in phenylketonuria (PKU) was investigated on carnitine metabolism in rats and in patients with PKU. Of phenylacetic acid (PEAA), phenylpyruvic acid and homogentisic acid the PEAA was found to be the most effective in inhibiting carnitine biosynthesis in rats. Following 60 min, a single intraperitoneal dose of PEAA the relative conversion rate, i. e. the hydroxylation, of tracer [Me-(3)H]butyrobetaine to [Me-(3)H]carnitine decreased from 62.2+/-6.00% to 39.4+/-5.11% (means+/-S.E.M., P<0.01) in the liver, in the only organ doing this conversion in rats. The conversion of loading amount of unlabeled butyrobetaine to carnitine was also markedly reduced. The impaired hydroxylation of butyrobetaine was reflected by a reduced free and total carnitine levels in the liver and a reduced total carnitine concentration in the plasma. PEAA decreased the hepatic level of glutamic acid and alpha-ketoglutaric acid (alpha-KG), suggesting a mechanism for the reduced flux through the butyrobetaine hydroxylase enzyme, because alpha-KG is an obligatory co-enzyme. In the plasma and urine of PKU patients on unrestricted diet, markedly decreased total carnitine levels were detected. In the liver of PEAA-treated rats and urine of PKU patients, a novel carnitine derivative, phenacetyl-carnitine was verified by HPLC and gas chromatography-mass spectrometry.

An alternative purification protocol for producing hepatitis B virus X antigen on a preparative scale in Escherichia coli.

A truncated variant of the hepatitis B virus X gene (HBx) was cloned into the fusion expression vector of pGEX-3X (Pharmacia), resulting in a GST-HBx fusion gene construction (pGEX-3XXBF). This plasmid was transformed into and expressed by the Escherichia coli strain DH5. More than 80% of the expressed fusion protein was found in the insoluble fraction (inclusion body) of the cell lysate. The fusion protein was selectively extracted from the inclusion bodies with 8 M urea at pH 6.5, and it was refolded by diluting 3-fold with deionized distilled water at 4 degrees C. The in vitro cleavage of the refolded fusion protein by factor Xa at about 2-3 mg ml-1 in the presence of 2.66 M urea at pH 6.5 was complete. The final steps of purification involved precipitation of the cleaved proteins with ammonium sulphate, solubilization in guanidine hydrochloride and separation on a Superdex 75 FPLC column. With this approach, following an inclusion body strategy and a beneficial in vitro refolding, a predominantly hydrophobic and highly disulphide-bonded protein was produced in preparative scale for subsequent diagnostic use.

A longitudinal study of phenylketonuria based on the data of the Budapest Screening Center.

Between 1975 and 1994 about 1.5 million neonates were screened by Guthrie tests at the phenylketonuria (PKU) Centre in Budapest. In this period 160 children with PKU were found. The corresponding incidence rate is about 1:9000. In a cumulative longitudinal design we investigated 56 patients with classical PKU between the ages of 3 months and 20.5 years. Treatment started at a mean age of 16.9 days (SD = 8.8). On average, pretreatment phenylalanine (Phe) levels were 1956 mumol/l (SD = 864), measured with the fluorometric method. Their Phe levels, physical growth, intellectual development, and performances in achievement tests were analysed. For the entire sample 45% of half year median Phe levels were in the recommended range, 33% represented poor dietary control, 22% were classified as intermediate. Physical growth was not significantly different from the Hungarian average. Mean verbal as well as nonverbal IQs were in the middle of the normal range. For an extended sample of 131 patients not followed longitudinally, mean recent IQ was 99.6 (SD = 16.3). Data concerning educational career were available for 107 patients. Normal schools were attended by 88 patients, 19 were in need of special elementary education. From 40 patients who had already finished elementary school, 17 underwent secondary school education, 1 is a university student, 15 are skilled workers, 4 were semi-skilled, and 4 are unemployed for reasons unrelated to their intellectual status.

Acidic fibroblast growth factor (FGF) but not basic FGF induces sleep and fever in rabbits.

Acidic fibroblast growth factor (FGF) and basic FGF belong to a growth factor family. Interleukin-1, another member of that family, is involved in sleep regulation. FGFs and interleukin-1 share structural and functional features. We therefore determined whether acidic FGF and basic FGF were somnogenic. Male New Zealand White rabbits were provided with electroencephalographic (EEG) electrodes, a brain thermistor, and a lateral intracerebroventricular (icv) cannula. The animals were injected icv with isotonic NaCl (control) and on separate days with one of three doses of acidic or basic FGF (0.01, 0.1, or 1.0 micrograms) or with heat-treated acidic FGF (1.0 micrograms). The EEG, brain temperature, and motor activity were recorded for 23 h. The biological activity of basic FGF was determined in vitro by its ability to induce DNA synthesis in rat aortic smooth muscle cells. Acidic FGF induced prolonged dose-related increases in non-rapid eye movement sleep beginning in the 1st postinjection h and continuing for 12-23 h after the treatment. Acidic FGF also induced fevers of approximately 1 degree C after the 1.0 micrograms dose. Both activities of acidic FGF were lost after heat treatment. In contrast, basic FGF lacked somnogenic and pyrogenic activity, although it did induce DNA synthesis. Current results suggest that acidic FGF is part of the complex cytokine network in brain involved in sleep regulation.

Experience based on 800,000 newborn screening tests of the Budapest Phenylketonuria Centre.

800 000 newborns were screened for hyperphenylalaninaemia by the Guthrie-test in the Budapest PKU Centre in the 10 and a half years since 1 May, 1973. The blood samples were taken from mature newborns on the fifth and from premature babies on the fourteenth day of life. All infants exhibiting a level equal to or exceeding 12 mg/dl were telegraphically invited to the Centre and those having a level of 15 mg/dl or higher were put on an appropriate diet. The patients were classified according to the result of the phenylalanine tolerance 73 were found to have classical phenylketonuria and 15 had atypical phenylketonuria. The total incidence of phenylketonuria was thus 1: 9091. The mean age at introduction of diet was 30 +/- 15 days during the first period, while 21 +/- 11 days during the second period. Infants having an initial value of 4-12 mg/dl were kept under continuous control; among them 69 were found to have benign hyperphenylalaninaemia (HPA). The PKU/HPA ratio amounted to 1.28. Both screening and care were carried out by the Centre, and the practice of care is described in detail. A preliminary evaluation of the therapeutical results with a view of the patients' social class is offered. Phenylalanine levels during the diet were greatly influenced by the familial background and the sociocultural environment.