Enhanced Controlled Transdermal Delivery of Ambroxol from the EVA Matrix.

To avoid the systemic adverse effects that might occur after oral administration, transdermal delivery of ambroxol was studied as a method for maintaining proper blood levels for an extended period. Release of ambroxol according to concentration and temperature was determined, and permeation of drug through rat skin was studied using two chamber-diffusion cells. The solubility according to PEG 400 volume fraction was highest at 40% PEG 400. The rate of drug release from the EVA matrix increased with increased temperature and drug loading doses. A linear relationship existed between the release rate and the square root of loading rate. The activation energy (Ea) was measured from the slope of the plot of log P versus 1000/T and was found to be 10.71, 10.39, 10.33 and 9.87 kcal/mol for 2, 3, 4 and 5% loading dose from the EVA matrix, respectively. To increase the permeation rate of ambroxol across rat skin from the EVA matrix, various penetration enhancers such as fatty acids (saturated, unsaturated), propylene glycols, glycerides, pyrrolidones, and non-ionic surfactants were used. The enhancing effects of the incorporated enhancers on the skin permeation of ambroxol were evaluated using Franz diffusion cells fitted with intact excised rat skin at 37° using 40% PEG 400 solution as a receptor medium. Among the enhancers used, polyoxyethylene-2-oleyl ether increased the permeation rate by 4.25-fold. In conclusion, EVA matrix containing plasticizer and permeation enhancer could be developed for enhanced transdermal delivery of ambroxol.

Different contribution of co-stimulatory molecules B7.1 and B7.2 to the immune response to recombinant modified vaccinia virus ankara vaccine expressing prM/E proteins of Japanese encephalitis virus and two hepatitis B virus vaccines.

This study clarifies the role of co-stimulatory molecules B7.1 and B7.2 in the immune response to 3 types of vaccines: a/ recombinant modified Vaccinia virus Ankara (MVA) (vJH9) expressing prM/E proteins of Japanese encephalitis virus (JEV), b/ recombinant yeast-expressed Hepatitis B virus (YHBV), c/ human plasma-derived Hepatitis B virus (PHBV). We constructed plasmids expressing B7.1 and B7.2 molecules and found that the expression level of B7.2 protein in transfected CHO-k1 cells was higher than that of B7.1 protein. Mice were co-injected with vaccines vJH9, YHBV and PHBV and plasmids expressing B7.1 or B7.2, respectively, and specific antibody titers for each vaccine were monitored at days 7, 14 and 28 post injection (p.i.). In mice injected with vJH9 vaccine and both B7 plasmids, plasmid B7.2 induced a higher anti-JEV immune response than plasmid B7.1. This implies that the stimulation of the B7.2 immune pathway may be a feasible method of boosting protective immunity against a recombinant viral vaccine. Both B7 molecules were able to induce a specific anti-HBV immune response using YHBV vaccine. On the other hand, B7 molecules had little effect to the specific antibody induction in PHBV vaccination. These results suggested that the contribution of B7.1 and B7.2 molecules in an immune response depended on the character and status of the presenting antigen.

Detection of hepatitis A virus from clotting factors implicated as a source of HAV infection among haemophilia patients in Korea.

To investigate the causal relationship of blood clotting factors and hepatitis A virus (HAV) infection in haemophilia patients during 1998-1999 in Korea, we performed a 1:3 matched case-control study and molecular detection of HAV from clotting factors and patients. The epidemiological investigation showed that one lot of clotting factor VIII was related epidemiologically to patients with hepatitis A with an odds ratio of 35.0, or 38.4 when adjusted for the interval between injections. We examined 17 sera collected from seven patients and 124 lots of blood clotting factors (factor VIII and factor IV) by HAV reverse transcriptase-polymerase chain reaction (RT-PCR). HAV RNA was detected in five clotting factors and six sera. The HAV sequence of one of the factor VIII samples was identical to the sequences found in three patients' sera. Findings from the laboratory and epidemiological studies suggested that the clotting factor was causally related to HAV infection in three haemophilia patients.

A seroprevalence study of poliovirus antibody among primary schoolchildren in Korea.

We aimed to determine the seroprevalence of poliovirus antibody in Korea by using the cell culture neutralization method recommended by the WHO. A total of 500 sera collected from children at eight primary schools in Kyunggi province were used for this study. We found that 82.2% of children were positive for all three types of poliovirus and antibody-positive rates for types I, II and III were 94.4, 96.6 and 86.8% respectively, indicating that seropositive rates for types I and II were considerably higher than for type III (P<0.0001). This result implies that the type III component of the oral polio vaccine should be evaluated further. Although a greater number of children, including young infants, need to be tested for seroprevalence, this study still provides us with valuable information on the effectiveness of vaccination against polioviruses in Korea.

Genetic analysis of the VP1 region of human enterovirus 71 strains isolated in Korea during 2000.

We have isolated Human enterovirus 71 (EV71) from stool and CSF samples taken from patients with acute flaccid paralysis, herpangina, or hand, foot and mouth disease in 2000. Both the cell culture-neutralization test and RT-PCR were used to detect enteroviruses. Rhabdomyosarcoma (RD), HEP2c, and BGM cells were used for the isolation of viruses, and serotypes were determined by the neutralization test using EV71-specific antiserum. For genomic analysis, we amplified a 437-bp fragment of the 5'-noncoding region of the enterovirus genome and a 484-bp fragment of the VP3/VP1 region of EV71 by RT-PCR, with positive results. Products amplified using an EV71-specific primer pair were sequenced and compared with other isolates of EV71. Analysis of the nucleotide sequences of the amplified fragments showed that the EV71 isolates from patients were over 98% homologous and belonged to the genotype C.

Short report: genetic heterogeneity of Japanese encephalitis virus assessed via analysis of the full-length genome sequence of a Korean isolate.

We determined the full-length genome sequence of Japanese encephalitis virus (JEV) K94P05 isolated in Korea. Sequence analysis showed that the 10,963-nucleotide-long RNA genome of K94P05 was 13 or 14 nucleotides shorter than the genome of other JEV isolates because of a deletion in the 3' noncoding region of K94P05. Compared with sequences of other JEV isolates, the full-length nucleotide sequence showed 89.0-89.6% homology, and the deduced amino acid sequence showed between 96.4-97.3% homology. A region of approximately 60 nucleotides immediately downstream of the open reading frame stop codon of K94P05 showed high sequence variability as compared with other JEV isolates. K94P05 formed a distinct group within a phylogenetic tree established with the full-length genome sequences. Cross-neutralization studies showed that polyclonal antibodies to Korean isolates were 3 times better at neutralizing the Korean isolates than antibodies to Nakayama-NIH. These findings suggest that Korean JEV K94P05 is genetically and antigenically distinct from other Asian JEV isolates.

Sequence analysis of hemagglutinin and nucleoprotein genes of measles viruses isolated in Korea during the 2000 epidemic.

To characterize the genetic properties of currently circulating measles viruses in Korea, the complete nucleotide sequences of hemagglutinin (H) protein and nucleoprotein (N) genes of Korean viruses were analyzed. The entire genes of H and N were directly amplified by RT-PCR from each clinical specimen and sequenced. Sequence analyses of H and N genes indicated that all Korean viruses had a high degree of homology (>99.8%) when compared with each other. The Korean viruses differed from other wild-type viruses by as much as 6.8% in the H gene and 6.5% in the N gene at the nucleotide level. The deduced amino acid variability was up to 6.4% for the H protein and up to 6.5% for the N protein. Phylogenetic analyses of nucleotide sequences and deduced amino acid sequences of the H and N genes revealed that all Korean viruses were grouped into the clade H1.

Light, carbon dioxide, and octenol-baited mosquito trap and host-seeking activity evaluations for mosquitoes in a malarious area of the Republic of Korea.

Two field trials for commercially available and experimental mosquito traps variously baited with light, carbon dioxide, octenol, or combinations of these were evaluated in a malarious area at Paekyeon-Ri near Tongil-Chon (village) and Camp Greaves, Paju County, Kyonggi Province, Republic of Korea. The host-seeking activity for common mosquito species was determined using hourly aspirator collections from a human- and propane lantern-baited Shannon trap. The total number of mosquitoes and number of each species captured during the test were compared using 8 x 8 and 5 x 5 Latin square designs based on trap location. Significant differences were observed for the total number of mosquitoes collected in the 8 x 8 test, such that counterflow geometry (CFG) with CO2 > or = CFG with CO2 and octenol > or = Shannon trap > or = Mosquito Magnet with octenol > American Biophysics Corporation (ABC) light trap with light, CO2 (500 ml/min), and octenol > or = ABC light trap with light and dry ice > or = ABC light trap with light and CO2 > ABC light trap with light only. A concurrent 5 x 5 test found significant differences in trap catch, where Mosquito Magnet with octenol > New Jersey light trap > or = EPAR Mosquito Killer with CO2 > or = ABC light trap with light and dry ice > Centers for Disease Control (CDC) light trap (manufactured by John W. Hock) with light and octenol. Significant differences in trap catch were noted for several species including: Aedes vexans, Anopheles sinensis, An. yatsushiroensis, An. lesteri, Culex pipiens, and Cx. orientalis. Traps baited with octenol captured significantly fewer Cx. pipiens than those not baited with octenol. Likewise, no Cx. orientalis were captured in octenol-baited traps. Host-seeking activity showed a similar bimodal pattern for all species captured. Results from these field trap evaluations can significantly enhance surveillance efforts. Significantly greater numbers of mosquitoes were captured with mosquito traps using counterflow technology (e.g., Mosquito Magnet and CFG traps) when compared to standard light and carbon dioxide-baited traps. Additionally, field evaluations demonstrate that various traps can be utilized for isolation and detection of arboviruses and other pathogens.

Introduction of HIV type 1 subtype E virus into South Korea.

Subtype E HIV-1 is the most prevalent strain in Southeast Asia. Although subtype B is prevalent in Korea, geographical distance and increases in travel may lead to the spread of subtype E in Korea. Therefore, we tried to identify and monitor the patterns of HIV subtype E virus introduction into Korea. The divergence of nucleotide sequences within the envelope region (V3 to V5) of Korean subtype E isolates ranged from 4.3 to 14.6% (n = 8; mean, 9.5 +/- 2.8%). In pairwise comparisons of subtype E isolates between Korea and other regions, the divergence of nucleotide sequences between 8 Korean and 16 Asian subtype E variants ranged from 1.3 to 15.2% (mean, 7.8 +/- 2.6%), whereas the divergence of nucleotide sequences between 8 Korean and 2 African variants ranged from 11.7 to 20.7% (mean, 15.4 +/- 2.2%). A phylogenetic tree showed that Korean subtype E isolates cluster with the Asian isolates but far from the African isolates. These epidemiological and molecular epidemiological data suggest that HIV-1 subtype E strains have been transmitted into Korea from endemic areas of Southeast Asia rather from Africa.

Antibody responses in humans to an inactivated hantavirus vaccine (Hantavax).

Hantaviruses cause haemorrhagic fever with renal syndrome (HFRS) and result in severe human morbidity and mortality. Safe and effective vaccines are needed urgently in order to reduce the incidence of human illness. Hitherto studies of hantavirus vaccine efficiency have been limited to individuals at low risk of infection. In this study the immune response to an inactivated hantavirus vaccine was measured in 64 human volunteers at high risk of infection by virtue of residence and occupation. 30 d after vaccination, 79% of subjects developed a significant hantavirus antibody titre as measured by immunofluorescence (IFA) and 62% by enzyme linked immunosorbent assay (ELISA). Seroconversion rates increased to 97% one month after the booster dose. Neutralising antibody titres paralleled this trend with 13% of vaccine recipients producing neutralising antibody one month after the first dose and 75% of vaccine recipients responding one month after boosting. Antibody titres had declined by one year, however, with only 37% and 43% of sera positive by IFA and ELISA, respectively. Re-vaccination at this time produced a vigorous anamnestic response with 94% and 100% of vaccine recipients yielding positive antibody titres. Only 50% of the sampled population, however, produced neutralising antibodies following the booster dose one year later. The vaccine was well tolerated and there were no apparent differences in the responses of males and females. However, further improvement of this vaccine is necessary in order to induce a more longlasting humoral immune response.

Identification of IY81149 and its metabolites in the rat plasma using the on-line HPLC/ESI mass spectrometry.

Reversed-phase high-performance liquid chromatography/mass spectrometry (HPLC/MS) with an electrospray ionization (ESI) interface was applied to the identification of metabolites of IY 81149 in the rat plasma. Fragments obtained using collision-induced dissociation (CID) in both positive and negative modes were utilized to elucidate the structure of metabolites. The eluent from the conventional HPLC column was split and directly introduced into an ESI-mass spectrometer for the identification of the structures. The CID technique allowed the sensitive identification of sulfonyl-IY81149 and hydroxy-IY81149 from the rat plasma.

The esthetics of the smile: a review of some recent studies.

PURPOSE: This article reviews recent research on the esthetics of the smile, covering the attractiveness of the smile, the effect of aging on the smile, oral condition and the smile, personality and smile, and smile exercises. MATERIAL AND METHODS: The subjects were Koreans with normal occlusion. Photographs of a full smile were taken and the esthetic quality of the subjects' smiles was estimated. Smile scores were correlated with oral condition, personality, the practice of smile exercises, and elements of the smile, such as the position of the lip in a smile. The personality of the subjects was assessed by means of a Sixteen Personality Factor Questionnaire. Gibson's smile exercises were used to investigate the effect of smile exercise. RESULTS: In an attractive smile, the full shape of the maxillary anterior teeth was shown between the upper and lower lip, the upper lip curved upward or was straight, the maxillary anterior incisal curve was parallel to the lower lip, and teeth were displayed to the first molar. The amount of maxillary incisal exposure gradually decreased with age, accompanied by a gradual increase in mandibular incisal exposure. Personality traits such as warmth, calmness, extroversion, and low anxiety were closely related to an attractive smile. Smile exercises were an effective means of improving the esthetic level of the smile if patients exercised continuously. CONCLUSION: An attractive smile is important for esthetic treatment. The lip position, oral condition, personality traits, and smile exercise affect the esthetics of the smile.

Protection against lethal Japanese encephalitis virus infection of mice by immunization with the highly attenuated MVA strain of vaccinia virus expressing JEV prM and E genes.

Genes encoding the glycosylated precursor of the membrane (prM) and envelope (E) proteins of a Korean strain of Japanese encephalitis virus (JEV) were inserted into the genome of the host-range restricted, highly attenuated, and safety-tested MVA strain of vaccinia virus. MVA recombinants containing the JEV genes, under strong synthetic or modified H5 vaccinia virus promoters, were isolated. Synthesis of JEV prM and E proteins was detected by immunofluorescence microscopy, flow cytometry, and polyacrylamide gel electrophoresis. Mice inoculated and boosted by various routes with either of the MVA recombinants produced JEV neutralizing antibodies, that had titres comparable with those induced by an inactivated JEV vaccine, as well as haemagglutination-inhibiting antibodies. Mice immunized with 2 x 10(6) infectious units of MVA/JEV recombinants by intramuscular or intraperitoneal routes were completely protected against a 10(5) LD50 JEV challenge at 9 weeks of age.

Envelope gene sequence variation among Japanese encephalitis viruses isolated in Korea.

The nucleotide (nt) sequences of the envelope (E) gene of 4 Japanese encephalitis virus (JEV) isolates from Korea (K82PO1, K87P39, K91P55 and K94PO5) were determined and the deduced amino acid (aa) sequences were compared within themselves and with the published sequences of 16 other JEV strains originating from other parts of Asia. Homologies of 87.2 - 95.6% at the nt level and 95.8 - 98.0% at the aa level among the Korean JEV isolates were found. aa positions 89, 129, 220, 225, 327, 366, 456 and 477 characterized the Korean isolates. According to the phylogenetic analysis based on the E gene nt sequence, the Korean isolates formed distinct subgroup consisting of at least 2 genetic types.

Stem cell factor protects bone marrow-derived cultured mast cells (BMCMC) from cytocidal effect of nitric oxide secreted by fibroblasts in murine BMCMC-fibroblast coculture.

The survival of mast cells are dependent on two kinds of growth factors, one derived from T cells (IL-3) and another derived from fibroblasts (stem cell factor [SCF]). The 3T3 fibroblast cell line derived from WCB6F(1-)+/+ mouse embryos (+/+ 3T3 fibroblasts) supported the proliferation of bone marrow-derived cultured mast cells (BMCMC) in the PWM-stimulated spleen cell conditioned medium (PWM-SCM), whereas the 3T3 fibroblast cell line from WCB6F1-Sl/Sld mouse embryos (Sl/Sld 3T3 fibroblasts) did not. To study the role of nitric oxide (NO) on the growth of mast cells in BMCMC-fibroblast coculture, we used a NO synthase inhibitor, NG-monomethyl-L-arginine (NGMMA). NGMMA recovered survival and maintained proliferation of mast cells in BMCMC-Sl/Sld 3T3 fibroblasts coculture. Sl/Sld 3T3 fibroblasts as well as 3T3 fibroblasts from NIH(-)+/+, BALB(-)+/+ or Swiss(-)+/+ mouse embryos secreted NO in PWM-SCM, but not in alpha-MEM. SCF protected BMCMC from cytotoxicity of exogenous NO in IL-3-supplemented alpha-MEM. We concluded that SCF might protect BMCMC from cytocidal effect of NO in BMCMC-fibroblasts coculture.

Antigenic and genetic analysis of Japanese encephalitis viruses isolated from Korea.

The characteristics of the five Korean isolates of Japanese encephalitis (JE) virus were compared with those of the already reported JE virus strains from Japan and China using the hemagglutination test and polymerase chain reaction direct sequencing of the JE virus genomes (capsid/premembrane, envelope region). The hemagglutination patterns of all the isolates were distinctly different from the Nakayama-NIH strain. The optimal pH of hemagglutination of all the Korean isolates was 6.6-7.0 and the reaction range was broader than that of the Nakayama-NIH strain. The 198 nucleotide sequences in the capsid/premembrane gene region of the five Korean strains indicated that they were classified into the third genotype group, the JE strains from the countries in the temperate zone including the Nakayama-NIH, JaOArS982, and Beijing-1 strains. Four of the five Korean isolates formed a unique phylogenetic tree within the third genotype group, although the last one was genetically highly related to the Nakayama-NIH strain. The 251 nucleotide sequences in the envelope region of the five isolates were more divergent than the capsid/premembrane region. Four of the five isolates showed a large nucleotide divergency as compared with the JaOArS982 strain (< or = 12.4%), but the last one was similar to the JaOArS982 strain (98% of nucleotide homology). These results suggest the evolutionary divergence of the JE viruses isolated in Korea from the Japanese and Chinese strains and that there may exist at least two antigenically different JE virus strains in Korea.

Incidence and predictors of postextubation laryngeal edema in pediatric patients with congenital heart disease.

Laryngeal edema developed in 10.1% of studied patients with congenital heart disease after cardiac surgery. The 181 patients were divided into two groups; those with laryngeal edema (group 1) and those without laryngeal edema (group 2). The mean ages in group 1 and 2 were 10 and 22.9 months. Group 1 patients were younger on average than those of group 2 (p < 0.05). The differences in the cardiopulmonary bypass time and anesthesia time between the two groups were not statistically significant. The duration of intubations and ventilatory support before and after the onset of laryngeal edema and the period of the ICU stay were longer in group 1 than in group 2 (P < 0.05). A predictor of postextubation laryngeal edema was not found in our patients from above mentioned parameters. We conclude that the higher incidence of laryngeal edema may be due to young age (most were under 1 year of age), and duration of intubation and ventilatory support.

Factors affecting stability of isozymes of creatine phosphokinase.

Human brain-type (BB) creatine phosphokinase (CPK) and skeletal muscle (MM) CPK were purified from autopsy tissues. The stability of each isozyme with and without the reducing agent mercaptoethanol in human plasma, saline solution, and saline solution with bovine serum albumin was studied. Human BB CPK, at 37 C, without added mercaptoethanol, lost 50% of its activity in 15 minutes during incubation in plasma. It was much more stable at both room temperature and 4 C. BB CPK was also unstable in saline solution plus bovine serum albumin in the absence of mercaptoethanol but was much more stable in the presence of mercaptoethanol. Plasma factors with molecular weights less than 1,000 were potent inhibitors of CPK activity. Electrophoresis following incubation of BB CPK in plasma without mercaptoethanol revealed a new band with CPK activity with the electrophoretic mobility close to that of cardiac muscle (MB) CPK, but no band with the electrophoretic mobility of MM CPK was noted. A new band whose electrophoretic mobility was intermediate between that of MM CPK and that of MB CPK appeared after 21 hours of incubation of MM CPK in human plasma. Modifications of methods for handling blood samples in order to increase the possibility of preserving CPK isozymes in human plasma are proposed. Immunologic methods for determining CPK isozymes may provide more definitive answers as to the nature of the isozyme species present in human sera in various diseases.

Enzymic, electrophoretic and immunoreactive properties of labeled MM and BB isoenzymes of human creatine kinase.

Purified MM and BB isoenzymes of human creatine kinase (CK) were labeled with Bolton-Hunter reagent. Labeling process did not affect their enzymic activity, although the labeled enzymes lost enzymic activity in storage. The labeled BB isoenzyme progressively changed its electrophoretic mobility, while labeled MM isoenzyme did not. Both labeled isoenzymes, however, maintained their immunoreactivity with their respective antisera. These results suggest that the enzymic and the immunoreactive sites of each CK isoenzyme are different and that BB isoenzyme, not MM isoenzyme, is electrophoretically unstable.

Radioimmunoassay for MM and BB isoenzymes of creatine kinase substantiated by clinical application.

We have developed a radioimmunoassay technique that is highly specific for measuring the MM isoenzyme of creatine kinase. The specificity of the radioimmunoassay for the BB isoenzyme was poor. In patients with treated Duchenne-type muscular dystrophy or untreated hypothyroidism, the MM isoenzyme, but not the BB isoenzyme value, was consistently above above normal. In the radioimmunoassay for the BB isoenzyme the antisera might cross react with other materials and the inactivated isoenzyme, but not with MM or MB isoenzymes.