Hepatocytes corrected by gene therapy are selected in vivo in a murine model of hereditary tyrosinaemia type I.

Current strategies for hepatic gene therapy are either quantitatively inefficient or suffer from lack of permanent gene expression. We have utilized an animal model of hereditary tyrosinaemia type I (HT1), a recessive liver disease caused by deficiency of fumarylacetoacetate hydrolase (FAH), to determine whether in vivo selection of corrected hepatocytes could improve the efficiency of liver gene transfer. As few as 1,000 transplanted wild-type hepatocytes were able to repopulate mutant liver, demonstrating their strong competitive growth advantage. Mutant hepatocytes corrected in situ by retroviral gene transfer were also positively selected. In mutant animals treated by multiple retrovirus injections >90% of hepatocytes became FAH positive and liver function was restored to normal. Our results demonstrate that in vivo selection is a useful strategy for hepatic gene therapy and may lead to effective treatment of human HT1 by retroviral gene transfer.

Pharmacological correction of neonatal lethal hepatic dysfunction in a murine model of hereditary tyrosinaemia type I.

Hereditary tyrosinaemia type I, a severe autosomal recessive metabolic disease, affects the liver and kidneys and is caused by deficiency of fumarylacetoacetate hydrolase (FAH). Mice homozygous for a FAH gene disruption have a neonatal lethal phenotype caused by liver dysfunction and do not represent an adequate model of the human disease. Here we demonstrate that treatment of affected animals with 2-(2-nitro-4-trifluoro-methylbenzyol)-1,3-cyclohexanedione abolished neonatal lethality, corrected liver function and partially normalized the altered expression pattern of hepatic mRNAs. The prolonged lifespan of affected animals resulted in a phenotype analogous to human tyrosinaemia type I including hepatocellular carcinoma. The adult FAH-/- mouse will serve as useful model for studies of the pathophysiology and treatment of hereditary tyrosinaemia type I as well as hepatic cancer.

Genetic correction of sickle cell disease: insights using transgenic mouse models.

Sickle cell disease is a hereditary disorder characterized by erythrocyte deformity due to hemoglobin polymerization. We assessed in vivo the potential curative threshold of fetal hemoglobin in the SAD transgenic mouse model of sickle cell disease using mating with mice expressing the human fetal Agamma-globin gene. With increasing levels of HbF, AgammaSAD mice showed considerable improvement in all hematologic parameters, morphopathologic features and life span/survival. We established the direct therapeutic effect of fetal hemoglobin on sickle cell disease and demonstrated correction by increasing fetal hemoglobin to about 9-16% in this mouse model. This in vivo study emphasizes the potential of the SAD mouse models for quantitative analysis of gene therapy approaches.

Impaired energy homeostasis in C/EBP alpha knockout mice.

Mice homozygous for the targeted deletion of the c/ebp alpha gene, which expresses the CCAAT/enhancer-binding protein alpha (C/EBP alpha), did not store hepatic glycogen and died from hypoglycemia within 8 hours after birth. In these mutant mice, the amounts of glycogen synthase messenger RNA were 50 to 70 percent of normal and the transcriptional induction of the genes for two gluconeogenic enzymes, phosphoenolpyruvate carboxykinase and glucose-6-phosphatase, was delayed. The hepatocytes and adipocytes of the mutant mice failed to accumulate lipid and the expression of the gene for uncoupling protein, the defining marker of brown adipose tissue, was reduced. This study demonstrates that C/EBP alpha is critical for the establishment and maintenance of energy homeostasis in neonates.

Dach1 mutant mice bear no gross abnormalities in eye, limb, and brain development and exhibit postnatal lethality.

Drosophila dachshund is necessary and sufficient for compound eye development and is required for normal leg and brain development. A mouse homologue of dachshund, Dach1, is expressed in the developing retina and limbs, suggesting functional conservation of this gene. We have generated a loss-of-function mutation in Dach1 that results in the abrogation of the wild-type RNA and protein expression pattern in embryos. Homozygous mutants survive to birth but exhibit postnatal lethality associated with a failure to suckle, cyanosis, and respiratory distress. The heart, lungs, kidneys, liver, and skeleton were examined to identify factors involved in postnatal lethality, but these organs appeared to be normal. In addition, blood chemistry tests failed to reveal differences that might explain the lethal phenotype. Gross examination and histological analyses of newborn eyes, limbs, and brains revealed no detectable abnormalities. Since Dach1 mutants die shortly after birth, it remains possible that Dach1 is required for postnatal development of these structures. Alternatively, an additional Dach homologue may functionally compensate for Dach1 loss of function.

Ex vivo hepatic gene therapy of a mouse model of Hereditary Tyrosinemia Type I.

Previously, this lab has reported the use of hepatocyte transplantation and in vivo gene therapy for the correction of a mouse model of Hereditary Tyrosinemia Type I (HT1). Here, we demonstrate repopulation of fumarylacetoacetate hydrolase (FAH)-deficient livers with cultured hepatocytes. Correction of the disease phenotype was achieved by retrovirally transducing cultured FAH- hepatocytes ex vivo, followed by transplantation and selective repopulation. Treated mice were phenotypically normal and had corrected plasma amino acid levels and liver function tests. Our results demonstrate that efficient hepatic repopulation using ex vivo genetically manipulated hepatocytes is feasible.

Adenovirus-mediated gene therapy in a mouse model of hereditary tyrosinemia type I.

Mice lacking the enzyme fumarylacetoacetate hydrolase (FAH) have symptoms similar to humans with the disease hereditary tyrosinemia type I (HT1). FAH-deficient mice were injected with a first-generation adenoviral vector expressing the human FAH gene and followed for up to 9 months. Nontreated FAH mutant control mice died within 6 weeks from fulminant liver failure, whereas FAH adenovirus-infected animals survived until sacrifice at 2-9 months. Nine of 13 virus-treated animals developed hepatocellular cancer. Immunohistochemical analysis revealed a mosaic of FAH-deficient and FAH-positive cells in all animals and liver function tests were improved compared to controls. Even mice harvested 9 months after viral infection had > 50% FAH-positive cells. These results demonstrate the strong selective advantage of FAH-expressing cells in an FAH-deficient liver but also illustrate the danger of carcinomas arising from FAH-deficient hepatocytes in HT1.

Retrovirus-mediated in vivo gene transfer in the replicating liver using recombinant hepatocyte growth factor without liver injury or partial hepatectomy.

Retrovirus-mediated gene delivery into hepatocytes in vivo provides long-term gene expression, which is of great importance for treating most genetic and metabolic disorders. However, clinical application has not been realized because of the requirement for prior 70% partial hepatectomy or chemical (toxic) liver injury to initiate hepatocyte replication at the time of retroviral gene transduction. In this paper, we describe a novel gene delivery system that uses recombinant hepatocyte growth factor (rHGF) prior to retrovirus-mediated in vivo gene transfer in the liver without partial hepatectomy or liver injury. A single retroviral infusion through the portal vein following five systemic injections (via the tail vein) of 100 microg/kg rHGF resulted in a 10.4% 5-bromo-2'-deoxyuridine (BrdU) labeling index (BLI) and 0.14% retroviral gene transduction efficiency (RGTE) in hepatocytes, which were 6.3- and 12.9-fold higher than those of controls, respectively. Modest additional increases in BLI and RGTE (13.4% and 0.22%, respectively) were seen after five systemic injections of 500 microg/kg rHGF. The correlation between BLI and RGTE was statistically confirmed regardless of treatment. When rats received multiple retroviral infusions through a cannulated portal vein following five portal injections of 100 microg/kg rHGF, RGTE was dramatically increased (1.3%) and in some areas of the liver exceeded more than 10%. There was no evidence of liver injury in any animal. This approach has great potential for clinical application in terms of avoiding invasive procedures or liver injury.

Cyclosiloxanes produce fatal liver and lung damage in mice.

To examine the toxicity of cyclosiloxanes (CSs), the predominant low molecular weight cyclic silicones found in breast implants, we injected female CD-1 mice intraperitoneally with different doses of distillate (3.5-35 g/kg body weight) containing cyclosiloxane D3 (hexamethylcyclotrisiloxane; CS-D3), cyclosiloxane D4 (octamethylcyclotetrasiloxane; CS-D4), cyclosiloxane D5 (decamethylcyclopentasiloxane; CS-D5), and cyclosiloxane D6 (dodecamethylcyclohexasiloxane; CS-D6). The distillate was found to be lethal and all the mice injected with 35 g/kg died within 5-8 days. The median lethal dose (LD50) for distillate was estimated to be approximately 28 g/kg. These mice developed inflammatory lesions of the lung and liver as well as liver cell necrosis with elevated serum levels of alanine aminotransferase, aspartate aminotransferase, and lactic acid dehydrogenase. Administration of CS-D4 alone also produced lethality in these mice with an LD50 of 6-7 g/kg. CS-D4-treated mice also exhibited pulmonary and hepatic lesions and elevated serum enzymes. Analysis of LD50 data indicates that CS-D4 is about as toxic as carbon tetrachloride or trichloroethylene. We measured hydroxyl radical formation in CS-D4-treated mice and found increases of approximately 20-fold in liver and approximately 7-fold in lung on day 4 following injection. Our findings are significant because in vitro experiments have demonstrated that CSs can migrate out of breast implants, and in mouse experiments CSs have been shown to be widely distributed in many organs after a single subcutaneous injection and to persist for at least a year.

Randomized controlled trial of clarithromycin and ethambutol in the treatment of Crohn's disease.

BACKGROUND: A mycobacterial infection may be the cause of Crohn's disease in some patients. Measurement of intestinal permeability may identify Crohn's disease patients with a high likelihood of relapse and may quantify the severity of intestinal injury. AIM: To assess the effect of 3 months of clarithromycin and ethambutol on the disease activity and intestinal permeability in patients with Crohn's disease at high risk of relapse. METHODS: Patients with Crohn's disease, with a lactulose-mannitol permeability test above 0.03, were randomly assigned to receive either clarithromycin, 500 mg twice daily, and ethambutol, 15 mg/kg daily, or identically appearing placebo for 3 months in addition to their regular therapy. The Harvey-Bradshaw index and the lactulose-mannitol test were assessed in a blind fashion every 3 months for 12 months. RESULTS: Thirty-one patients were randomized to receive either drugs (n=15) or placebo (n=16). The groups were similar in age, sex, duration of disease, location of disease, past complications and disease severity. Specifically, there was no difference between the drug or placebo groups in the mean Harvey-Bradshaw index (4.8 vs. 4.4), number with active disease (33% vs. 44%) and mean lactulose-mannitol test (0.06 vs. 0.10). During the 12-month follow-up period, there were no consistent, statistically significant differences in the mean Harvey-Bradshaw index or lactulose-mannitol test between treatment and placebo groups. Individual patients showed either improvement or worsening of these indices, but these were not related to study medication. Specifically, no 'cures' were noted with anti-mycobacterial treatment. CONCLUSIONS: Three months of treatment with clarithromycin and ethambutol does not benefit Crohn's disease patients who are receiving standard medical therapy.

Conjugated bilirubin versus direct bilirubin in neonates.

Conjugated and direct bilirubin were measured in 288 samples from 107 neonates less than 15 days old. Retrospective analysis of the medical records showed that 53 neonates were hepatobiliary-normal, 42 patients had no obvious evidence of hepatobiliary disease but had received total parenteral nutrition, and 12 were clearly hepatobiliary-abnormal. Neither the mean values nor the distributions of either the conjugated bilirubin, as measured by a multilayered slide, or the direct bilirubin, measured by a solution diazo procedure, differed when comparing the hepatobiliary-normal population to that receiving total parenteral nutrition. However, as would be predicted, the hepatobiliary-abnormal population differed significantly from both of these groups using either direct bilirubin or conjugated bilirubin results. Samples obtained from hepatobiliary-abnormal neonates were reviewed in chronologic sequence if direct bilirubin results differed from conjugated bilirubin with respect to classification of normality/abnormality. In two of three neonates with developing cholestasis, conjugated bilirubin exceeded its upper limit of normal earlier than did direct bilirubin. Conjugated bilirubin returned to normal earlier than direct bilirubin for two neonates with an improving clinical status. Conjugated bilirubin measurement was judged to be more responsive to developing or resolving cholestasis then direct bilirubin. In addition, conjugated bilirubin measurement from the slide is known to agree well with that determined by high performance liquid chromatography fractionation of bilirubin and is less susceptible to interference than is direct bilirubin measurement, characteristics that recommend its use over direct bilirubin analysis in a clinical setting.

High-performance liquid chromatography in the diagnosis of hemoglobinopathies and thalassemias. Report of three cases.

High-performance liquid chromatography is a technic that has recently been applied to the diagnosis of hemoglobinopathies and thalassemias. Its advantages over other methods include increased sensitivity, resolution and simplicity, as well as speed. In this report, the authors present an HPLC procedure that uses a weak cation exchange column and a gradient elution system for the diagnosis of hemoglobinopathies and thalassemias. The authors illustrate the utility of this procedure by reporting three cases in which the technic enabled them to make the correct diagnosis, which by traditional methods would have been missed or equivocal.

Unconjugated hyperbilirubinemia is overestimated in neonates with cholestasis. A more reliable method is proposed.

Unconjugated bilirubin was determined in 458 serum samples from 160 neonatal and pediatric patients by using two approaches. The first approach was to calculate indirect bilirubin by subtracting direct bilirubin, as measured by a diazo solution assay, from total bilirubin quantified by a Jendrassik-Grof procedure. The second approach was actually to measure the unconjugated bilirubin fraction using a multilayered slide. For samples containing little or no conjugated bilirubin, correlation between the calculated indirect bilirubin value and the unconjugated bilirubin measured by the slide was judged acceptable. Samples containing increased levels of conjugated bilirubin, however, yielded discrepancies between the approaches including differences of up to 5-10 mg/dL and more. Moreover, the magnitudes of the differences correlated with the amounts of conjugated bilirubin present in the samples. The unconjugated bilirubin values given by the slide were found to correlate more closely than the calculated indirect values with results obtained by an HPLC procedure that may be regarded as the most reliable method available. Therefore, we find that the slide provides a more accurate measure of unconjugated bilirubin than does the indirect bilirubin value in specimens from pediatric patients having evidence of cholestasis. The authors also believe that the magnitude of the difference in the values could make a difference in therapeutic strategies.

Diagnosis of Hemoglobinopathies by High-Performance Liquid Chromatography.

This report describes a unique cation exchange high-performance liquidchromatography capable of separating more than 40 frequently encountered human hemoglobins and variants within 12 min. Some of these variants are unresolvable by the conventional electrophoretic methods and would thus lead to an incorrect diagnosis of hemoglobinpathy. The method provides high sensitivity, superior resolution and accurate quantitation of hemoglobin concentrations. It can also be fully automated thus make it an ideal methodology for the diagnosis of hemoglobin disorders in a routine clinical laboratory.

The effect of gestational age and fetal indomethacin levels on the incidence of constriction of the fetal ductus arteriosus.

OBJECTIVE: To determine the effects of gestational age and fetal serum indomethacin levels on constriction of the ductus arteriosus after maternal indomethacin administration. METHODS: Twenty-five pregnant Rh-sensitized patients were given a 50-mg oral dose of indomethacin 6 hours before fetal serum indomethacin levels were determined at the time of 50 diagnostic or therapeutic funipunctures. The ductus arteriosus was evaluated with Doppler ultrasound immediately before 40 of the procedures. Constriction of the ductus arteriosus was defined as a peak diastolic flow greater than 35 cm/second. Least-squares regression and multiple regression were used for statistical analysis. RESULTS: The peak diastolic velocity of the fetal ductus arteriosus after maternal indomethacin ingestion was constant at 25 cm/second before 27 weeks, increased between 27-30 weeks to a mean of 39 cm/second, and was stable thereafter (R2 = 0.35; P < .05). There was no significant correlation between constriction of the ductus and fetal serum indomethacin levels (P = .17). CONCLUSIONS: The constrictive effect of maternal indomethacin ingestion on the fetal ductus arteriosus begins as early as 27 weeks' gestation. Constriction of the ductus arteriosus is independent of fetal serum indomethacin levels.

Influence of short-term indomethacin therapy on fetal urine output.

Eight pregnancies ranging from 27-32 weeks' gestation were treated for preterm labor with oral indomethacin. The dosage regimen was 25 mg every four hours in four patients and 25 mg every six hours in the other four patients. The maximum duration of indomethacin therapy was 72 hours. In three patients, fetal ductus arteriosus constriction mandated discontinuation of indomethacin at 24 hours. Sonographic assessment of hourly fetal urine output was performed before therapy, at multiple regular intervals during therapy, and 24 hours after the last dose of indomethacin. A dramatic decline was noted from the mean baseline fetal urine output of 11.2 mL/hour. The mean fetal urine output at five, 12, and 24 hours during therapy was 2.2, 1.8, and 1.8 mL/hour, respectively (P less than .05). Twenty-four hours after completion of indomethacin therapy, the mean fetal urine output was 13.5 mL/hour. Poor correlation (r = 0.14, P less than .05) was noted between maternal serum indomethacin levels and hourly fetal urine output. This significant decline in urine output is consistent with other results in neonatal and adult animals and humans. Furthermore, it implies a role for prostaglandins in controlling urine output during fetal life.

Ferritin, transferrin and iron concentrations in the cerebrospinal fluid of multiple sclerosis patients.

The concentrations of ferritin, transferrin and iron were measured in the cerebrospinal fluid (CSF) of multiple sclerosis (MS) and control patients. Ferritin levels were significantly elevated in the CSF of chronic progressive active MS patients (4.71+/-0.54 ng/ml) compared to levels in normal individuals (3.07+/-0.17 ng/ml). MS patients with active or stable relapsing-remitting disease had ferritin levels that were comparable to those found in normal individuals. There were no significant differences in transferrin or iron levels in the CSF between MS and normal individuals. Both ferritin and transferrin levels were elevated in patients that had high CSF IgG values but not in patients with a high IgG index. Since ferritin binds iron, the increase of CSF ferritin levels in chronic progressive MS patients could be a defense mechanism to protect against iron induced oxidative injury. Ferritin levels could be a laboratory measure that helps to distinguish between chronic progressive and relapsing-remitting MS.

Enzymatic measurement of serum glycerol using a COBAS-BIO centrifugal analyzer.

An enzymatic method for the measurement of glycerol using glycerol kinase coupled to pyruvate kinase and lactate dehydrogenase was adapted to a COBAS-BIO centrifugal analyzer. Routine standardization is not required under the optimized conditions since the calculation factor derived from the measurement of pure glycerol standard was found to be identical to the theoretical value. Excellent correlation between the method and the DuPont ACA triglyceride method was obtained. The coefficients of variation for within-run and day-to-day precision were less than 4%. Only 2 microL of serum is required, making this an excellent means for monitoring therapeutic glycerol administration in premature infants with hydrocephalus. The method has the sensitivity to measure glycerol concentrations as low as 0.1 mmol/L. This, together with the rapid analysis time (28 specimens in 10 min), makes it suitable for the correction of glycerol interference in the measurement of serum triglyceride.

Diagnosis of hemoglobinopathies: electrophoresis vs. HPLC.

BACKGROUND: Alkaline cellulose acetate and acidic citrate agar electrophoreses are the most widely utilized methods for hemoglobin analysis. However, due to their limited resolution, incorrect or unresolved diagnosis of common hemoglobinopathies are sometimes encountered. METHODS: Isoelectric focusing provides excellent resolution but is labor intensive and lacks accurate quantitation. High-performance liquid chromatographic methods have been developed for either screening or confirmation of hemoglobinopathies with relatively high sensitivity or specificity. Through the years, we have developed, refined and optimized an HPLC procedure using a porous silica coated with polyaspartic acid to improve the elution time of hemoglobin analysis while maintaining the high sensitivity and resolution necessary for both screening and confirmatory purposes. RESULTS: The method is capable of separating more than 45 commonly encountered hemoglobin variants within 12 min. These include Barts, H, A1C, Raleigh, Hope, I, F, Camden, N-Baltimore, I-High Wycombe, I-Paris, J-Baltimore, N-Seattle, Grade, Fannin-Lubbock, Malmo, South Florida, A, Chicago, G-Georgia, Lepore-Baltimore, P-Galveston, G-Coushatta, Lepore-Boston, E, Zurich, Osu Christiansborg, A2, G-Philadelphia, Korle Bu, Russ, E-Saskatoon, Richmond, D-Punjab, Deer Lodge, Koln, Montgomery, S, Q-Thailand, G-San Jose, A2', Hasharon, Q-India, Tampa, Constant Spring, SG-hybrid, C-Harlem, O-Arab, British Columbia, and C. The method provides not only the identification of the aforementioned hemoglobin and variants but also an accurate quantitation of their concentrations, particularly Hb F and A2, which are useful for the diagnosis of HPFH and beta-thalassemia, respectively. CONCLUSIONS: The simplicity of the sample preparation, superior resolution of the method, and accurate quantitation of hemoglobin concentration, combined with complete automation, make this an ideal methodology for the routine diagnosis of hemoglobin disorders in a clinical laboratory.

Evaluation of the Kodak EKTACHEM clinical chemistry slide for the measurement of bilirubin in newborns.

Measurement of neonatal bilirubin using the Jendrassik-Grof method (x) and the EKTACHEM NBIL assay (y) was compared over a 6-month period in a total of 1191 specimens from 483 patients less than 30 days of age. Linear regression analysis of the data yields a slope of 0.937, an intercept of 0.387, and Sy,x of 0.55 and a correlation coefficient of 0.983 for a total of 1032 specimens from patients less than or equal to 14 days of age and a slope of 1.090, an intercept of 0.002, an Sy,x of 1.03 and a correlation coefficient of 0.950 for a total of 159 specimens from patients greater than 14 days of age. The best correlation between EKTACHEM NBIL assay and the reference Jendrassik-Grof method was observed in samples from patients less than or equal to 14 days of age. Data from patients older than 14 days showed a higher proportional bias and a lower correlation coefficient between the methods. High performance liquid chromatographic analysis demonstrated that patients greater than 14 days of age had a higher incidence of elevated delta-bilirubin. Linearity extends to 200 mg/l. The NBIL assay provides a rapid, precise micromethod that is less sensitive than the Jendrassik-Grof method to the in vitro photo degradation of bilirubin and is not subject to the interference from hemoglobin and lipids. Because delta-bilirubin is not measured by this method, it is only recommended for newborns less than or equal to 14 days of age.