Biomedical subjects

Dong-Hun Lee

Publications and source records attributed to Dong-Hun Lee.

13 recordsLinked to original sources

PubMed2006

Attempted therapeutic immunization in a chimpanzee chronic HBV carrier with a high viral load.

BACKGROUND: We previously reported successful therapeutic immunization in a chimpanzee having a relatively low viral load, which was immunized with recombinant plasmid hepatitis B surface antigen (HBsAg) DNA and boosted with recombinant HBsAg encoding canarypox virus. In the present study, we attempted to confirm these findings in an animal with a high virus load. METHODS AND RESULTS: We tested three immunization strategies successively over a 3-year period. In the first of these, we administered four monthly injections of DNA encoding HBsAg + PreS2 + hepatitis B core antigen (HBcAg) + DNA encoding interleukin (IL)-12, (given 3 days later), and boosted with canarypox expressing all of the above HBV genes 6 months after initial immunization. No reduction in viral load was observed. In the second trial, we administered lamivudine for 8 weeks, and then began monthly DNA-based immunization with plasmids expressing the above viral genes; however, viral loads rebounded 1 week after termination of lamivudine therapy. In a third trial, we continued lamivudine therapy for 30 weeks and immunized with vaccinia virus expressing the above viral genes 18 and 23 weeks after the start of lamivudine therapy. Again viral loads rebounded shortly after cessation of lamivudine treatment. Analysis of cell-mediated immune responses, and their avidity, revealed that DNA-based immunization produced the strongest enhancement of high avidity T-cell responses, while recombinant vaccinia immunization during lamivudine therapy enhanced low avidity responses only. The strongest low and high avidity responses were directed to the middle surface antigen. CONCLUSIONS: Three strategies for therapeutic immunization failed to control HBV viremia in a chronically infected chimpanzee with a high viral load.

Animals↗

PubMed2005

Protection against chronic hepatitis C virus infection after rechallenge with homologous, but not heterologous, genotypes in a chimpanzee model.

An open question for hepatitis C virus (HCV) vaccine development is whether the various genotypes of this virus protect against the development of chronic infection after heterologous infection with different genotypes. We approached this question by challenging chimpanzees that had recovered from HCV genotype 1a or 1b infection with 6 heterologous genotypes as well as with a homologous genotype (for chimpanzees originally infected with genotype 1a). All 9 chimpanzees rechallenged with a homologous genotype developed self-limited infections. Of 11 chimpanzees challenged with 100 chimpanzee infectious doses of heterologous genotypes, 6 developed self-limited infections, with peak viral loads in acute-phase serum that were ~5-fold lower than those seen during primary infections. One chimpanzee (which had recovered from genotype 1b infection and was rechallenged with genotype 6a) did not develop viremia but did show an anamnestic cell-mediated immune response after rechallenge. Four of the 11 chimpanzees rechallenged with heterologous genotypes developed chronic infections with the genotypes used for rechallenge. These findings suggest that a universally protective HCV vaccine may need to incorporate epitopes from multiple genotypes.

Animals↗

PubMed2005

Sustained E2 antibody response correlates with reduced peak viremia after hepatitis C virus infection in the chimpanzee.

Immune correlates of protection against hepatitis C virus (HCV) infection are not well understood. Here we investigated 2 naive and 6 immunized chimpanzees before and after intravenous challenge, 12 weeks after the last immunization, with 100 50% chimpanzee infectious doses (CID(50)) of heterologous genotype 1b HCV. Vaccination with recombinant DNA and adenovirus vaccines expressing HCV core, E1E2, and NS3-5 genes induced long-term HCV-specific antibody and T-cell responses and reduced peak viral load about 100 times compared with controls (5.91 +/- 0.38 vs. 3.81 +/- 0.71 logs, respectively). There was a statistically significant inverse correlation between peak viral loads and envelope glycoprotein 2 (E2)-specific antibody responses at the time of challenge. Interestingly, one vaccinee that had sterilizing immunity against slightly heterologous virus generated the highest level of E2-specific total and neutralizing antibody responses as well as strong NS3/NS5-specific T-cell proliferative responses. The other four vaccinees with low levels of E2-specific antibody had about 44-fold reduced peak viral loads but eventually developed persistent infections. In conclusion, vaccine-induced E2-specific antibody plays an important role in prevention from nonhomologous virus infection and may provide new insight into the development of an effective HCV vaccine.

Animals↗

PubMed2005

Individual donor nucleic acid amplification testing for detection of West Nile virus.

We have developed an economical, high-throughput nucleic acid amplification test (NAT) for blood-borne viruses, suitable for use in the screening of plasma samples from individual blood donors. This assay system includes a semiautomated procedure, using 96-well glass fiber plates for the extraction of viral nucleic acids from plasma and "universal beacon" technology which permits the detection of all genotypes of highly variable viruses (e.g., human immunodeficiency virus and hepatitis C virus). In this detection system, two fluorescent- detection technologies were employed successfully in a single tube: molecular beacon for West Nile virus (WNV) detection using a 6-carboxyfluorescein fluorophore and TaqMan for internal control detection using a VIC fluorophore. To establish proof of concept, we focused on the development of a robust individual donor NAT for WNV. The assay showed no reactivity to 15 other viruses tested or to 420 blood donor samples from the WNV pre-epidemic season. No cross-contamination was observed on an alternating positive-/negative-well test. The sensitivity (limit of detection, 95%) of the assay for WNV is between 3.79 and 16.3 RNA copies/ml, depending on which material was used as a standard. The assay detected all positive blood donation samples identified by the Roche WNV NAT. The assay can be performed qualitatively for screening and quantitatively for confirmation.

Blood Donors↗

PubMed2004

A novel continuous toxicity test system using a luminously modified freshwater bacterium.

An automated continuous toxicity test system was developed using a recombinant bioluminescent freshwater bacterium. The groundwater-borne bacterium, Janthinobacterium lividum YH9-RC, was modified with luxAB and optimized for toxicity tests using different kinds of organic carbon compounds and heavy metals. luxAB-marked YH9-RC cells were much more sensitive (average 7.3-8.6 times) to chemicals used for toxicity detection than marine Vibrio fischeri cells used in the Microtox assay. Toxicity tests for wastewater samples using the YH9-RC-based toxicity assay showed that EC50-5 min values in an untreated raw wastewater sample (23.9 +/- 12.8%) were the lowest, while those in an effluent sample (76.7 +/- 14.9%) were the highest. Lyophilization conditions were optimized in 384-multiwell plates containing bioluminescent bacteria that were pre-incubated for 15 min in 0.16 M of trehalose prior to freeze-drying, increasing the recovery of bioluminescence and viability by 50%. Luminously modified cells exposed to continuous phenol or wastewater stream showed a rapid decrease in bioluminescence, which fell below detectable range within 1 min. An advanced toxicity test system, featuring automated real-time toxicity monitoring and alerting functions, was designed and finely tuned. This novel continuous toxicity test system can be used for real-time biomonitoring of water toxicity, and can potentially be used as a biological early warning system.

Biological Assay↗

PubMed2004

Glenohumeral arthropathy after arthroscopic anterior shoulder stabilization.

PURPOSE: We present 5 cases of iatrogenic arthropathy after arthroscopic Bankart reconstruction using a metallic suture anchor. TYPE OF STUDY: Retrospective case series. METHODS: Five patients with pain and crepitus on motion were referred to our institution for further evaluation of the previous procedure on anterior shoulder instability. Screw-type metallic suture anchors were used in all cases. All patients were men, with an average age of 23 years (range, 21 to 26). Surgical records on previous procedure were reviewed, and the clinical symptoms were evaluated using a visual analogue scale (VAS), the Simple Shoulder Test (SST), and the Rowe scoring system. The secondary surgery for each patient was performed at an average of 12 months (range, 7 to 20) after the initial arthroscopic stabilization, except in one patient who wanted to postpone the revision surgery. RESULTS: Protrusion of the anchor tip was seen in all and chondral defects in the humeral head with some degree of synovitis were also seen. Slight differences between preoperative and postoperative pain were seen, but almost no improvement in function, including range of motion, stability, and average Rowe score were seen after the second procedure. Patients who underwent revision surgery were dissatisfied with the final outcomes. CONCLUSIONS: Careful attention should be paid when using a metallic suture anchor. A secure, buried placement of the anchor is required in arthroscopic Bankart reconstruction. Poorly placed suture anchors may damage the glenohumeral joint, and if these are not corrected either at arthroscopic surgery or shortly after, the results can be suboptimal. If a patient complains of unusual mechanical symptoms after using anchors, radiographs should be performed. This kind of serious complication can be discovered earlier to prevent the severe destruction of the glenohumeral joint. LEVEL OF EVIDENCE: Level IV.

Adult↗

PubMed2004

Comparative analysis of cyanobacterial communities from polluted reservoirs in Korea.

Cyanobacteria are the dominant phototrophic bacteria in water environments. Here, the diversity of cyanobacteria in seven Korean reservoir waters where different levels of algal blooms were observed during the summer of 2002, was examined by T-RFLP analysis. The number of T-RF bands in the HaeIII T-RFLP profiles analyzed from those water samples ranged from 20 to 44. Of these, cyanobacteria accounted for 6.1 to 27.2% of the total bacteria. The water samples could be clustered into 2 groups according to the Dice coefficient of the T-RF profiles. The eutrophic Dunpo and oligotrophic Chungju reservoirs were selected, and several representative clones from both reservoir waters analyzed for the nucleotide sequences of their 16S rDNA. The major clones were found to belong to the Microcystis and Anabaena species in the waters from the Dunpo and Chungju reservoirs, respectively, which was in agreement with the T-RFLP result. That is, the Microcystis and Anabaena species were dominant in the eutrophic and polluted Dunpo and oligotrophic Chungju reservoir waters, respectively. These results indicated that there is a correlation between prevalence of cyanobacterial species and levels of pollution in reservoir waters.

Animals↗

PubMed2004

Genetic organization of the dhlA gene encoding 1,2-dichloroethane dechlorinase from Xanthobacter flavus UE15.

Xanthobacter flavus strain UE15 was isolated in wastewater obtained from the Ulsan industrial complex, Korea. This strain functions as a 1,2-dichloroethane (1,2-DCA) degrader, via a mechanism of hydrolytic dechlorination, under aerobic conditions. The UE15 strain was also capable of dechlorinating other chloroaliphatics, such as 2-chloroacetic acid and 2-chloropropionic acid. The dhlA gene encoding 1,2-DCA dechlorinase was cloned from the genomic DNA of the UE15 strain, and its nucleotide sequence was determined to consist of 933 base pairs. The deduced amino acid sequence of the DhlA dechlorinase exhibited 100% homology with the corresponding enzyme from X. autotrophicus GJ10, but only 27 to 29% homology with the corresponding enzymes from Rhodococcus rhodochrous, Pseudomonas pavonaceae, and Mycobacterium sp. strain GP1, which all dechlorinate haloalkane compounds. The UE15 strain has an ORF1 (1,356 bp) downstream from the dhlA gene. The OFR1 shows 99% amino acid sequence homology with the transposase reported from X. autotrophicus GJ10. The transposase gene was not found in the vicinity of the dhlA in the GJ10 strain, but rather beside the dhlB gene coding for haloacid dechlorinase. The dhlA and dhlB genes were confirmed to be located at separate chromosomal loci in the Xanthobacter flavus UE15 strain as well as in X. autotrophicus GJ10. The dhlA and transposase genes of the UE15 strain were found to be parenthesized by a pair of insertion sequences, IS1247, which were also found on both sides of the transposase gene in the GJ10 strain. This unique structure of the dhlA gene organization in X. flavus strain UE15 suggested that the dechlorinase gene, dhlA, is transferred with the help of the transposase gene.

Bacterial Proteins↗

PubMed2004

Monitoring of bacterial community in a coniferous forest soil after a wildfire.

Changes in the soil bacterial community of a coniferous forest were analyzed to assess microbial responses to wildfire. Soil samples were collected from three different depths in lightly and severely burned areas, as well as a nearby unburned control area. Direct bacterial counts ranged from 3.3-22.6 x 10(8) cells/(g.soil). In surface soil, direct bacterial counts of unburned soil exhibited a great degree of fluctuation. Those in lightly burned soil changed less, but no significant variation was observed in the severely burned soil. The fluctuations of direct bacterial count were less in the middle and deep soil layers. The structure of the bacterial community was analyzed via the fluorescent in situ hybridization method. The number of bacteria detected with the eubacteria-targeted probe out of the direct bacterial count varied from 30.3 to 84.7%, and these ratios were generally higher in the burned soils than in the unburned control soils. In the surface unburned soil, the ratios of alpha-, beta- and gamma-proteobacteria, Cytophaga-Flavobacterium group, and other eubacteria groups to total eubacteria were 9.9, 10.6, 15.5, 9.0, and 55.0%, respectively, and these ratios were relatively stable. The ratios of alpha-, beta- and gamma-proteobacteria, and Cytophaga-Flavobacterium group to total eubacteria increased immediately after the wildfire, and the other eubacterial proportions decreased in the surface and middle layer soils. By way of contrast, the composition of the 5 groups of eubacteria in the subsurface soil exhibited no significant fluctuations during the entire period. The total bacterial population and bacterial community structure disturbed by wildfire soon began to recover, and original levels seemed to be restored 3 months after the wildfire.

Bacteria↗

PubMed2004

Monitoring of soil bacterial community and some inoculated bacteria after prescribed fire in microcosm.

The soil bacterial community and some inoculated bacteria were monitored to assess the microbial responses to prescribed fire in their microcosm. An acridine orange direct count of the bacteria in the unburned control soil were maintained at a relatively stable level (2.0 approximately 2.7 x 10(9) cells/g(-1).soil) during the 180 day study period. The number of bacteria in the surface soil was decreased by fire, but was restored after 3 months. Inoculation of some bacteria increased the number of inoculated bacteria several times and these elevated levels lasted several months. The ratios of eubacteria detected by a fluorescent in situ hybridization (FISH) method to direct bacterial count were in the range of 60 approximately 80% during the study period, with the exception of some lower values at the beginning, but there were no definite differences between the burned and unburned soils or the inoculated and uninoculated soils. In the unburned control soil, the ratios of alpha-, beta- and gamma-subgroups of the proteobacteria, Cytophaga-Flavobacterium and other eubacteria groups to that of the entire eubacteria were 13.7, 31.7, 17.1, 16.8 and 20.8%, respectively, at time 0. The overall change on the patterns of the ratios of the 5 subgroups of eubacteria in the uninoculated burned and inoculated soils were similar to those of the unburned control soil, with the exception of some minor variations during the initial period. The proportions of each group of eubacteria became similar in the different microcosms after 6 months, which may indicate the recovery of the original soil microbial community structure after fire or the inoculation of some bacteria. The populations of Azotobacter vinelandii, Bacillus megaterium and Pseudomonas fluorescens, which had been inoculated to enhance the microbial activities, and monitored by FISH method, showed similar changes in the microcosms, and maintained high levels for several months.

Bacillus↗

PubMed2003

Exposure to low infective doses of HCV induces cellular immune responses without consistently detectable viremia or seroconversion in chimpanzees.

In hepatitis C virus (HCV) infection, there is accumulating data suggesting the presence of cellular immune responses to HCV in exposed but seemingly uninfected populations. Some studies have suggested cross-reactive antigens rather than prior HCV exposure as the main reason for the immune responses. In this study we address this question by analyzing the immune response of chimpanzees that have been sequentially exposed to increasing doses of HCV virions. The level of viremia, as well as the immune responses to HCV at different times after virus inoculation, were examined. Our data indicate that HCV infective doses as low as 1-10 RNA (+) virions induce detectable cellular immune responses in chimpanzees without consistently detectable viremia or persistent seroconversion. However, increasing the infective doses of HCV to 100 RNA (+) virions overcame the low-inoculum-induced immune response and produced high-level viremia followed by seroconversion.

Animals↗

PubMed2002

Lack of evidence for HIV type 1-related SIVcpz infection in captive and wild chimpanzees (Pan troglodytes verus) in West Africa.

Serum from 387 chimpanzees (Pan troglodytes verus), caught in the wild or bred in captivity, was tested for antibody to HIV-1 and HIV-2, using second- and third-generation enzyme immunoassays. Six samples were repeatedly positive; however, only one of these was Western blot positive. Serial sera drawn before and after the Western blot-positive samples were seronegative, and thus we conclude that this sample represented specimen contamination, or mislabeling. Thus, none of the 387 Pan troglodytes verus from West Africa were spontaneously infected with SIVcpz. Chimpanzees are known to be exquisitely susceptible to infection with HIV-1 when experimentally inoculated, and thus our findings suggest that HIV-1-related viruses do not exist in Pan troglodytes verus in the wild. As it has been convincingly shown that SIVcpz exists in wild Pan troglodytes troglodytes in Central Africa, this suggests that HIV-1 arose in Central Africa, but not in West Africa.

Africa, Central↗

PubMed2002

Stabilized viral nucleic acids in plasma as an alternative shipping method for NAT.

BACKGROUND: Preservation of the integrity of viral nucleic acids in blood specimens during shipping and handling is crucial for NAT and viral load monitoring. An economical and convenient method is described for nucleic acid stabilization by using an RNA stabilizing solution (RNAlater, Ambion) in plasma that is designed for the shipment of samples to tropical countries. STUDY DESIGN AND METHODS: HCV, HIV, and HBV FFP were compared with RNAlater-treated plasma and dried plasma spots (DPSs) after incubation at 37 degrees C, which was chosen as an upper limit of ambient shipping temperature, for up to 28 days. HCV-infected chimpanzee plasma was shipped at either room temperature after RNAlater treatment or as frozen plasma in liquid nitrogen from Liberia to New York City. They were then compared for HCV RNA levels. The nucleic acid stabilities were determined by quantitative PCR by using a molecular beacon assay on a sequence detection system (ABI 7700, PE-Biosystems) and by visualizing the PCR components on an acrylamide gel. RESULTS: Quantitative PCR data showed that a 60:40 or greater ratio of RNAlater:plasma volume successfully stabilized HCV RNA and HIV RNA in plasma for up to 28 days at 37 degrees C. HBV DNA in plasma was stable for up to 14 days at 37 degrees C without any stabilizing solution. DPSs on filter paper stabilized viral nucleic acids, but the recoveries were 3 to 10 times less than those with frozen plasma. The integrity of the 5' UTR region of HCV RNA in RNA later-treated chimpanzee plasma was intact when its PCR component was viewed on an acrylamide gel. CONCLUSION: The DPS method stabilized nucleic acids, at least with the extraction method used, was less sensitive than use of RNAlater, and required tedious manual handling. RNAlater provides a convenient way of stabilizing viral nucleic acid in plasma at ambient temperature during sample transportation.

5' Untranslated Regions↗