OBJECTIVEThis study is performed to investigate the cell topographies and biomechanical properties of two different types of temporomandibular joint (TMJ) discs from goats by using JPK Nano Wizard 3 biological atomic force microscopy (AFM). This process provides a guideline for selecting seed cells for TMJ disc tissue engineering.

METHODSTMJ disc cells from primary goats were cultured by monolayer culture method. AFM was used to contact scan the topographies of the two types of TMJ disc cells under physiological environment. Approximately 20 chondrocyte-like and fibroblast-like cells were selected randomly to plot the force-versus-distance curves of the cytoplasm and nucleus. Young's modulus and adhesion were analyzed by JPK Data Processing.

RESULTSThe triangle-shapednucleus of the chondrocyte-like cell occupied a large portion of the cell. Cytoskeleton was arranged dendritically on the surface. Pseudopodia were extended from cell edges. The spindle-shaped nucleus of the fibroblast-like cell occupied a significantly larger region compared with the cytoplasmic region. Cytoskeleton was arranged regularly. Cell edges were smooth with less pseudopodia extended. No difference was found in the surface roughness between the two types of cells. According to the force-versus-distance curves, the Young's moduli of the two types of cells were not statistically different (P>0.05), but differences were found in the cytoplasmic regions (P=0.047). No statistical difference was found in the adhesions between the two types of cells (P>0.05).

CONCLUSIONThe AFM topography and curves were compared and analyzed. The two types of TMJ disc cells exhibited significantly different topographies, but only slight difference in their mechanical abilities.

Objective To investigate the therapeutic effects of givinostat , a histone deacetylase inhibitor (HDACI), on the development of chronic asthma with airway inflammation , airway remodeling and airway hyperresponsiveness ( AHR) .Methods BALB/C mice were randomly divided into control group , asthma group, dexamethasone group and givinostat group (n=12 per group).AHR was assessed.Total cell numbers and differential counts , interleukin-4 ( IL-4 ) , interleukin-5 ( IL-5 ) and interferon-γ( IFNγ) levels in the bronchoalveolar lavage fluid ( BALF) were measured in the above 4 groups.The pathology of lung tissue was evaluated .Immunohistochemical ( IHC) staining and Western blot were used to detect αsmooth muscle actin(α-SMA) and transforming growth factor-β1(TGFβ1).Results Compared with the asthma only group, givinostat treatment relieved airway resistance (2.96 ±1.01 vs 6.50 ±0.79,P<0.05).Total inflammatory cells [(33.04 ±5.62) ×104/ml vs (98.04 ±9.27) ×104/ml,P<0.01], eosinophil cells [(9.17 ±2.33) ×104/ml vs(37.64 ±6.98) ×104/ml, P <0.01], IL-4 [(10.12 ±2.98)ng/ml vs (16.88 ±2.78)ng/ml,P<0.05] and IL-5 [(27.09 ±3.62)ng/ml vs (37.86 ±7.34)ng/ml, P<0.05] levels were all reduced in givinostat group , while IFNγ[ ( 91.86 ±23.73 ) pg/ml vs ( 60.49 ±11.88 ) pg/ml, P>0.05] was enhanced in BALF.Inflammatory cell infiltration around the airway was reduced , with decreased inflammatory cell score [(1.60 ±0.69) points vs (3.40 ±0.68) points, P <0.01] and inflammatory cell number (111.65 ±31.41 vs 601.25 ±186.85, P<0.01).The goblet cell metaplasia [(26.36 ±2.33)%vs (57.21 ±11.56)%] and collagen deposition area [(52.77 ±7.58)μm2/μm vs (111.81 ±12.40)μm2/μm] were obviously reduced (P<0.01).The expressions of α-SMA and TGFβ1 in the lung tissue were both significantly decreased ( P<0.01 ) .Conclusion Givinostat treatment can reduce airway inflammation , airway remodeling and airway hyperresponsiveness in chronic asthma .Its effect is comparable to that of glucocorticoid hormone treatment .

BACKGROUND:Our preliminary studies have shown that basic fibroblast growth factor can induce the differentiation of bone marrow mesenchymal stem cells into disc cells of the temporomandibular joint, and for basic fibroblast growth factor, 10μg/L is superior to 5μg/L in col agen synthesis.
OBJECTIVE:To observe ultrastructural changes of bone marrow mesenchymal stem cells after being induced by different concentrations of basic fibroblast growth factor.
METHODS:We cultured primary sheep bone marrow mesenchymal stem cells and selected passage 3 and 4 cells at good growth state. Bone marrow mesenchymal stem cells were stimulated with 5 and 10μg/L basic fibroblast growth factor and their growth state was observed under inverted phase contrast microscope. Uninduced cells served as controls. The slides with cellcrawling pieces were stained with Safranin O, picrosirius and type I col agen immunohistochemistry at days 7, 14 and 21, respectively. Simultaneously we col ected the cells at day 21 to observe the ultrastructural changes of bone marrow mesenchymal stem cells.
RESULTS AND CONCLUSION:After being induced with different concentrations of basic fibroblast growth factor, bone marrow mesenchymal stem cells were able to differentiate into disc cells of the temporomandibular joint;and after being induced with 10μg/L basic fibroblast growth factor, cells were more like fibroblast-like cells of the temporomandibular joint disc. These findings indicate that bone marrow mesenchymal stem cells have morphological basis for differentiation to the fibroblast-like cells of the temporomandibular joint disc.

PURPOSE: To report two cases of ocular ischemia following scleral encircling. METHODS: A 21-year-old man with glaucoma and a 76-year-old woman without any medical problem were transferred to our department for surgery to treat retinal detachment. After retrobulbar anesthesia and limbal peritomy of conjunctiva, the 4-rectus muscles were isolated. Scleral encircling was performed with No. a 42 band (4.0 mm in width) after cryotherapy done completely around retinal tear. RESULTS: Following surgery, One patient experienced ophthalmic artery occlusion and while the other patient experienced central retinal artery occlusion. Vision was not restored in either cases despite IV injection of 250 ml of 15% mannitol solution and anterior chamber paracentesis. CONCLUSIONS: In the cases where patients are of old age or suffer from glaucoma, we strongly recommend that the surgeons perform the scleral encircling carefully.
Aged ; Anesthesia ; Anterior Chamber ; Conjunctiva ; Cryotherapy ; Female ; Glaucoma ; Humans ; Ischemia* ; Mannitol ; Muscles ; Ophthalmic Artery ; Paracentesis ; Retinal Artery Occlusion ; Retinal Detachment ; Retinal Perforations ; Young Adult

OBJECTIVE: To investigate the cases of intracranial abnormal brain MRI findings even in the negative brain CT scan after mild head injury. METHODS: During a 2-year period (January 2009-December 2010), we prospectively evaluated both brain CT and brain MRI of 180 patients with mild head injury. Patients were classified into two groups according to presence or absence of abnormal brain MRI finding even in the negative brain CT scan after mild head injury. Two neurosurgeons and one neuroradiologist validated the images from both brain CT scan and brain MRI double blindly. RESULTS: Intracranial injury with negative brain CT scan after mild head injury occurred in 18 patients (10.0%). Headache (51.7%) without neurologic signs was the most common symptom. Locations of intracranial lesions showing abnormal brain MRI were as follows; temporal base (n=8), frontal pole (n=5), falx cerebri (n=2), basal ganglia (n=1), tentorium (n=1), and sylvian fissure (n=1). Intracranial injury was common in patients with a loss of consciousness, symptom duration >2 weeks, or in cases of patients with linear skull fracture (p=0.00013), and also more frequent in multiple associated injury than simple one (35.7%>8.6%) (p=0.105). CONCLUSION: Our investigation showed that patients with mild head injury even in the negative brain CT scan had a few cases of intracranial injury. These findings indicate that even though the brain CT does not show abnormal findings, they should be thoroughly watched in further study including brain MRI in cases of multiple injuries and when their complaints are sustained.
Basal Ganglia ; Brain ; Craniocerebral Trauma ; Head ; Headache ; Humans ; Magnetic Resonance Imaging ; Magnetics ; Magnets ; Multiple Trauma ; Neurologic Manifestations ; Prospective Studies ; Skull Fractures ; Unconsciousness

PURPOSE: This study aimed to identify the true extent of non-responsiveness in full-term infants born from HBsAg-negative or HBsAg-positive mothers and vaccinated against hepatitis B virus (HBV) at 0, 1, and 6 months of age and to evaluate the effect of revaccination among non-responders. METHODS: The study included 716 full-term infants born in 2004-2007. Of 716, 662 infants (A group) were born to HBsAg- negative mothers and 54 infants (B group: 50, except HBsAg-positive infants) were born to HBsAg-positive mothers. All infants were administered DNA recombinant vaccines at 0, 1, and 6 months of age. B group infants received hepatitis B immunoglobulin at birth. Anti-HBs titers were tested at 7-12 and 9-15 months in A and B groups, respectively. Three revaccination doses were administered to non-responders whose anti-HBs titers were under 10 mIU/ml; revaccinated infants were retested at 1-3 months after last vaccination. The association between HBeAg seropositivity of mother and the failure of HBV immunoprophylaxis was evaluated. RESULTS: The seroconversion rates after primary hepatitis B vaccination were higher in A group (94.1%) than in B group (78%, P<0.001). The seroconversion rates were high in revaccinated infants (A group non-responders: 96.9%, B group non- responders: 87.5%). The failure of HBV immunoprophylaxis was significantly associated with maternal HBeAg seropositivity (P<0.001). CONCLUSION: The seroconversion rates after primary hepatitis B vaccination were low in B group infants. Revaccination of non-responders in B group was very effective. Therefore, anti-HBs testing and revaccination of B group is very important. Revaccination of non-responders in A group was also very effective. Thus, testing the immune status of infants born to HBsAg-negative mothers even after primary hepatitis B vaccination should be considered. However, to realize this, further studies on the cost-effectiveness of anti-HBs testing in healthy full-term infants are necessary.
DNA ; Hepatitis ; Hepatitis B ; Hepatitis B e Antigens ; Hepatitis B Surface Antigens ; Hepatitis B virus ; Humans ; Immunization, Secondary ; Immunoglobulins ; Infant ; Mothers ; Parturition ; Vaccination ; Vaccines, Synthetic

OBJECTIVE: Treatment with cyclosporine(CsA) for a long-term period may induce renal glomerulosclersosis and interstitial fibrosis. Reactive oxygen species(ROS) seems to be involved in the process of glomerulosclersosis and interstitial fibrosis. We investigated the effect of CsA on the generation of ROS and extracellular matrix accumulation in cultured human mesangial cells. We also studied the relationship between ROS formation and extracellular matrix and the effect of antioxidant on ROS formation and/or extracellular matrix degradation. METHODS: Mesangial cells were treated with varying dose of Cyclosporine(0, 2.5, 5, 7.5 and 10microgram/ mL) and also with cyclosporine(5microgram/mL) plus N- acetylcysteine(1mM). ROS was measured by flow cytometric analysis. mRNA expression of MMP-2, TIMP-2, MT1-MMP and collagen III was assessed by RT-PCR method. MMP-2 activity was measured by gelatin zymography. RESULTS: No significant difference was noted in cell viability with each CsA concentration. CsA inhibited the cell proliferation in a dose dependent manner and induced the expression of ROS. Antioxidant NAC reversed the effect of cyclosporine. CsA had no effect on the mRNA expression of collagen III, MMP-2, TIMP-2, MT1-MMP. However CsA decreased the MMP-2 activity in a dose dependent manner, which was also reversed by NAC. CONCLUSION: Cyclosporine-induced ROS may be associated with the extracellular matrix accumulation, that is glomerulosclersosis and interstitial fibrosis by inhibiting the cell proliferation and by decreasing the degradation of extracellular matrix. Antioxidant, at least in vitro, may prevent some of the adverse effects of CsA on renal function.
Cell Proliferation ; Cell Survival ; Collagen ; Cyclosporine* ; Extracellular Matrix* ; Fibrosis ; Gelatin ; Humans* ; Matrix Metalloproteinase 14 ; Mesangial Cells* ; Oxygen ; RNA, Messenger ; Tissue Inhibitor of Metalloproteinase-2

OBJECTIVE: Cyclosporine(CsA) and tacrolimus, albeit different in structure, exert immunosuppressive effect by similar mechanism. Although most of clinical manifestations, including nephrotoxicity, are similar in the patients using these drugs, there are some differences including gum hyperplasia, neurotoxicity, and hepatic fibrosis between two drugs. There are several reports about association between reactive oxygen species(ROS) and CsA. In contrast, tacrolimus is known to decrease ROS in central nervous system. Thus, we investigated the possibility of different effects of tacrolimus and CsA on the generation of ROS, on the synthesis and degradation of collagen. METHODS: Experiments were done in primary cultured mesangial cells between 4th and 8th passages. CsA was added to the culture dishes in different concentration(making final CsA concentration of 0, 2, 4, 8 microgram/milliliter) and N-acetylcysteine(NAC) was also added in another mesangial cell culture at 4 microgram/milliliter of CsA concentration; tacrolimus was added in similar pattern(making final tacrolimus concentration of 0, 0.1, 0.2, 0.4 microgram/milliliter, NAC in 0.2 microgram/milliliter of tacrolimus concentration). RESULTS: No significant decrease in viability was noted in both cell groups, but growth retardation was weak in tacrolimus treated cells comparing with CsA treated cells. By flow cytometry, we could find the generation of ROS in CsA treated cells, but not in tacrolimus treated cells. In RT-PCR, there is no significant difference in m-RNA expression for a number of molecules including collagen III, MMP-2, TIMP-2, MT1-MMP in either CsA treated cells or tacrolimus cells. But in zymogram, MMP-2 activities were decreased at higher CsA concentration, then increased with addition of NAC. In tacrolimus cells, MMP2 activity was not changed at 0.1 and 0.2 microgram/milliliter; but, at the concentration of 0.4 microgram/milliliter, changed and not reversed by NAC. MMP-9 activity was similar in both cells. CONCLUSION: We could find ROS generation in CsA treated human mesangial cells, but not in tacrolimus treated cells. We think this difference resulted in the decrease of post-transcriptional MMP-2 activity in CsA treated cells and we also think tacrolimus cells in our experiments were not influenced by ROS. From these results, tacrolimus and CsA are different in the generation of ROS that have some effects in the matrix accumulation in mesangial cells. These result does not mean that tacrolimus is superior to CsA in nephrotoxicity, because nephrotoxicity is similar between two drugs. In conclusion, the mechanisms of nephrotoxicity are different between CsA and tacrolimus.
Central Nervous System ; Collagen ; Cyclosporine* ; Extracellular Matrix* ; Fibrosis ; Flow Cytometry ; Gingiva ; Humans* ; Hyperplasia ; Matrix Metalloproteinase 14 ; Mesangial Cells* ; Oxygen ; Tacrolimus* ; Tissue Inhibitor of Metalloproteinase-2

CCR3 is a chemokine receptor that mediates the accumulation of allergic inflammatory cells, including eosinophils and Th2 cells, at inflamed sites. The regulatory sequence of the CCR3 gene, contains two Runt-related transcription factor (RUNX) 1 sites and two PU.1 sites, in addition to a functional GATA site for transactivation of the CCR3 gene. In the present study, we examined the effects of the cis-acting elements of RUNX1 and PU.1 on transcription of the gene in EoL-1 eosinophilic cells and Jurkat T cells, both of which expressed functional surface CCR3 and these two transcription factors. Introduction of RUNX1 siRNA or PU.1 siRNA resulted in a modest decrease in CCR3 reporter activity in both cell types, compared with transfection of GATA-1 siRNA. Cotransfection of the two siRNAs led to inhibition in an additive manner. EMSA analysis showed that RUNX1, in particular, bound to its binding motifs. Mutagenesis analysis revealed that all point mutants lacking RUNX1- and PU.1-binding sites exhibited reduced reporter activities. These results suggest that RUNX1 and PU.1 participate in transcriptional regulation of the CCR3 gene.
Eosinophils ; Mutagenesis ; RNA, Small Interfering ; T-Lymphocytes ; Th2 Cells ; Transcription Factors ; Transcriptional Activation ; Transfection

Telomerase is a ribonucleoprotein enzyme that synthesizes telomeric DNA onto the ends of chromosomes. thereby preventing the replication-dependent shortening of these ends. Telomerase activity is detected in a wide range of cancers of various tissues, and its expression may be a critical step in tumor progression. Our objective was to determine if detection of telomerase activity may be an indicator for diagnosis of breast cancer and any association between telomerase activity and prognostic factors of breast cancer. Using a polymerase chain reaction-based telomerase activity assay, we examined telomerase activity in 30 breast cancer specimens (2 ductal carcinoma in situ, 28 invasive ductal carcinoma), 25 benign lesions (14 fibroadenomas, 11 fibrocystic diseases) and 24 normal breast tissues (13 adjacent to malignancy, 11 adjacent to benign lesion). Among surgically resected samples, telomerase activity was detected in 23 (77%) of 30 breast cancers. While telomerase activity was not detected in any of 11 specimens of fibrocystic disease and 11 adjacent normal tissues to benign lesion, surprisingly low levels of telomerase activity were detected in 5 (36%) of 14 fiboadenomas and 1 (7%) of 13 adjacent normal tissues to malignancy. There was no significant difference in expression of telomerase among prognostic factors of breast cancer. In summary, telomerase activity in breast cancer may be useful in diagnosis of breast cancer. We found no correlation between telomerase activity and stage, tumor size or LN status. Mechanisms of telomerase expression are still under investigation; therefore, the significance of telomerase expression in malignant tumors and their progression remains to be determined.
Breast Neoplasms* ; Breast* ; Carcinoma, Intraductal, Noninfiltrating ; Diagnosis ; DNA ; Fibroadenoma ; Humans* ; Ribonucleoproteins ; Telomerase*