Detection and Quantification of Residual Cellular DNA in the Production of Recombinant HPV-16 L1 Virus-Like Particles

A number of recombinant proteins isolated from cell sources are being produced for biopharmaceuticals. Although most biopharmaceuticals are highly purified, there is a safety concern that such recombinant products could be contaminated with impurities including adventitious virus, mycoplasma, endotoxin and oncogenic DNA. Residual DNA in recombinant biopharmaceuticals is a potential risk factor and must be evaluated and removed to meet the regulatory guidelines. Recombinant HPV type 16 L1 VLPs, recombinant protein produced in Spodoptera frugiperda (Sf) 9 insect cells, is a HPV subunit vaccine candidate which has been studied as a preventive vaccine of cervical cancers. In this study, we performed detection and quantification of residual cellular DNA in the production of recombinant HPV type 16 L1 VLPs. HPV-16 L1 VLPs were purified by processes including detergent lysis, sonication treatment, sucrose cushion centrifugation, CsCl equilibrium density centrifugation, and DNase treatment which was added to inactivate residual cellular DNA after CsCl centrifugation step. We have developed a precise assay based on a dot-blot hybridization using digoxigenin random primed labeling DNA probes for the detection and quantification of residual cellular DNA during the purification process and final products. Detection limit of residual cellular DNA was 0.1 ng in this assay and the amount of residual cellular DNA in the final product was 0.5 ng~1 ng per 100 microgram of protein. This study describes safer and more sensitive methods alternative to radioactive techniques employed for residual cellular DNA quantification of biopharmaceuticals produced by recombinant protein technology and presents method validation data demonstrating precision and reproducibility.

Granulocyte Transfusion in Severe Neutropenic Pediatric Patients / 대한소아혈액종양학회지

PURPOSE: The use of granulocyte transfusion (GT) which had been diminished since 1980s has been recently interested on the base of clinical efficacy approved by transfusion of granulocyte collected after administration of granulocyte-colony stimulating factor (G-CSF) to donors. So we studied the clinical efficacy of GT in severe neutropenic pediatric patients with severe infections. METHODS: Twelve patients with malignant hematologic disorders and solid tumors in Ajou University Hospital from March 1997 to February 2001 were indicated for GT. These patients had continuous neutropenia-related infections despite appropriate antibiotics, antifungal or IV immunoglobulin therapy. GTs were carried out 12 hrs after collection of granulocytes using leukapheresis from donors stimulated by G-CSF. RESULTS: The median number of GT is 3 (2~7), and the mean dose of granulocyte is 3.51+/-4.21 10(10)/m(2) and mean volume of granulocyte is 220 +/-22.92 mL. The median duration of the use of G-CSF and antibiotics or antifungal agents is 6 (3~14) days and 3 (1~9) days. Ten of 12 patients had favorable responses (FR), and 2 patients had unfavorable responses (UR). Two patients who had FR died of acute respiratory distress syndrome (ARDS), complication of GT. CONCLUSION: GT is effective treatment for severe neutropenic pediatric patients with severe infections. Enough amount of granulocytes could be collected after administration of G-CSF without dexamethasone to donors. However, ARDS which is an adverse effect of GT, is considered in pulmonary compromised patients.

A Case of Bartter Syndrome with Muscle Weakness and Short Stature

Bartter syndrome is a rare disorder characterized by the association of hypokalemic hypochloremic metabolic alkalosis, hyperreninemia, hyperaldosteronemia, short stature and nephrocalcinosis. This disorder presents with hyperplasia of juxtaglomerular apparatus on renal biopsy. We experienced a case of late-onset Bartter syndrome with nephrocalcinosis in a 9-year-old boy, whose chief pictures were muscle weakness, short stature, persistent sterile pyuria and microscopic hematuria. We report this case with a brief review of related literatures.

Changes in Mean Platelet Volume and Platelet Distribution Width after Therapy in Childhood Idiopathic Thrombocytopenic Purpura

PURPOSE: Mean platelet volume(MPV) and platelet distribution width(PDW) are useful parameters in evaluating disorders of platelets. In cases with idiopathic thrombocytopenic purpura(ITP), they change as platelet count increases. In this study, we compared the values of MPV and PDW in ITP patients at diagnosis with those of normal children. We also studied whether the early changes in MPV may predict the clinical course. METHODS: From December 1995 to May 2001, 71 patients with ITP were admitted to Ajou University Hospital. They were treated with IVIg 400 mg/kg for five days and MPV, PDW, platelet count were analysed. Normal control group(n=38) was compared. The study group was divided into acute and chronic forms, and also divided into group A, good early responders whose platelets increased more than 100,000/microL within 5 days and group B who did not. RESULTS: Mean value of MPV at diagnosis in ITP patients was lower than the normal control group(P<0.05). In group A, MPV was abruptly increased on the first day after IVIg and then started to decrease. But in group B, MPV was steadily increased until the fourth day after IVIg. In the normal control group, there were inverse correlations between platelet count and MPV(r= -0.415, P<0.05), but in ITP patients, there were positive relationships between platelet count and MPV(r=0.646, P<0.05) at diagnosis. CONCLUSIONS: MPV at diagnosis of ITP was lower than the normal control. MPV and PDW could not predict the course of ITP patients, but MPV could distinguish good early responders. More research is needed to find out the reasons of decreased MPV at diagnosis of ITP.

Development of a Screening Kit for Early Diagnosis and Prevention of Wilson's Disease

PURPOSE: Wilson's disease is an autosomal recessive disorder characterized by copper accumulation in the liver, brain, and other organs due to defected copper metabolism. The incidence of Wilson's disease is approximately one in 30,000 population in the world, more common than phenylketonuria in Korea. The early diagnosis or presymptomatic diagnosis of Wilson's disease is critical in order for them to live a normal life. However, unfortunately, there are no commercial kits available for Wilson's disease screening in the world yet. METHODS: We developed a mass-screening kit for the purpose of early diagnosis and prevention of Wilson's disease using sandwich ELISA method. This kit can handle a large number of samples at the same time by using filter paper as in newborn screening. Using the polyclonal or monoclonal anti-ceruloplasmin antibodies, this kit determines the plasma ceruloplasmin levels-one of the main markers for Wilson's disease. RESULTS: The plasma levels of the ceruloplasmin were considerably lower in the Wilson's disease (4.5+/-1.6 mg/dL) group compared to normal controls(22.1+/-1.4 mg/dL), sufficient to be used for mass screening. In addition, the results using this screening kit showed 100% positive and negative concordance rates with the test results obtained from immuno-turbidimetry analysis which is the currently used in most test centers for ceruloplasmin measurement in the serum or plasma after centrifugation. CONCLUSION: Taken together, we successfully developed a screening kit which is very effective for the early diagnosis and prevention of Wilson's disease. By using simple filter paper method for sample collection, this kit provides suitable mass screening. We suggest the screening for Wilson's disease at the age of 3-5 years.

Smoking patterns and factors associated with smoking in Korean adult women

BACKGROUND: Women smoker faces increased risk for heart disease, stroke, cancers, and respiratory diseases as well as a number of gender specific risks. Maternal smoking has been linked to harmful effects on their offspring such as increased incidence of premature, stillbirth and sudden infant death syndrome, etc. Recently, the rate of female adolescent smoking in Korea is increasing, and the rate of adult woman is expected to increase. This study is aimed at obtaining basic information about smoking patterns and factors associated with smoking in Korean adult women. METHODS: From May 1995 to April 1999, a self-administered questionnaire was distributed to the female visitors of Health Promotion Center in Seoul National University Hospital. The number of smokers was 492. The control group(984 nonsmokers) was selected by 1:2 age group matched random sampling. Smokers and control group were divided into three age groups-young age(20-39 years), middle age(40-59 years), old age(60 years and over)-and analysed the resalts using SPSS and EpiInfo software. RESULTS: The most frequent amount of cigarette smoking was below 10 cigarettes in all age groups. The duration of smoking was below 10 years in most young and middle age groups. In the old age group the duration of smoking was longer than others. Variables positively associated with smoking included alcohol drinking, being engaged in trade service, being unmarried or divorced or separated or parted for ever, high stress status, dissatisfation of life in young age group. The variable of no exercise was added to them in middle age group. In old age group alcohol drinking, no exercise, no school education were the positive meaningful factors associated with smoking in multivariant logistic regression analysis. CONCLUSIONS: The modifiable variables associated with smoking in Korean adult women included alcohol drinking, high stress status, dissatisfation of life and no exercise.

Investigation on the Immunity to Pertussis in the Korea

Acellular pertussis vaccine has been used widely in Korea since 1984. However, because many of the former generations were not inoculated with pertussis vaccine, they may infect infants with pertussis. With this background, we investigated the prevalence of pertussis antibodies in all age groups. Enzyme-linked immunosorbent assay (ELISA) to assess IgG antibodies to pertussis toxin (PT) and filamentous hemagglutinin (FHA) and bacterial agglutination (BA) to assess antibodies to agglutinogen were compared on 842 serum samples which were donated from 11 hospitals in Seoul area. In comparison with age groups under 20 years, antibodies of adults against PT and FHA were maintained. But antibodies against agglutinogen showed no pattem in all age groups. Antibodies to PT were correlated with antibodies to FHA. There was no significant difference in antibody levels between male and female (p<0.05).

The Changes of the Bone Mineral Density by Treatment Modality in Patients with Turner Syndrome / 대한소아내분비학회지

PURPOSE:Decreased bone mineral density(BMD) has been reported in girls with Turner syndrome. Estrogen therapy is recommanded to improve sexual infantilism and decreased BMD. Short stature is also characteristic finding in patients with Turner syndrome. Treatment modality for short stature has included estrogen, anabolic steroids and growth hormone(GH). Recently GH therapy in GH deficient children could increase BMD in addition to improve short stature. We observed the treatment effects on bone mineral density in patients with Turner syndrome. METHODS: Bone Mineral Density in second to fourth lumbar spine area were measured by dual energy X-ray absorptiometry in 56 girls with Turner syndrome, before and after growth hormone and/or estrogen. All Turner girl was confirmed by clinical and chromosomal examination. RESULTS: 1) There was no significant difference in BMD according to karyotype. 2) The mean BMD of untreated Turner syndrome was 0.752+/-0.122g/cm2. 3) The mean BMD before and after GH treatment were 0.620+/-0.028g/cm2, 0.793+/-0.093g/cm2 respectively. The mean BMD before and after estrogen treatment were 0.761+/-0.125g/cm2, 0.918+/-0.141g/cm2 respectively. In combined group, the BMD were 0.752+/-0.087g/cm2 and 0.939+/-0.134g/cm2. Growth hormone was also effective to improve BMD as well as estrogen. But the changes of BMD were more significant in estrogen and combined group(p<0.05). 4) A significant positive correlation was found between age and BMD(p<0.05). CONCLUSIONS:Estrogen therapy can accelerate epiphysial maturation and compromise final height. Growth hormone therapy in Turner girls was effective for improvement bone mineral density as well as growth improvement. But growth hormone and estrogen combined therapy or Estrogen therapy is more effective to improve bone mineral density in Turner syndrome. Estrogen replacement can be delayed for a while on growth hormone treatment and the appropriated time of estrogen therapy should be elucidated.

A Case of Juvenile Xanthogranuloma Associated with Neurofibromatosis

We report a case of juvenile xanthogranuloma associated with neurofibromatosis. The patient was a 10-month-old female who had multiple yellowish papules on the face and shoulders A Biopsy of these lesions revealed foamy histiocytes with an admixture of inflammatory cells and Touton giant cells. Multiple, more than 6, cafe-au-lait spots, measuring more than 1.5cm in diameter were scattered on the trunk and buttocks Retardation of physical growth and development was observed.

A Case of Juvenile Xanthogranuloma Associated with Neurofibromatosis

We report a case of juvenile xanthogranuloma associated with neurofibromatosis. The patient was a 10-month-old female who had multiple yellowish papules on the face and shoulders A Biopsy of these lesions revealed foamy histiocytes with an admixture of inflammatory cells and Touton giant cells. Multiple, more than 6, cafe-au-lait spots, measuring more than 1.5cm in diameter were scattered on the trunk and buttocks Retardation of physical growth and development was observed.