Corneal Repair with Adhesive Cell Sheets of Fetal Cartilage-Derived Stem Cells.

BACKGROUND: Corneal scarring or disease may lead to severe corneal opacification and consequently, severe loss of vision due to the complete loss of corneal epithelial cells. We studied the use of epithelial cell sheets differentiated from fetal cartilage-derived stem cells (FCSC) to resurface damaged cornea. METHODS: The FCSC were isolated from the femoral head of immature cartilage tissue. The ability of the FCSCs to differentiate into corneal epithelial cells was evaluated using differentiation media at 2 days and 7 days post-seeding. A sheet fabricated of FCSCs was also used for the differentiation assay. The results of the in vitro studies were evaluated by immunocytochemistry and Western blots for corneal epithelial cell markers (CK3/12 and Pax6) and limbal epithelial stem cell markers (ABCG2 and p63). To test the material in vivo, an FCSC-sheet was applied as a treatment in a chemically burned rabbit model. The healing ability was observed histologically one week after treatment. RESULTS: The in vitro experiments showed morphological changes in the FCSCs at two and seven days of culture. The differentiated cells from the FCSCs or the FCSC-sheet expressed corneal epithelial cells markers. FCSC were create cell sheet that successfully differentiated into corneal epithelial cells and had sufficient adhesion so that it could be fused to host tissue after suture to the ocular surface with silk suture. The implanted cell sheet maintained its transparency and the cells were alive a week after implantation. CONCLUSION: These results suggest that carrier-free sheets fabricated of FCSCs have the potential to repair damaged corneal surfaces.

Visual Outcomes after Bilateral Implantation of an Extended Depth of Focus Intraocular Lens: A Multicenter Study.

PURPOSE: To evaluate visual performance after bilateral implantation of an extended depth of focus (EDOF) intraocular lens (IOL). METHODS: This multicenter, prospective, observational study included 100 patients who underwent bilateral cataract surgery with a toric or non-toric EDOF IOL (Tecnis Symfony), and 96 patients completed the final assessment at 4 to 6 months. Binocular corrected distance visual acuity and uncorrected distance visual acuity (UDVA), uncorrected intermediate visual acuity (UIVA), and uncorrected near visual acuity (UNVA), spectacle independence, visual symptoms, and patient satisfaction were evaluated. RESULTS: Mean decimal visual acuity results showed a binocular corrected distance visual acuity of 1.10 ± 0.18, UDVA of 1.04 ± 0.17, UIVA of 0.96 ± 0.16, and UNVA of 0.68 ± 0.18. Binocular UDVA and UIVA were 0.8 (decimal) or better in 98% and 94% of patients, respectively. Binocular UNVA was 0.63 (decimal) or better in 76% of patients. Overall, 76% of the patients achieved spectacle independence across all distances, and more than 85% reported no or mild dysphotoptic phenomena. On a scale of 0 to 10, the median patient satisfaction score was 9 for far, 9.5 for intermediate, and 8 for near vision. CONCLUSIONS: The Symfony EDOF IOL provided excellent distance, intermediate visual outcome, and functional near visual acuity. The visual results were associated with prominent levels of spectacle independence and patient satisfaction.

Difference of auditory brainstem responses by stimulating to round and oval window in animal experiments.

ABSTACT To ensure the safety and efficacy of implantable hearing aids, animal experiments are an essential developmental procedure, in particular, auditory brainstem responses (ABRs) can be used to verify the objective effectiveness of implantable hearing aids. This study measured and compared the ABRs generated when applying the same vibration stimuli to an oval window and round window. The ABRs were measured using a TDT system 3 (TDT, USA), while the vibration stimuli were applied to a round window and oval window in 4 guinea pigs using a piezo-electric transducer with a proper contact tip. A paired t-test was used to determine any differences between the ABR amplitudes when applying the stimulation to an oval window and round window. The paired t-test revealed a significant difference between the ABR amplitudes generated by the round and oval window stimulation (t = 10.079, α < .0001). Therefore, the results confirmed that the biological response to round window stimulation was not the same as that to oval window stimulation.

Pd/C-Catalyzed Carbonylative Esterification of Aryl Halides with Alcohols by Using Oxiranes as CO Sources.

A carbonylative esterification reaction between aryl bromides and alcohols, promoted by Pd/C and NaF in the presence of oxiranes, has been developed. In this process, oxiranes serve as sources of carbon monoxide by their conversion to aldehydes through a palladium-promoted Meinwald rearrangement pathway. Intramolecular versions of this process serve as methods for the synthesis of lactones and phthalimides.

Effects of flow-induced shear stress on limbal epithelial stem cell growth and enrichment.

The roles of limbal epithelial stem cells (LESCs) are widely recognized, but for these cells to be utilized in basic research and potential clinical applications, researchers must be able to efficiently isolate them and subsequently maintain their stemness in vitro. We aimed to develop a biomimetic environment for LESCs involving cells from their in vivo niche and the principle of flow-induced shear stress, and to subsequently demonstrate the potential of this novel paradigm. LESCs, together with neighboring cells, were isolated from the minced limbal tissues of rabbits. At days 8 and 9 of culture, the cells were exposed to a steady flow or intermittent flow for 2 h per day in a custom-designed bioreactor. The responses of LESCs and epithelial cells were assessed at days 12 and 14. LESCs and epithelial cells responded to both types of flow. Proliferation of LESCs, as assessed using a BrdU assay, was increased to a greater extent under steady flow conditions. Holoclones were found under intermittent flow, indicating that differentiation into transient amplifying cells had occurred. Immunofluorescent staining of Bmi-1 suggested that steady flow has a positive effect on the maintenance of stemness. This finding was confirmed by real-time PCR. Notch-1 and p63 were more sensitive to intermittent flow, but this effect was transient. K3 and K12 expression, indicative of differentiation of LESCs into epithelial cells, was induced by flow and lasted longer under intermittent flow conditions. In summary, culture of LESCs in a bioreactor under a steady flow paradigm, rather than one of intermittent flow, is beneficial for both increasing proliferation and maintaining stemness. Conversely, intermittent flow appears to induce differentiation of LESCs. This novel experimental method introduces micro-mechanical stimuli to traditional culture techniques, and has potential for regulating the proliferation and differentiation of LESCs in vitro, thereby facilitating research in this field.

1,5-Anhydroglucitol reflects postprandial hyperglycemia and a decreased insulinogenic index, even in subjects with prediabetes and well-controlled type 2 diabetes.

To examine the serum 1,5-anhydroglucitol (AG) levels as a surrogate measure of postprandial hyperglycemia (PPH) and insulin secretion in a wide range of hyperglycemia, we compared the relationship between the glycemic index during a 75g oral glucose tolerance test (OGTT) and the insulinogenic index and 1,5-AG according the overall glycemic state. Fasting serum 1,5-AG levels were lower in the type 2 diabetic group (18.0+/-7.0microg/mL) than in the normal glucose tolerance (NGT, 25.4+/-4.0microg/mL), impaired fasting glucose (IFG, 24.6+/-6.2microg/mL), and impaired glucose tolerance (IGT, 22.1+/-6.2microg/mL) groups and were clearly correlated with glycemic values from the OGTT. 120-min post-challenge plasma glucose (PPG(120)) emerged as an independent predictor for 1,5-AG levels after multiple linear regression analysis (beta=-0.554, P<0.001). Additionally, 1,5-AG levels were significantly correlated with PPG(120) in each quartile of A1C, and the coefficients increased with higher A1C quartiles. Subjects with low 1,5-AG levels had both increased insulin resistance and decreased insulin secretion. Decreased 1,5-AG levels are closely correlated with PPH and decreased insulin secretion capacity across a wide range of glycemia, even in relatively well-controlled diabetes.

Quantification of AML1-ETO fusion transcript as a prognostic indicator in acute myeloid leukemia.

BACKGROUND AND OBJECTIVES: In spite of the high complete remission rate that chemotherapy achieves in acute myeloid leukemia with AML1-ETO gene rearrangement, relapse is a major cause of treatment failure in this condition. We aimed to determine a predictor of relapse with the real-time quantitative reverse transcription polymerase chain reaction (RQ-PCR) of AML1-ETO chimeric mRNA. DESIGN AND METHODS: We serially monitored AML1-ETO fusion transcripts using RQ-PCR in 113 bone marrow or peripheral blood samples from 21 patients with AML1-ETO-positive acute myeloid leukemia and analyzed the prognostic relevance of the results. RESULTS: Higher transcript levels at diagnosis were associated with a higher probability of relapse (p=0.038 in all patients and p=0.001 in adult patients). A decrease of less than 3-log at the time of achieving complete remission was also associated with a higher risk of relapse (p=0.035 in all patients and p=0.011 in adult patients). RQ-PCR detected the reappearance of AML1-ETO fusion transcripts in both peripheral blood and bone marrow during apparent complete remission. Detection of the transcripts preceded hematologic relapse by one to three months. The transcript levels in peripheral blood correlated with those in bone marrow at the same time point. INTERPRETATION AND CONCLUSIONS: Our findings indicate that regular monitoring of AML1-ETO chimeric transcript levels by RQ-PCR on bone marrow or peripheral blood samples could be extremely useful for the selection of high-risk patients and be an early predictor of relapse.

Differentiation of Anaplasmataceae through partial groEL gene analysis.

The nucleotide sequences (287 bp) of the partial groEL gene from 14 reference strains of Anaplasmataceae were determined and compared. A partial groEL gene is useful for the identification and characterization of Anaplasmataceae, in spite of its short nucleotide sequences.

Rapid and simple identification of Orientia tsutsugamushi from other group rickettsiae by duplex PCR assay using groEL gene.

In this study, two new duplex PCR methods based on the groEL gene were developed and investigated for the diagnosis of rickettsiae. The first duplex PCR assay amplified the 229-bp and the 366-bp DNAs of 6 strains including typhus group (TG) and spotted fever group (SFG) rickettsiae, and 5 scrub typhus group (STG) rickettsiae, respectively. The second duplex PCR assay amplified the 397-bp and the 213-bp DNAs of 6 Rickettsia strains and 5 STG strains. These duplex PCR methods could simultaneously perform the rapid identification of rickettsiae and the differential diagnosis of STG and other group rickettsiae in a single reaction.

Protective effects of bovine colostrum on non-steroidal anti-inflammatory drug induced intestinal damage in rats.

The aim of this study was to examine whether bovine colostrum was able to prevent the NSAID induced small intestinal damage in animals. The animal model population of the study consisted of 4 groups: control group, diclofenac group, diclofenac with 10% low fat milk group and diclofenac with 5% colostrum group. The animals with milk or colostrum were fed with 10% low fat milk or 5% colostral solution for 5 days before the administration of diclofenac. Gut injuries were induced by administration of a single dose of diclofenac (100 mg/kg orally). Epithelial permeability values (24 hour urinary excretion of 51Cr-ethylenediaminetetraacetic acid [51Cr-EDTA]), enteric aerobic bacterial counts, serum biochemical profiles and pathologic findings of distal ileum were measured. Diclofenac caused a marked increase in the intestinal permeability, enteric bacterial numbers and intestinal villous damage, and enteric protein and albumin loss. Combined administration of bovine colostrum reduced the increase in intestinal permeability, enteric bacterial overgrowth, protein losing enteropathy and mucosal villous damage of the small intestine induced by diclofenac. Bovine colostrum may have a beneficial effect in prevention of NSAID induced small intestinal injuries.

Relationship of serum osteoprotegerin levels with coronary artery disease severity, left ventricular hypertrophy and C-reactive protein.

OPG (osteoprotegerin) is an inhibitor of osteoclastogenesis and recent work suggests it has a role in atherosclerosis. Therefore we measured serum OPG levels in patients with coronary artery disease, compared the serum OPG levels among the different groups according to the number of stenotic vessels and determined whether there was any correlation with aortic calcification, LV (left ventricular) mass index and serum CRP (C-reactive protein) levels. Subjects (n=100; mean age, 57 years) who underwent coronary angiograms were enrolled. Blood pressure, body mass index, fasting blood glucose, lipid profiles and CRP levels were measured and the LV mass indices were calculated using ECGs. Serum OPG levels were measured by ELISA. The presence of calcification in the aortic notch was checked by a chest X-ray. The subjects were divided into four groups according to the number of stenotic vessels. The mean serum OPG levels increased significantly as the number of stenotic vessels increased, and the mean serum OPG levels were higher in the group with three-vessel disease compared with the groups with no- or one-vessel disease. The mean serum CRP level was significantly higher in the group with three-vessel disease compared with the groups with no-, one- and two-vessel disease. Age and LV mass index showed significant positive correlations with serum OPG levels, although significance was lost after an adjustment for age. Serum CRP levels were positively correlated with serum OPG levels even after an adjustment for age. There were no differences in serum OPG levels according to the presence of fasting hyperglycaemia or aortic calcification. In conclusion, serum OPG level was related to the severity of stenotic coronary arteries and serum CRP levels. LV mass indices showed no significant correlation with OPG levels. The precise mechanism for the role of OPG in atherosclerosis needs to be investigated further.

Rapid identification of Borrelia burgdorferi sensu lato by PCR-restriction fragment length polymorphism analysis of groEL gene.

Thirty-one reference strains and 23 Korean isolates of the genus Borrelia were identified through the PCR-RFLP analysis using the groEL gene. This will be useful for the rapid differentiation of B. burgdorferi sensu lato and complements one of the 5S-23S intergenic spacers.

Evaluation of groEL gene analysis for identification of Borrelia burgdorferi sensu lato.

The nucleotide sequences of the groEL genes, the flagellin genes, and the 16S rRNA genes from 22 reference strains of Borrelia were compared. groEL sequence analysis is useful not only in interspecies differentiation but also in intraspecies differentiation of Borrelia afzelii and Borrelia garinii isolates.

Identification of the Coxiella sp. detected from Haemaphysalis longicornis ticks in Korea.

Two Haemaphysalis longicornis ticks were found positive in PCR assay of com-1 gene to detect Coxiella burnetii DNA from 100 ticks. The nucleotide sequences of com-1 and 16S rRNA gene were determined from 2 ticks and compared with those of other C. burnetii strains. The results suggest that H. longicornis harbor Coxiella sp. bacteria in Korea. Furthermore, icd, cbhE', and cbbE' genes are C. burnetii specific genes whereas com-1 gene is Coxiella genus specific gene. This study gives the first documentation to prove the existence of Coxiella sp. in tick collected in Korea.

Differentiation of rickettsiae by groEL gene analysis.

The nucleotide sequences (534 to 546 bp) of the groEL gene, which encodes the 60-kDa heat shock protein GroEL, from 15 rickettsial strains were determined and compared. In the phylogenetic tree created by the unweighted pair group method with arithmetic averages and the neighbor-joining method, rickettsial strains could be distinguished from Ehrlichia strains. Five spotted fever group strains, four typhus group strains, and six scrub typhus group (STG) strains were differentiated as distinct entities. Unlike gltA and ompA gene analyses, differentiation between members of the genus Rickettsia and the STG rickettsiae by groEL gene analysis was possible. In comparison with 16S rRNA gene analysis, the groEL gene has a higher degree of divergence among the rickettsiae. We therefore successfully developed rapid differentiation methods, PCR-restriction fragment length polymorphism analysis and a species-specific PCR, based on the groEL gene sequences. Four Korean isolates were identified by these methods and groEL gene analysis. The results suggest that the groEL gene is useful for the identification and characterization of rickettsiae.

Identification of the spotted fever group rickettsiae detected from Haemaphysalis longicornis in Korea.

Seven Haemaphysalis ticks were found positive in PCR assay of gltA gene to detect the spotted fever group (SFG) rickettsiae DNA from 100 ticks. The nucleotide sequence of 16S rRNA gene was determined from 5 ticks and compared to those of other Rickettsia strains. The nucleotide sequence from 4 ticks showed high homologies (99.7 to 100%) with that of R. japonica YH, and that from 1 tick (tick no. 48) was identical with that of R. rickettsii R, suggesting that SFG rickettsiae exists in Korea. This is the first documentation of SFG rickettsiae in Korea.

Differentiation of Borrelia burgdorferi sensu lato through groEL gene analysis.

The nucleotide sequences (310 bp) of the groEL gene, which encode the 60-kDa heat shock protein GroEL from 31 reference strains of Borrelia were determined and compared. More than 92.3% similarity was observed among Borrelia burgdorferi sensu lato strains. In the phylogenetic tree constructed with the maximum-likelihood method, each species of B. burgdorferi sensu lato was differentiated as a distinct entity. We developed polymerase chain reaction-restriction fragment length polymorphism analysis using a specific single amino acid variation [N(213) (AAT)-->S (AGC or AGT)] between B. burgdorferi sensu stricto strains and the other B. burgdorferi sensu lato strains. These results showed that the groEL gene is useful for differentiation of B. burgdorferi sensu lato.