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.

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.

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.

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.

Postoperative adjuvant therapy in early invasive cervical cancer patients with histopathologic high-risk factors.

The purpose of this study is to evaluate the efficacy of postoperative adjuvant therapy in preventing treatment failure after primary treatment with surgery in early invasive cervical cancer patients associated with the following histopathologic high-risk factors: lymph node metastasis (either macroscopic or microscopic), parametrial extension, lymphovascular permeation and depth of invasion > or =10 mm. Postoperative adjuvant concurrent chemoradiotherapy (PCCRT), postoperative adjuvant chemotherapy (PCT), or postoperative adjuvant radiotherapy (PRT) alone was administered to the 80 early invasive cervical cancers with at least one of the high-risk factors. Each of 61 patients received three to six cycles of chemotherapy at intervals of approximately 3 weeks. Twenty three patients were treated with PCCRT, 38 patients were treated with PCT alone, and 19 patients received PRT. The 5-year survival rates of patients with macroscopic lymph node metastasis were 66.7% and 35.7% in PCCRT and PRT, respectively. With microscopic lymph node metastasis, the 5-year survival rates were 83.3%, 60.0%, and 70.1% in PCCRT, PCT, and PRT, respectively. With parametrial extension, the 5-year survival rate was 58.1% in PCCRT. The 5-year survival rates of patients with lymphovascular permeation were 100%, 90.9%, and 66.7% in PCCRT, PCT, and PRT, respectively. With depth of invasion > or =10 mm, the 5-year survival rates were 100% and 91.3%, in PCCRT and PCT, respectively. PCCRT appears to be superior to PRT or PCT alone in early invasive cervical cancer patients with histopathologic high-risk factors.

A broad-spectrum caspase inhibitor blocks concanavalin A-induced hepatitis in mice.

Fulminant hepatic failure (FHF) is a clinical syndrome resulting from massive death of liver cells or sudden and severe impairment of liver function. The causes of FHF are diverse and the overall mortality is very high. Recently, it became clear that apoptosis of hepatocytes is the critical cause of acute hepatic failure in FHF. It is well known that a family of cysteine proteases called caspase is one of the key mediators of the apoptotic pathway. Thus, caspases are attractive potential targets for the treatment of disorders resulting from excessive apoptosis. In this report, we examined the activity of a new caspase inhibitor, Xyz 033 mp. This nonpeptide inhibitor showed broad-spectrum caspase-inhibiting activity and protected primary rat hepatocytes from apoptotic death. In a mouse model of FHF induced by concavalin A (Con A), Xyz 033 mp suppressed elevated AST and ALT and specifically reduced IL-1 beta concentration. Also, Xyz 033 mp rescued mice from lethal experimental hepatitis induced by Con A. In addition, histological examinations indicated that Xyz 033 mp protected hepatocytes from the fatal apoptogenic effect of Con A. These results suggest that Xyz 033 mp may be a candidate therapeutic agent for FHF caused by massive apoptotic death of hepatocytes.

Association of focal adhesion kinase with fibronectin and paxillin is required for precartilage condensation of chick mesenchymal cells.

We show that tyrosine phosphorylation of FAK was increased as precartilage condensation occurred, followed by a subsequent decrease in proliferation of in vitro micromass culture of wing bud mesenchymal cells. FAK was associated with fibronectin and paxillin, which were maximal at day 3 of culture. FAK was also associated with signaling molecules such as PLC-gamma and PI3-kinase through c-Src. The beta1 integrin antibody and several inhibitors of signaling molecules such as herbimycin A, U73122, LY294002, as well as cytochalasin D, an actin depolymerizing agent, remarkably decreased tyrosine phosphorylation of FAK and its association with fibronectin and paxillin during condensation. resulting in a marked inhibition of condensation and chondrogenesis. Taken together, our findings suggest that beta1 integrin-mediated interaction of mesenchymal cells and fibronectin signals to accelerate the precartilage condensation through tyrosine phosphorylation of FAK and its association with paxillin. This signaling pathway is required for precartilage condensation and subsequent cartilage nodule formation in chondrogenesis.

Concurrent chemotherapy and radiotherapy in invasive cervical cancer patients with high risk factors.

The aim of this study was to evaluate the survival of 395 previously untreated cervical cancer patients with at least one high risk factor following concurrent chemoradiation and to assess the toxicities. Two different chemotherapy regimens were used for concurrent chemoradiation. In the patients with squamous cell carcinoma, 100 mg/m2 of cisplatin was infused intravenously, followed immediately by five consecutive daily administrations of 5-fluorouracil, 1,000 mg/m2/day, each infused intravenously over 24 hr. As for the patients with adenocarcinoma, 70 mg/m2 of cisplatin, 250 mg/m2 of cytoxan and 45 mg/m2 of adriamycin were administered intravenously on days 1, 2, and 3, respectively. The 5-year survival rate was 54.4% with stage III and IV, 62.6% with lymph node metastasis on computed tomogram or MRI, 77.9% with stage I-II disease with lesion size > or =4 cm, and 50.3% with small cell carcinoma or adenocarcinoma. Side effects from concurrent chemoradiation such as nausea, vomiting, and alopecia were present in all 395 cases. Anemia, leukopenia, thrombocytopenia, hepatotoxicity, and nephrotoxicity were observed to varying degrees, but there was no toxic death. This study suggests that cisplatin-based concurrent chemoradiation in treating cervical cancer patients with high risk factors is effective and relatively well tolerated, with acceptable toxicity.

Disruption of actin cytoskeleton induces chondrogenesis of mesenchymal cells by activating protein kinase C-alpha signaling.

Disruption of actin cytoskeleton with cytochalasin D has been known to induce chondrogenic differentiation of chick embryo limb bud mesenchymal cells. However, the mechanism(s) for the induction of chondrogenesis by cytochalasin D is not yet clearly known. In the present study, we examined possible involvement of protein kinase C (PKC) and extracellular signal-regulated protein kinase (Erk-1) in chondrogenesis of mesenchymal cells induced by disruption of actin cytoskeleton. Disruption of actin cytoskeleton with cytochalasin D or latrunculin B induced chondrogenesis of mesenchymal cells cultured at subconfluent cell density, as determined by type II collagen expression. Among the expressed PKC isoforms, cytochalasin D dramatically increased expression and activation of PKCalpha in a dose-dependent manner, and inhibition or downregulation of PKCalpha blocked cytochalasin D-induced chondrogenesis. Cytochalasin D also downregulated Erk-1 phosphorylation that is associated with chondrogenesis. Our results, therefore, suggest that disruption of actin cytoskeleton induces chondrogenesis of mesenchymal cells by activating PKCalpha and by inhibiting Erk-1 signaling.

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.

Spontaneous apoptosis as a predictor of radiotherapy in patients with stage IIB squamous cell carcinoma of the uterine cervix.

The purpose of this study was to investigate the correlation between the spontaneous apoptotic index (SAI) determined from pretreatment biopsy specimens with the various clinical outcomes of patients with FIGO stage IIB squamous cell carcinoma of the uterine cervix in a retrospective analysis. Forty-eight patients treated with curative radiotherapy between 1989 and 1993 were evaluated. Pretreatment biopsy specimens of those patients were scored for apoptosis, mitosis, and proliferating cell nuclear antigen (PCNA) immunohistochemical staining. The range of the SAI was 0.2-4.7% (median 1.1%). Patients whose tumours had a SAI above the median had better local control (p = 0.0062) and overall survival (p = 0.0053) than those with a lower SAI. Furthermore, the SAI was marginally significant on local control by a multivariate Cox regression analysis (p = 0.0571). There was no correlation between the SAI and proliferation (mitosis and PCNA).

N-substituted-3-arylpyrrolidines: potent and selective ligands at serotonin 1A receptor.

3-Arylpyrrolidines are synthesized through the coupling of N-benzyl-3-(methanesulfonyloxy)pyrrolidine with diarylcuprates. Pharmacological evaluation of a series of N-substituted-3-arylpyrrolidines toward several neurotransmitter receptors indicated that some of them are good ligands for serotonin 1A receptor. Particularly, N-[(N-saccharino)butyl]pyrrolidines were found to be potent and selective ligands. A preliminary biological evaluation for several selected compounds indicated that they are potentially effective antianxiety and antidepressant agents.

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.

Modulation of MAP kinase signaling and growth characteristics by the overexpression of protein kinase C in NIH3T3 cells.

This study was performed to examine effects of the overexpression of protein kinase C (PKC) isoforms (i.e., beta I, beta II, gamma, delta, eta, and zeta) on mitogen-activated protein (MAP) kinase (Erk-1 and -2) signaling and growth characteristics of NIH3T3 cells. Phorbol ester (PMA) activated endogenous and ectopically expressed PKC alpha, beta I, beta II, gamma, delta, epsilon, and eta. Overexpression of the examined PKC isoforms enhanced PMA-induced MAP kinase activation. Potentiation of MAP kinase activation was also observed upon stimulation of cells with platelet-derived growth factor (PDGF) although there was no indication for the activation PKC isoforms by PDGF. Inhibition of PKC blocked PMA- but not PDGF-induced MAP kinase activation. Thus, potentiation of PDGF-induced MAP kinase activation appears to be independent to PKC activity, while PMA-induced MAP kinase activation requires PKC activity. The ability of PKC isoforms to potentiate MAP kinase activation is not related to the growth characteristics of cells because individual PKC isoforms differentially regulated maximum density and proliferation of cells.

Hepatic metastases from carcinoma of the uterine cervix.

OBJECTIVE: To investigate the patterns of hepatic involvement and the outcome of patients with hepatic metastases from carcinoma of the uterine cervix. METHODS: Of 1665 patients with carcinoma of the uterine cervix, 20 patients with hepatic metastases were detected clinically during the course of the disease. Clinical presentation and detailed patterns of hepatic involvement were retrospectively reviewed for these patients. Comparative analysis between patterns of heaptic metastases and survival data was also undertaken. RESULTS: Hepatic metastasis from carcinoma of the uterine cervix were nearly always accompanied by uncontrolled locoregional diseases and/or extrahepatic metastases, whereas only 1 patient developed an isolated hepatic metastasis. Ninety percent of the hepatic metastases were metachronously detected. The median time from the appearance of primary carcinoma to detection of hepatic metastases was 39 months, but late metastases after 5 years were not uncommon. Metastatic lesion in 16 patients consisted of multiple tumors distributed in either one or both anatomical lobes, whereas only 4 patients had a solitary lesion confined to a single lobe. Patients with hepatic metastases were unlikely to survive 2 years with a median survival of 10 months. CONCLUSION: Favorable patterns of hepatic metastases in patient with carcinoma of the uterine cervix were not major determinants of favorable survival if components of extrahepatic disease were concomitantly present.

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

Interaction of phospholipase C gamma 1 via its COOH-terminal SRC homology 2 domain with synaptojanin.

The role of the phospholipase C gamma 1 (PLC gamma 1) in signal transduction was investigated by characterizing its interactions with proteins that may represent components of a novel signalling pathway. A 145-kDa protein that binds SH2 domain of PLC gamma 1 was purified from rat brain. The sequence of peptide derived from the purified binding protein now identify it as synaptojanin, a nerve terminal protein that has been implicated in the endocytosis of fused synaptic vesicles and shown to be a member of the inositol polyphosphate 5-phosphatase family. We demonstrate here stable association of PLC gamma 1 with synaptojanin, a protein that not only binds carboxyl terminal SH2 domain of PLC gamma 1, but also inhibits PLC gamma 1 activity.

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.

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.