Preconceptional oral vaccination prevents experimental biliary atresia in newborn mice.

INTRODUCTION: Biliary atresia (BA) in humans resembles BA induced in Balb/c-mice by Rhesus Rotavirus (RRV). In mice, susceptibility to BA is ascribed to the lack of maternally derived immune protection. This study investigated whether vaccination of dams against RRV protected their offspring from developing BA. MATERIALS AND METHODS: Before mating, female mice were vaccinated orally with RotaTeq or Rotarix. Pups (n=243) from both test groups and a control group were intraperitoneally infected with RRV. Sacrifice of the animals was scheduled for days 7, 14 and 21 after infection. Then, gross and mircoscopia findings of the liver and the hepatoduodenal ligament gave evidence of BA, and hepatic viral load was tested by virus-specific real-time PCR, as well as plaque forming units. RESULTS: Two weeks after infection, the incidence of cholestasis was 100% in controls, 77% in pups of RotaTeq-vaccinated dams, and 56% in pups of Rotarix-vaccinated dams. However, in contrast to controls (incidence of BA: 82%) most pups in the test groups recovered (incidence of BA in pups of RotaTeq-vaccinated dams 11%; incidence of BA in pups of Rotarix-vaccinated dams 3%). Hepatic viral load was identical at various time-points in all pups, suggesting that differences in RRV clearance did not underlie this effect. CONCLUSION: In this mouse model, oral vaccination with RotaTeq and Rotarix prevented most RRV-induced BA. This provides a new approach to a better understanding of both the pathomechanism of BA development and the capabilities of the innate immune system. It also suggests a first approach for prophylaxis against BA.

In situ expression of the costimulatory molecules CD80 and CD86 on langerhans cells and inflammatory dendritic epidermal cells (IDEC) in atopic dermatitis.

The functional expression of costimulatory molecules on antigen-presenting cells may be a key event in the pathogenesis of atopic dermatitis (AD). Recently, the expression of CD86 (B7-2/B70) has been demonstrated on CD1a+ epidermal dendritic cells (DC) in AD lesions by immunohistological and functional analysis. Therefore, we sought to further characterize the in situ expression of costimulatory molecules on these cells, considering the two subpopulations of (1) CD1a+++/CD11b- Langerhans cells (LC) containing Birbeck granules and (2) CD1a+/CD11b+++ inflammatory dendritic epidermal cells (IDEC), devoid of Birbeck granules, from AD and other inflammatory skin diseases. Flow cytometry, skin mixed lymphocyte reactions (SMLR) and immunohistological analysis were performed, and showed that IDEC and not LC are the relevant cells expressing the costimulatory molecules CD80 and CD86 in situ. This expression varied with the underlying diagnosis, with AD showing the highest expression of both CD80 and CD86 in situ. Furthermore, the expression of CD80, CD86 and CD36 were significantly correlated. With short-term culture, both CD80 and CD86 were further upregulated on LC and IDEC. Finally, anti-CD86 antibody reduced the stimulatory activity of epidermal DC. These results indicate that costimulatory molecules on LC and IDEC might play a role in the pathogenesis of AD.

Hepatocyte growth-factor-scatter factor can stimulate post-operative tumor-cell proliferation in childhood hepatoblastoma.

Rapid growth of residual tumor after partial hepatectomy has been observed during the period of liver regeneration in children with malignant embryonal hepatoblastoma. The aim of this study was to elucidate the role of hepatocyte growth-factor-scatter factor (HGF-SF) in this phenomenon. Markedly increased serum levels of HGF-SF up to 15 ng/ml were found in 13/18 patients after liver resection and in 6/16 patients with regressive tumors after chemotherapy, in comparison with 15 patients with non-pre-treated hepatoblastoma and 20 healthy children of the same age group. In the tumors, epithelial tumor cells highly expressed the HGF-SF receptor c-met, as shown by immunohistochemistry and m-RNA RT-PCR. The hepatoblastoma cell lines HepT1, HepT3 and HUH6 reacted with significantly increased proliferation to rhHGF-SF in these concentrations (1-15 ng/ml). In the tumors, HGF-SF was found to be expressed in the stromal fibroblasts. In culture, hepatoblastoma cells (HepT3, HUH6) stimulated secretion of the factor by human fibroblasts, indicating the paracrine fashion of intratumoral HGF-SF production. Cultured hepatoblastoma cells ceased to proliferate at 20-50 ng/ml HGF-SF, and they underwent cell death at >/=100 ng/ml. In contrast, the hepatocellular-carcinoma cell line HepG2 decreased growth under HGF-SF in a dose-dependent manner. We conclude that post-operatively secreted and intratumorally produced HGF-SF can function as a growth factor for hepatoblastoma, while the same agent has a cytostatic effect in unphysiologically high concentrations.

Recombinant AAV-2 harboring gfp-antisense/ribozyme fusion sequences monitor transduction, gene expression, and show anti-HIV-1 efficacy.

Vector-mediated delivery of potentially antivirally active genes is a key step in somatic gene therapy including therapeutic approaches against AIDS. A crucial technical prerequisite is to monitor DNA transfer into target cells. Here, we describe recombinant infectious particles derived from the adeno-associated virus type 2 (AAV-2) that are suitable to deliver effective HIV-1-directed antisense and ribozyme genes into target cells. To monitor transduction, we designed and tested a number of fusions between indicator-coding sequences of luciferase or gfp with effective HIV-1-directed antisense or ribozyme sequences. The combination of an indicator function and an antiviral func- tion in cis allows successful identification of transduced cells and measurement of effects on the replication of HIV-1 in antisense/ribozyme-expressing cells only. The fusion genes were shown to express the indicator genes. Inhibition of HIV-1 replication mediated by the antisense/ribozyme portion of the fusion transcripts was similar to parental constructs and neither acute nor long-term toxicity of fusion genes and their gene products was observed. These results suggest the use of rAAV constructs described here as tools to study the transducibility of target cells, gene expression and efficacy of HIV-1-directed antisense and ribozyme genes.

Postnatal development of urea synthesis capacity in preterm infants with intrauterine growth retardation.

The postnatal development of the urea-synthesizing capacity was studied in 21 preterm infants with intrauterine growth retardation (IUGR) and compared with results found in 12 infants without IUGR as controls. The urea-synthesizing capacity was estimated by the ratio Q of 15N abundance of ammonia and urea in 6-hour urine samples collected after enteral administration of 3 mg [15N]H4Cl/kg body weight. The measurements were performed on the first day when a protein intake of 3.0-3.5 g/kg/day and an energy intake of 120 kcal/kg/day were tolerated (study day 1: postnatal 14-21 days) and on the day of discharge from the hospital (study day 2: postnatal age 39-56 days). The group of infants with IUGR was subdivided in one group of infants who developed catch-up growth (n = 12) and one group who did not demonstrate catch-up growth (n = 9). On study day 1, the Q values of the IUGR infants without catch-up growth were significantly higher than those of the IUGR infants with catch-up growth (13.4 +/- 2.3 vs. 9.2 +/- 2.2) or of the control infants without IUGR. During the time period from study day 1 to study day 2 the Q values of the IUGR infants with catch-up growth decreased significantly (9.2 +/- 2.2 vs. 4.8 +/- 2.0; p < 0.001) and were in the range of the control infants without IUGR. In contrast, the Q values of the IUGR infants without catch-up growth did not significantly change during the study period (13.4 +/- 2.3 vs. 11.3 +/- 2.8; p = 0.097). On both study days there was a significant correlation between the Q values and the degree of IUGR (study day 1: r = 0.652, p < 0.01; study day 2: r = 0.842, p < 0.001). The data indicate that the urea-synthesizing capacity of preterm infants increases during early postnatal life and that severe IUGR may impair this development. Metabolic investigations using urea as marker for evaluation of optimal quantity or quality of dietary proteins should carefully be interpreted when infants with severe IUGR are studied.

Clinical relevance of point mutations in the cytoplasmic domain of the granulocyte colony-stimulating factor receptor gene in patients with severe congenital neutropenia.

Recently, point mutations in the gene of the granulocyte colony-stimulating factor (G-CSF) receptor have been reported in two patients with severe congenital neutropenia who developed acute myeloid leukemia (AML). We investigated the frequency of these specific G-CSF receptor mutations in patients with congenital neutropenia undergoing treatment with r-metHuG-CSF (Filgrastim) and the clinical relevance of these mutations. Nucleotides 2306 to 2561 including the critical region (nucleotides 2384-2429) from the intracellular domain of the G-CSF receptor gene were amplified by reverse transcriptase-polymerase chain reaction. Detection of point mutations was performed with specific restriction enzyme analysis, as well as sequencing of PCR products. Both genomic DNA and cDNA from neutrophils and mononuclear cells were analyzed from 28 patients with severe congenital neutropenia. Four of 28 patients with congenital neutropenia displayed a point mutation in the tested cytoplasmic region of the G-CSF receptor gene. The point mutations replace a glutamine codon by a stop codon of the G-CSF receptor gene. Among these four congenital neutropenia patients with a mutated G-CSF receptor, two developed AML. All four patients were investigated regularly and no correlation between occurrence of G-CSF receptor mutation and time or dose of r-metHuG-CSF treatment was found. No point mutations in the G-CSF receptor critical domain could be detected in cells from the other 24 congenital neutropenia patients. Furthermore, we tested six family members of the two patients with AML including mothers and fathers, one sister, and one brother who suffers from congenital neutropenia, as well. All family members displayed a normal G-CSF receptor gene. After the acquisition of the G-CSF receptor mutations, the congenital neutropenia patients continued to respond to G-CSF therapy with an increase in absolute neutrophils in the peripheral blood. We conclude that the point mutations in the critical region of the intracellular part of the G-CSF receptor occur spontaneously and are not inherited. From our data, we suggest that the described G-CSF receptor point mutations do not alter the response to treatment with r-metHuG-CSF and are not the cause of severe congenital neutropenia.

Hepatic biotransformation capacity in low-birth-weight infants as measured with the [15N]methacetin urine test: influences of gestational age, postnatal age, and intrauterine growth retardation.

The influences of the gestational age (range: 28-36 weeks) and the postnatal age (range: 6-100 days) on the biotransformation capacity of the liver were studied in 51 preterm appropriate-for-gestational-age infants and in 20 preterm small-for-gestational-age infants using the [15N]methacetin urine test. Methacetin is a test drug assessing a two-step pathway of biotransformation including monooxygenation and conjugation. After oral administration of 3 mg [15N]methacetin/kg body-weight, the cumulative 15N excretion in urine during the consecutive 9 h was measured and used as a marker of microsomal biotransformation capacity. In preterm appropriate-for-gestational-age infants, the biotransformation capacity increases with gestational age as well as with postnatal age, but the strongest correlation could be found between cumulative [15N] excretion and postmenstrual age. Intrauterine growth retardation results in lower biotransformation capacity (26.3 +/- 11.3 vs. 36.1 +/- 9.6% [15N] excretion, expressed as percentage of intake) and disturbed postnatal development of this hepatic function. The data indicate that normal intrauterine development is a prerequisite for normal postnatal development of the biotransformation capacity, which might have consequences for the metabolism and efficacy of certain drugs in small-for-gestational-age infants.

Dexamethasone inhibits tumor necrosis factor-induced granulocyte colony-stimulating factor production in human endothelial cells.

Dexamethasone (10(-5)-10(-7) M) is able to suppress the tumor necrosis factor (TNF)-induced production of granulocyte colony-stimulating factor (G-CSF) in human umbilical vein endothelial cells (HUVEC). Using Western-blot analysis and bioassay for the evaluation of G-CSF protein and activity, a significant decrease in TNF-induced production could be found in cells cultured in the presence of dexamethasone as compared to TNF stimulation in the absence of dexamethasone. No inhibition by dexamethasone was seen in endothelial cells stimulated with interleukin 1 beta (IL-1 beta; 10 U/ml). Addition of IL-1 to cultures stimulated with TNF in the presence of dexamethasone could overcome the inhibitory effects of corticosteroids. Suppression of G-CSF production can, at least in part, explain the functional abnormalities of granulocytes found in patients treated with glucocorticosteroids.

Effects of two antibiotics on hepatic function in low birth weight infants: ampicillin vs. cefotaxime.

In 21 low birth weight infants with two regimens of antibiotic therapy during the first 3 days of life possible hepatotoxic side effects were studied 8 days after the last administration of the tested drugs. Fourteen of the infants were treated with ampicillin/gentamicin and 7 received cefotaxime/gentamicin. The serum concentrations of total bile acids, the activities of transaminases in serum and the cumulative 15N excretion in urine after administration of 3 mg of 15N-labeled methacetin/kg of body weight were used as markers of hepatotoxic side effects. Neither the concentrations of total bile acids (22.6 +/- 12.1 and 19.4 +/- 10.8 mM, respectively) nor the activities of transaminases (alanine aminotransferase, 0.27 +/- 0.06 vs. 0.30 +/- 0.09 mumol/second/liter; aspartate aminotransferase, 0.46 +/- 0.11 vs. 0.49 +/- 0.10 mumol/second/liter) were different between the two groups. In contrast the cumulative 15N excretion in urine was significantly lower in the group treated with cefotaxime/gentamicin than in the group treated with ampicillin/gentamicin (17.2 +/- 6.4 vs. 33.0 +/- 5.1% of intake; P less than 0.01) and also lower than the reported age-related reference values. On the 28th day of life no differences could be found between the cumulative 15N excretion in the urine of the infants treated with cefotaxime/gentamicin and the reported age-related reference values of this test. The results indicate a limited capacity of the monooxygenase system of the liver of low birth weight infants during the first weeks of life and a specific reversible influence of cefotaxime on this hepatocellular system. Further investigations are required to evaluate the clinical relevance of this drug-specific inhibition of the hepatic monooxygenase pathway.

Development of urea-synthesizing capacity in preterm infants during the first weeks of life.

The urea-synthesizing capacity of the liver was studied in 20 healthy preterm infants during the first month of life. The urea-synthesizing capacity was estimated by the ratio of 15N abundances of ammonia and urea in the 6-hour urine after administration of 3 mg 15N-labelled ammonium chloride/kg body weight. The ratio increases with increasing protein intakes from the 2nd to the 3rd week of life. On protein intakes of more than 3 g/kg/protein day from the 3rd week to the end of the 2nd month of life, the ratio decreases suggesting a maturation of the urea cycle during the first weeks of life.

[15N]methacetin urine test: a method to study the development of hepatic detoxification capacity.

The [15N]methacetin urine test was used to study human O-demethylase activities to characterize the maturation of hepatic detoxification capacity. The study involved 43 healthy subjects aged 1 day-47 years. The urinary 15N elimination rates were measured following oral administrations of an aqueous [15N]methacetin solution. Age-dependent normal values of hepatic drug elimination capacity were established. Parameters were the 15N elimination half-life and cumulative elimination of the 15N dose as a percentage over a 9 h period. The maximum elimination rate (% dose/h) and peak time can give additional information. The 15N method is a simple, non-invasive and non-radioactive liver function test avoiding disadvantages of 14C and 13C breath tests. The [15N]methacetin test is suitable and useful in studying the hepatic development at birth and pathological changes of the microsomal detoxification capacity in early childhood.

Influence of intrauterine growth retardation on parameters of liver function in low birth weight infants.

To establish nutritional management of low birth-weight infants according to their individual metabolic situation, hepatocellular partial function was studied in 13 appropriate (AGA) and 11 small-for-gestational-age (SGA) low birthweight (LBW) infants during the first weeks of postnatal life. The concentrations of total bile acids and of alpha-amino-nitrogen in serum, the renal excretion of urea and ammonia and the renal excretion of 15N after enteral administration of 3 mg 15N-labeled methacetin/kg were measured. In comparison to AGA infants, SGA infants had elevated serum concentrations of total bile acids and of alpha-amino-nitrogen, decreased excretion of urea, increased excretion of ammonia in urine, and lower urinary 15N-excretion after enteral administration of 15N-labeled methacetin. The data suggest that hepatocellular functions are influenced by intrauterine growth retardation resulting in a reduced metabolic capacity in SGA infants. The metabolic differences between SGA and AGA infants should be considered in the nutritional management of LBW infants.

[Studies on dichlorvos (DDVP). III. Testing of dichlorvos for cocarcinogenic activity in mice]. / Untersuchungen mit Dichlorvos (DDVP). III. Prüfung von Dichlorvos auf kokanzerogene Wirkungen an Mäusen.

In order to clarify whether or not Dichlorvos (DDVP)--which did not exert carcinogenic effects in mice in our experiments--is cocarcinogenic, male and female mice of the strain C57Bl/6/Bln were subcutaneously injected with the carcinogen N-Nitrosodiethylamine (NDEA) and received in addition DDVP orally. For comparison NDEA and DDVP alone was administered to other groups of mice. The combined application (NDEA + DDVP) did not result in increased incidences of tumors and preneoplastic lesions as compared with the NDEA-treated groups of mice. The incidence of focal (transitional cell) hyperplasias of the urinary bladder epithelium was increased in the groups treated with NDEA + DDVP as compared to the groups with single compound treatment. There were no development of tumors and no differences in the latency periods of tumors which could be attributed to the combined treatment with NDEA + DDVP. Under these experimental conditions DDVP was not cocarcinogenic in mice.

Long-term care and management of Wilson's disease in the GDR.

Diagnosis, long-term management and family investigations of Wilson's disease are provided by selected clinical institutions in the GDR. From 187 patients detected since 1949, 111 are alive. In spite of the principal effectiveness of penicillamine treatment, confirmed by the disappearance of most of the central nervous system symptoms and successful professional rehabilitation of many patients, insufficient therapeutic discipline, psychosocial disturbances and penicillamine side-effects forcing its substitution by zinc or triethylenetetramine dihydrochloride in 14 cases need our further attention.

[The (15N) ammonium test in clinical research]. / Der [15N]-Ammoniumtest in der klinischen Forschung.

By use of the [15N]-ammoniumtest the liver function is investigated under influence of hormonal contraceptives in women and in liver diseases in children. With the described noninvasive nonradioactive isotope test the ammonia detoxification capability and the urea synthesis capacity of the liver is determined by measuring of the 15N-exceretion in ammonia and urea in the urine after oral administering of [15N]-ammonium chloride. The [15N]-ammonium test shows a significant influence of the hormonal contraceptives on the liver function and gives diagnostic evidence for liver diseases in children.

[15N]methacetin urine test to measure liver function: methodology for application in pediatrics.

A simple, noninvasive test is proposed to measure the activity of the hepatocellular monooxygenase system in vivo. Elimination rates of the stable isotope 15N are measured in urine after an oral dose of the 15N-labeled phenacetin homologue [15N]methacetin. Different forms of obtaining and expressing results are investigated in order to determine optimum methodology and differentiation for clinical application in pediatrics. The best discriminating power and reliability of results are seen with the measurement of 15N elimination half-life. For follow-up, a more easily measured parameter--eliminated dose over 9 h--is sufficient. In the latter case, only total nitrogen and 15N contents of a collective urine sample are measured.

[Studies on dichlorvos (DDVP). II. Testing of dichlorvos for carcinogenic activity in rats]. / Untersuchungen mit Dichlorvos (DDVP). II. Prüfung von Dichlorvos auf kanzerogene Wirkungen an Ratten.

The question has remained open, whether or not the pesticide Dichlorvos (DDVP) should be classified as a carcinogen. The results of a long-term experiment of testing DDVP in male and female C57Bl/6/Bln mice, recently published by us in this journal did not indicate the development of neoplastic lesions due to the administration of the compound. Now we report on results of long-term administration of DDVP to male and female BD IX/Bln rats. In the groups of male rats a increased incidence of proliferations of bile duct cells and of oval cells of the liver was observed, which was statistically significant (p less than 0.05) in the group of the higher dosed animals when compared with the group of the vehicle control male rats. Among the DDVP-treated female rats a significantly decreased incidence of tumors of the adrenal glands and of mammary tumors was observed as compared to the vehicle control group. Similar results were obtained in earlier experiments, when rats were treated with Trichlorfon, which easily convert to DDVP. In comparison to the corresponding control group DDVP-treated male rats showed a higher incidence of focal, hyperplasias of the urinary bladder, of focal hyperplasias of the pelvis and of transitional cell carcinomas of the renal pelvis. DDVP-treated female rats showed the opposite, namely lower incidences of these types of tumors, when compared with the control group. In our study on rats there were no neoplastic lesions found which could be attributed to the treatment of the animals with DDVP.

[Studies on dichlorvos (DDVP). I. Testing of dichlorvos for carcinogenic activity in mice]. / Untersuchungen mit Dichlorvos (DDVP). I. Prüfung von Dichlorvos auf kanzerogene Wirkung an Mäusen.

Up to now the question remained open, whether or not the pesticide Dichlorvos, should be classified as a carcinogen. We report on our results of long-time oral administration to male and female C57B1/6/Bln-mice of Dichlorvos to test the compound for carcinogenic activity. Dichlorvos significantly increased the incidence of focal hyperplasias (transitional cell hyperplasias) of the urinary bladder in male and female mice and decreased significantly the incidence of mixed lymphomas in mice of both sexes as compared with control animals (treated with solvent or untreated). There were no neoplastic lesions found including papillomas of the urinary bladder which could be attributed to the treatment of the animals with Dichlorvos.