Gamma-carboxylated isoforms of recombinant human protein S with different biologic properties

Human protein S (HPS), a regulator of hemostasis, is a vitamin K- dependent plasma protein with potential clinical utility. We have obtained high-level expression of the cDNA for HPS in two mammalian cell lines. Both cell lines secreted single chain recombinant HPS (rHPS) in serum-free medium as determined by Western blot analysis. The ability of the rHPS from both cell lines to act as a cofactor for human protein C (HPC) was determined; the rHPS secreted from the human 293 cell line had an activity six times that of the rHPS from the AV12-664 Syrian hamster cell line. Furthermore, the relative specific cofactor activity of rHPS from the 293 cell line was actually 2.5-fold higher than that of single-chain human plasma-derived HPS. Essentially all of the rHPS secreted from the 293 cell line exhibited a calcium-dependent elution profile on anion exchange chromatography, whereas only 25% to 35% of the hamster cell-derived rHPS exhibited this profile. However, the calcium-eluted rHPS from the AV12 cell line had a high specific cofactor activity, equivalent to that of the 293-derived rHPS. A NaCl- elutable rHPS fraction (calcium nondependent) was isolated from the recombinant AV12-664 cell line, further purified, and found to have reduced activity, only 40% that of the calcium-dependent rHPS. The only observable difference in the calcium-dependent and nondependent rHPS molecules was in the content of gamma-carboxyglutamic acid (Gla); the calcium-dependent material contained approximately 10 mol Gla/mol protein whereas the calcium-nondependent material contained only approximately 8 mol Gla/mol of protein. In addition, the calcium- nondependent rHPS had reduced ability to interact with phospholipid vesicles as evidenced by an eightfold increase in the apparent kd. Our data demonstrate the isolation of rHPS with high specific activity, and show that a reduction in as few as two Gla residues dramatically decreases its functional cofactor activity for HPC, due to a reduction in ability to interact with the phospholipid bilayer.     

Management of human immunodeficiency virus-associated thrombocytopenia with intravenous gamma globulin

A 17-month-old boy in whom immune-mediated thrombocytopenia (ITP) was the presenting manifestation of infection with human immunodeficiency virus (HIV) is being successfully managed with intermittent high-dose intravenous gamma globulin (IVIG) allowing maintenance of hemostatic platelet counts while avoiding the immunosuppression associated with other therapeutic modalities used to treat ITP. He continues to demonstrate marked responsiveness to IVIG, and has been maintained on weekly or bimonthly infusions for 12 months. The serendipitous documentation of HIV infection prior to IVIG therapy for immune-mediated thrombocytopenia in this child documents the importance of HIV testing prior to IVIG therapy to prevent erroneous assignment of IVIG as the vehicle responsible for transmission of HIV infection. This case history also documents the importance of HIV testing in the diagnostic evaluation of immune-mediated thrombocytopenias.     

Treatment of lymphohistiocytic erythrophagocytosis with VP-16 and aziridinylbenzoquinone

Lymphohistiocytic erythrophagocytosis (LE) is a usually fatal disease characterized by fever, organomegaly, hyperlipidemia, central nervous system involvement, and cellular immunodeficiency. Treatment with corticosteroids, cytotoxic chemotherapy, and blood exchange is unsuccessful. We have treated two children with the epipodophyllotoxin VP-16 and with intrathecal chemotherapy. Each patient had an initial complete response, and one remains in remission 36 months after therapy began. Aziridinylbenzoquinone (AZQ) therapy induced a complete response in a patient who relapsed during VP-16 therapy. A combination of VP-16 and intrathecal chemotherapy appears to be the most effective therapy for LE, and further evaluation of the role of AZQ is indicated.     

Parents' perceptions towards their child's use of inhaled medications for asthma therapy

Objective : Understanding patients' and their parents' perceptions towards asthma medication is important in developing strategies to ensure patient compliance. In this study, parents' perceptions towards their children's use of inhaled medication for asthma treatment were evaluated.
Methodology : A questionnaire was administered by interviewing parents of 210 asthmatic children attending the Department of Paediatrics, National University Hospital.
Results : Our results showed that 76/210 (36%) of parents either felt opposed to inhaler therapy and/or preferred oral medications. The main reasons for their reluctance to use inhalers were related to fear of dependence, side effects and overdosage, and the child's dislike for inhalers. A third of these parents felt that inhalers were only indicated for very severe asthma.
Conclusions : We conclude that a significant proportion of parents have reservations regarding the use of inhalers for the treatment of asthma. These factors should be taken into consideration when planning an effective asthma education programme.     

Abnormal anatomy of the lumbosacral region imaged by magnetic resonance in children with anorectal malformations

OBJECTIVE: To investigate the frequency of lumbosacral anomalies, the association with urogenital abnormalities, and the correlation with defaecation pattern by magnetic resonance imaging (MRI). METHODS: A prospective analysis was performed of routine MRI in patients with anorectal malformations. Between 1990 and 1994, MRI was performed in 43 such patients: 31 boys and 12 girls. Twenty four had a high anorectal malformation, 16 had a low anorectal malformation, and three had Currarino's triad. MRI was performed before reconstruction in 26, and postoperatively in 17. Urogenital anomalies were found in 21. RESULTS: Abnormalities of the spinal cord and spine were found with MRI in 20 patients (46.5%); caudal regression syndrome in 10, tethered cord in two, a combination of both in three, and other spinal anomalies in five. These anomalies were found in 30% of the patients with low anorectal malformations, and in 50% with high anorectal malformations. In patients with urogenital malformations, MRI more often showed spinal anomalies (13/21, 62%) than in patients without (7/22, 32%). In high anorectal malformations, defaecation was more often a problem in patients with spinal anomalies (12/15, 80%) than in patients without (2/8, 25%). CONCLUSIONS: Spinal anomalies in the lumbosacral region were found with MRI in 46.5% of patients with anorectal malformations. Since presence of these anomalies seems to be related to clinical outcome, MRI should be performed routinely in all such patients.     

Efficient construction of the securine A carbon skeleton

Securamine A is a structurally intriguing alkaloid possessing a pyrroloindole core joined via a modified isoprene subunit to a functionalized imidazole ring. Recent synthetic efforts in this laboratory have resulted in the efficient construction of key lactone 36, which undergoes tandem azide reduction/ring expansion to macrolactam 37. Macrolactam 37 possesses the complete macrocyclic core of securamine A.