Adeno-associated viral vector-mediated gene transfer of human blood coagulation factor IX into mouse liver

Recombinant adeno-associated virus vectors (AAV) were prepared in high titer (10(12) to 10(13) particles/mL) for the expression of human factor IX after in vivo transduction of murine hepatocytes. Injection of AAV-CMV-F.IX (expression from the human cytomegalovirus IE enhancer/promoter) into the portal vein of adult mice resulted in no detectable human factor IX in plasma, but in mice injected intravenously as newborns with the same vector, expression was initially 55 to 110 ng/mL. The expression in the liver was mostly transient, and plasma levels decreased to undetectable levels within 5 weeks. However, long-term expression of human F.IX was detected by immunofluorescence staining in 0.25% of hepatocytes 8 to 10 months postinjection. The loss of expression was likely caused by suppression of the CMV promoter, because polymerase chain reaction data showed no substantial loss of vector DNA in mouse liver. A second vector in which F.IX expression was controlled by the human EF1alpha promoter was constructed and injected into the portal vein of adult C57BL/6 mice at a dose of 6.3 x 10(10) particles. This resulted in therapeutic plasma levels (200 to 320 ng/mL) for a period of at least 6 months, whereas no human F.IX was detected in plasma of mice injected with AAV-CMV-F.IX. Doses of AAV-EF1alpha-F. IX of 2.7 x 10(11) particles resulted in plasma levels of 700 to 3, 200 ng/mL. Liver-derived expression of human F.IX from the AAV-EF1alpha-F.IX vector was confirmed by immunofluorescence staining. We conclude that recombinant AAV can efficiently transduce hepatocytes and direct stable expression of an F.IX transgene in mouse liver, but sustained expression is critically dependent on the choice of promoter.     

Gas-liquid chromatographic determination of azinphos ethyl in human plasma and in mouse plasma, tissue, and fat

A new method for the determination of azinphos ethyl (O,O-diethyl-S-(4-oxo-1,2,3-benzotriazin-3(4H)-ylmethyl) phosphorodithioate) in human plasma and in mouse plasma, tissue, and fat has been developed. The method is based on extraction with benzene or hexane and cleanup of fat and tissue samples by a minicolumn containing Florisil and sodium sulfate. Azinphos ethyl is eluted from the column with 10% acetonitrile in benzene and is concentrated to an appropriate volume for gas-liquid chromatographic analysis, using a 63Ni electron capture detector and a glass column containing 3% OV-1 on Gas-Chrom Q. The method is sensitive to 0.005 ppm in human plasma, 0.01 ppm in mouse plasma, 0.08 ppm in mouse liver, 0.05 ppm in mouse brain, and 0.10 ppm in mouse fat. The limit of detection is 2 pg; mean recoveries ranged from 96 to 98%.     

Racial difference in incidence of ABO hemolytic disease

In this review of 7,464 consecutive infants born at North Carolina Memorial Hospital, hemolytic disease from ABO incompatibility was found to be two to three times as common in black infants as in white infants. The statistical significance of the difference remained high as more restrictive criteria for ABO hemolytic disease were applied. ABO disease, serious enough to cause an indirect serum bilirubin of 15 mg/100ml or higher, had an incidence in black newborns as great as the incidence of Rh hemolytic disease in whites. In contrast, the general prevalence and severity of hyperbilirubinemia was not found to be higher in black newborns than in white infants. The difference cannot be attributed to differences in the prevalence of ABO blood groups between the two races. Policies of early discharge of newborns could be affected by the finding that ABO erythroblastosis is two to three times as common in black infants as in white infants.     

Etiology of corneal striae accompanying hydrogel lens wear

Vertical striae in the posterior cornea were produced experimentally in ten human subjects by depriving the anterior corneal surface of its normal oxygen supply and inducing corneal edema. These striae were similar in appearance and time of occurrence to those observed in gel lens wearers. Three subjects also wore gel lenses and developed vertical striae while wearing their lenses. These striae were eliminated by increasing the oxygen concentration at the anterior lens surface. The data confirm many clinical observations which have suggested that verical striae are caused by corneal edema accompanying gel lens wear.