Intravitreal delivery of AAV8 retinoschisin results in cell type-specific gene expression and retinal rescue in the Rs1-KO mouse.

X-linked juvenile retinoschisis (XLRS) is a neurodevelopmental abnormality caused by retinoschisin gene mutations. XLRS is characterized by splitting through the retinal layers and impaired synaptic transmission of visual signals resulting in impaired acuity and a propensity to retinal detachment. Several groups have treated murine retinoschisis models successfully using adeno-associated virus (AAV) vectors. Owing to the fragile nature of XLRS retina, translating this therapy to the clinic may require an alternative to invasive subretinal vector administration. Here we show that all layers of the retinoschisin knockout (Rs1-KO) mouse retina can be transduced efficiently with AAV vectors administered by simple vitreous injection. Retinoschisin expression was restricted to the neuroretina using a new vector that uses a 3.5-kb human retinoschisin promoter and an AAV type 8 capsid. Intravitreal administration to Rs1-KO mice resulted in robust retinoschisin expression with a retinal distribution similar to that observed in wild-type retina, including the expression by the photoreceptors lying deep in the retina. No off-target expression was observed. Rs1-KO mice treated with this vector showed a decrease in the schisis cavities and had improved retinal signaling evaluated by recording the electroretinogram 11-15 weeks after the application.

Crossover reactions between synthetic replicators yield active and inactive recombinants.

Self-replicating molecules can be synthesized through the covalent linkage of two complementary subunits to give a self-complementary structure. Complementarity refers to sizes, shapes, and the weak intermolecular forces involved in molecular recognition between the two subunits. In order to provide a model system for evolution at the molecular level, "crossover" or recombination experiments were staged with synthetic replicators. These reactions gave rise to new structural types. The ability (or inability) of the new recombinants to catalyze their own formation is shown to be a consequence of their molecular shapes.

Reciprocal expressions of cyclin E and cyclin D1 in hepatocellular carcinoma.

Deregulation of the cell cycle by overexpression of G1 cyclins, cyclin E and cyclin D1 genes, has been demonstrated to be a prerequisite for the development of human cancer. Recently, cyclin E is proposed to be sufficient for the progression of the G1 cell cycle without cyclin D1. Here we show that the proposed model system was specifically present in human hepatocellular carcinoma (HCC) unlike other human cancers. Of 31 HCC tissues analyzed, 21 (67.7%) exhibited an overexpression of cyclin E protein. In contrast to cyclin E gene expression, cyclin D1 expression was strongly downregulated in 19 (61.2%) HCCs. Interestingly, 65% of HCC tissues with overexpression of the cyclin E gene exhibited downregulation of cyclin D1, suggesting reciprocal deregulation of these cyclins in the G1 progression of the cell cycle. Southern blot analysis proved the amplification of cyclin E gene in HCC with a high level of overexpression. The present findings suggest that the reciprocal deregulation of cyclin E lacking cyclin D1 expression might play a role in G1 progression and the development of HCC.

Regulation of chondrogenic differentiation of mesenchymes by protein kinase C alpha.

Chondrogenesis of chick limb bud mesenchymes requires the expression and activation of protein kinase C (PKC). This study was performed to identify PKC isoform(s) involved in the regulation chondrogenic differentiation of mesenchymes. Multiple PKC isoforms including alpha, epsilon, zeta and lambda/iota were expressed in mesenchymes derived from chick limb buds. Among the expressed PKC isoforms, the levels of PKC alpha and epsilon were increased during chondrogenic differentiation of mesenchymes. The increase in the expression of these isoforms is more evident in the particulate membrane fraction compared with the cytosolic fraction. Chondrogenesis was blocked by either selective inhibition or down-regulation of PKC alpha. In addition, the degree of chondrogenesis was closely correlated with the expression levels of PKC alpha but not other PKC isoforms expressed in mesenchymes. Thus, the results indicate that only PKC alpha is required for the induction of chondrogenic differentiation

Economic burden of smoking in Korea.

OBJECTIVE: To support tobacco control policies in Korea by providing the estimated annual economic burden attributed to cigarette smoking. METHODS: The following two different approaches were used to estimate the cost: "disease specific" and "all causes". In the disease specific approach, we focused on estimating direct and indirect costs involved in treatments of cardiovascular, respiratory, and gastrointestinal diseases, and cancer as a result of smoking, by using an epidemiologic approach-the population attributable risk (PAR). To compute PAR, the relative risks of smoking in terms of physician visits, hospital admission, and death were estimated using the Cox proportional hazard model. In the all causes approach, we examined the differences in direct and indirect costs between smokers and non-smokers for all conditions and types of disease. The major data source was the Korea Medical Insurance Corporation cohort study, which had complete records of smoking status as of 1992 for 115 682 male and 67 932 female insured workers. RESULTS: By the disease specific approach, the estimated costs attributable to smoking in 1998 in Korea ranged from US 2269.42 million dollars (4.89 million dollars per 100,000 population; 0.59% of gross domestic product (GDP)) to 2956.75 million dollars (6.37 million dollars; 0.78% of GDP). The all causes approach yielded a minimum cost of 3154.75 million dollars (6.79 million dollars; 0.82% GDP) and a maximum of 4580.25 million dollars (9.86 million dollars; 1.19% GDP). CONCLUSION: The study confirms that smoking places a substantial economic burden on Korean society. In light of this, our study provides evidence for a strong need to develop a national policy to effectively control tobacco consumption in Korea.

Adjuvant therapy in cervical cancer patients with high risk factors.

Neoadjuvant and adjuvant chemotherapies are used adjunctively with surgery or radiation and are among the treatment options that are now employed for reducing treatment failure in early-stage cervical cancers with high-risk prognostic factors. Adjuvant therapies have been reported to significantly improve survival than would otherwise be possible with surgery or radiotherapy alone. However, for advanced cervical cancers, sequential or concurrent chemo-radiotherapy does not appear to significantly increase survival. The combination of radiotherapy with IFN-a2a and RA in the treatment of patients with locally advanced cervical cancer showed high response rates, however this should be confirmed in larger studies. Recent reports show that postoperative adjuvant radiotherapy has no benefit in survival, but that postoperative adjuvant chemotherapy has improved survival. Toxicities and the optimum number of cycles of neoadjuvant and adjuvant chemotherapy, as well as biologic therapy, will follow along with individualized treatment based on high-risk prognostic factors. Although more comprehensive studies and longer follow up will be required for complete evaluation of these adjuvant therapies, preliminary results are promising.

Cell-mediated immunity in patients with invasive carcinoma of the cervix.

Multiple in vitro immune parameters were investigated in thirty-four untreated patients with invasive carcinoma of the cervix and in twenty-five controls. The parameters measured were percentages and absolute counts of T and B cells, percentage of T cell subsets, lymphocyte response to phytohemagglutinin (PHA) and concanavalin A (Con A), natural killer (NK) activity, antibody-dependent cellular cytotoxicity (ADCC), and interleukin 2 (IL-2) activity. Patients with invasive cervical carcinoma, as compared with controls, showed a decrease in the percentage and count of T cells, a decrease in the percentage of helper-inducer (CD4+) T cells, decreased CD4+/CD8+ ratio, depressed lymphocyte response to PHA and Con A, and depressed NK and ADCC activities. There were no significant differences in these immune parameters between early and advanced tumor stages. The levels of total lymphocytes, monocytes, suppressor-effector (CD8+) T cells, and B cells were similar to those of the controls. IL-2 productivity in patients was lower than that in controls. In patients with invasive cervical carcinoma, a decrease in the percentage of CD4+ cells was associated with depressed PHA response and decreased IL-2 productivity was correlated with the reduced percentage of CD4+ cells and decreased NK activity. This study shows a significant defect in an important immune surveillance mechanism in patients with invasive carcinoma of the cervix and suggests that impaired IL-2 activity production may be related to quantitative and qualitative alterations in lymphocyte subpopulations which play a major role in immune surveillance against cervical cancer.

Analysis of risk factors for postmolar trophoblastic disease: categorization of risk factors and effect of prophylactic chemotherapy.

Early identification of high risk molar pregnancy is important in preventing the development of subsequent postmolar trophoblastic disease (PMTD). In the present study, evaluation of risk factors of developing PMTD, and indications for initiating prophylactic chemotherapy, and investigation of the effects of prophylactic chemotherapy were undertaken. One hundred and forty complete molar pregnancies treated at Yonsei University College of Medicine were retrospectively analyzed. Thirty-six cases of PMTD developed in these molar pregnancies during follow-up. Risk factors for PMTD were ranked according to frequency with which they were associated with PMTD. The patients with no risk factors were classified in the low-risk group, with one or two in the medium-risk group, and with three or more in the high-risk group. Prophylactic chemotherapy was administered to 14 of 52 low-risk, to 21 of 46 medium-risk, and to 17 of 42 high-risk patients. Among the high-risk patients, the time required for remission was significantly shorter in the group with prophylactic chemotherapy (13.5 weeks) than in the group without prophylactic chemotherapy (22.4 weeks). There were no differences in the duration until remission among the low- and medium-risk patients. Of the 52 patients who received prophylactic chemotherapy, 8 (15.4%) developed PMTD. Among the high-risk patients the occurrence of PMTD was significantly lower in the prophylactic chemotherapy group. Among the low-risk and medium-risk patients, there were no differences in the occurrence of PMTD between the chemoprophylaxis treated and untreated groups. Our results strongly support the use of prophylactic chemotherapy for patients that were designed under our high risk criteria. Prophylactic chemotherapy helps to prevent or reduce the risk of developing PMTD, and shorten the time required for complete remission in high-risk patients.

The effects of wild type p53 tumor suppressor gene expression on the normal human cervical epithelial cells or human epidermal keratinocytes transformed with human papillomavirus type 16 DNA.

The inactivation of p53 and p105RB by viral proteins or by mutations plays a key role in the oncogenesis of cervical carcinoma. The E6 and E7 proteins of HPV type 16 can bind to p53 and p105RB tumor suppressor gene products, respectively. In the present study, we tested a simple in vivo model that could explain the interactions between HPV E6 oncoprotein and p53 tumor suppressor protein. Our results showed that the life span of normal cervical epithelial cells was increased up to 4.5 times when transfected with expression vector containing E6/E7 ORF of HPV type 16. However, these cells did not divide after second crisis. Therefore, we employed an established human epidermal keratinocytes, RHEK-1. When transfected with an expression vector containing E6 ORF of HPV type 16, RHEK-1 cells showed anchorage independent growth character. When RHEK-E6 cells were transfected with wild type p53 expression vector, the growth rate of the RHEK-E6 cells was diminished. After 48 hours of transfection, many cells showed apoptotic signal but no more apoptotic signal was observed thereafter. These results suggested that the overexpression of the wild type p53 could overcome the dysfunction of the p53 on the cell cycle regulation imposed by E6 protein although not being of physiological condition.

The utilization of Doppler ultrasonography with color flow mapping in the diagnosis and evaluation of malignant trophoblastic tumors.

The Doppler ultrasound with color flow mapping image has been recently applied for the evaluation of gynecologic diseases, in particular, malignant trophoblastic tumors with the characteristic abundant blood flow. Doppler color flow mapping of uterine artery and intratumoral blood vessels was performed at a regular interval in all 26 patients including 3 cases of lost for follow up. Systolic/diastolic (S/D) ratio representing blood flow was measured in 19 cases of malignant trophoblastic tumors and 7 cases of hydatidiform mole diagnosed at the department of Obstetrics and Gynecology, Yonsei University, College of Medicine. The initial mean S/D ratio and standard deviation(SD) of uterine artery in 11 remitted and 5 non-remitted patients were 2.72 +/- 1.31 and 2.69 +/- 1.80, respectively. No significant difference was noted between two groups. However, the final S/D ratio of uterine artery in remitted group showed significantly higher values than non-remitted group, of which values were 6.23 +/- 2.38 and 3.08 +/- 1.54, respectively (P < 0.05). In aspect of blood flow changes in malignant trophoblastic tumors after chemotherapy, remitted group showed entirely disappeared blood flow, while non-remitted group had persistent blood flow. The mean S/D ratio and SD measured in hydatidiform mole patients were 5.43 +/- 1.65, of which value reflects higher resistance than malignant trophoblastic tumors. Also blood flow was not detected in all cases. This study suggests that color flow mapping Doppler ultrasound can be a useful method in diagnosing and monitoring the treatment in malignant trophoblastic tumors along with the conventional serum beta-hCG titration.

The effect of genetic counseling on performance of prenatal cytogenetic diagnosis.

Currently the number of pregnant women who have indications for, but do not receive, prenatal cytogenetic diagnosis is increasing. The purpose of this study was to review the prenatal cytogenetic services and to analyze the effect of genetic counseling on performance of the prenatal cytogenetic test. From January 1987 to July 1988, there were 2,796 deliveries at Severance Hospital, Yonsei Medical Center, of which 126 patients had indications for prenatal cytogenetic diagnosis. Chromosomal abnormalities were found in 5 patients (1, monosomy X; 1, trisomy 18; and 3, trisomy 21). Four patients were found in the group who had indications for prenatal cytogenetic diagnosis while only one was found in the group who did not (p less than 0.01). The most common indication for prenatal cytogenetic diagnosis was advanced maternal age (59%). The prenatal test rate was highest in patients whose indications were a previous child with chromosomal abnormality (100%) and parental translocation carrier (100%). Most (89%) of the patients were tested by amniocentesis between the 16th and 20th week of gestation. The two most common reasons for patients not receiving a prenatal cytogenetic diagnosis were late registration (41%) and absence of genetic counseling (34%).

Previous experience of induced abortion as a risk factor for fetal death and preterm delivery.

As part of a community-based study in Korea to evaluate the effects of previous induced abortion on length of gestation and pregnancy outcome of subsequent pregnancies, we analyzed data obtained from January 1979 to December 1981 on pregnancies reported to family health workers in Kang Hwa Island, Korea. The preterm, live-birth rates were not significantly associated with previous induced abortion. Overall, the life table-estimated fetal death rate for women enrolled at the eighth or earlier weeks of gestation was 13.7%, 10.2% for women with no previous induced abortion and 28.9% for women with previous induced abortion. The relative risk for fetal death for women who had undergone a previous abortion was 2.8; relative risk for parous women compared to nulliparous women was 3.4. After controlling for parity, previous induced abortion was not a significant variable for fetal death rate.

Pharmacological characterization of LB50016, N-(4-amino)butyl 3-phenylpyrrolidine derivative, as a new 5-HT1A receptor agonist.

LB50016 was characterized as a selective and potent 5-HT1A receptor agonist and evaluate its anxiolytic and antidepressant activities. It shows high affinity for 5-HT1A receptor, moderate affinity for alpha 2 adrenergic and 5-HT2A receptors and no significant affinity for other receptors tested. Hypothermia and increased serum corticosterone level were observed in LB50016-treated rats, which are mediated mostly by post synaptic 5-HT1A receptor activation. In the mouse forced swim model for depression, LB50016-elicited dose-dependent reductions in immobility time, showing ED50 of approximately 3 mg/kg i.p., which was blocked by pretreatment of NAN-190, 5-HT1A antagonist. In face-to-face test for anxiolytic activity in mice, estimated ED50 was 2 mg/kg, i.p. In isolation-induced aggression test with mice, fifty-fold increases in latency to attack were observed at 30 min and last up to 4 h after LB50016 treatment (3 mg/kg, i.p.). Taken together, LB50016-induced pharmacological activities are mediated by activation of 5-HT1A receptors, offering an effective therapeutic candidate in the management of anxiety and depression in humans.

Interferon-alpha 2a, 13-cis-retinoic acid and radiotherapy for locally advanced carcinoma of the cervix: a pilot study.

UNLABELLED: Standard treatment with radiotherapy for locally advanced cancer of the uterine cervix has a response rate of less than 50%. Recently, concurrent chemotherapy with radiotherapy was introduced into the clinic but is value remains controversial. Interferon and retinoic acid possess antiproliferative, immunomodulatory, and antineoplastic activities. The combination of interferon and retinoic acid has significant activity in patients with squamous cell carcinoma. These compounds may also potentiate radiation cytotoxicity. This pilot study aimed to assess the clinical efficacy and tolerability of the combination of interferon-alpha 2a, 13-cis-retinoic acid and radiotherapy. Patients with locally advanced carcinoma of the cervix were treated at Severance Hospital, Yonsei University College of Medicine. Fifteen patients received the combination of interferon-alpha 2a, 13-cis-retinoic acid and radiotherapy. Twelve patients treated in previous years with comparable radiotherapy and concurrent chemotherapy served as historical controls. RESULTS: (1) Interferon-alpha 2a, 13-cis-retinoic acid and radiotherapy resulted in a 47% response rate (33% complete remissions) while patients treated with concurrent chemoradiotherapy had a 42% response rate (17% complete remissions) (2). Major toxicity of interferon-alpha 2a, 13-cis-retinoic acid and radiotherapy was fever (60%). There was no grade 3 or 4 toxicity. CONCLUSION: Systemic interferon-alpha 2a, 13-cis-retinoic acid and radiotherapy is an active and tolerable therapy for locally advanced cervical cancer. Randomized studies are required to define the role of bioradiotherapy in the treatment of advanced cervical cancer.

Triple innominate osteotomy in young adults for the treatment of acetabular dysplasia: a 9-year follow-up study.

Fifty patients who underwent 60 triple innominate osteotomies were reviewed radiographically and clinically using a modified Harris hip score (HHS). Average patient age was 26 years (range: 13-48 years). At average 9-year follow-up (range: 5-14 years), 12 (20%) hips had been converted to total hip arthroplasty (THA) and 4 (7%) hips had incapacitating pain. Sixteen (27%) hips were considered failures. Average modified HHS at final follow-up was 67 (range: 28-91). Forty-nine (98%) of 50 patients reported they would recommend the procedure to others in the same situation. Radiographically, there was significant improvement in the center-to-edge angle of Wiberg and the acetabular angle of Sharp. There also was a statistically significant relationship between failure of the osteotomy and severity of preexisting hip arthrosis as measured by the Tonnis criteria. The results demonstrate triple innominate osteotomy was effective in eliminating pain, but the fact that 27% of hips required or will require THA indicates results may deteriorate with time.

Telomerase activity in pterygeal and normal conjunctival epithelium.

All pterygia have similar histologic features of solar degeneration seen in the skin such as acanthosis, keratosis, or hyperkeratosis. Although the pathogenesis of pterygia is still unclear, an association with solar exposure, in particular with UV radiation, has been reported. Telomerase activity has been found to be higher in some degenerative, precancerous, and cancerous skin lesions. We investigated telomerase activity in the epithelium and the stromal tissues of the pterygium. Pterygeal tissues were obtained from 30 patients. Telomerase activity was measured with TRAPeze-ELISA kit. Three of the 28 (10.7%) pterygeal stromal tissues demonstrated positive telomerase activity. Fourteen of the 27 (51.9%) epithelial tissues were positive in telomerase activity, whereas telomerase activity was positive in only 3 of 9 normal epithelia (33.3%). Telomerase activity in the pterygium-covered epithelium was increased as compared with that seen in the normal epithelium, but the increase was not statistically significant. In conclusion, telomerase activity was somewhat increased in pterygeal tissues. Telomerase activity may be involved in the pathogenesis of pterygium.