In vitro prediction of hepatotoxicity can enhance the performance of non-clinical animal testing for identifying chemical hazards. In this study, we assessed high-content analysis (HCA) using multi-parameter cell-based assays as an in vitro hepatotoxicity testing model using various hepatotoxicants and human hepatocytes such as HepG2 cells and human primary hepatocytes (hPHs). Both hepatocyte types were exposed separately to multiple doses of ten hepatotoxicants associated with liver injury whose mechanisms of action have been described. HCA data were obtained using fluorescence probes for nuclear size (Hoechst), mitochondrial membrane potential (TMRM), cytosolic free calcium (Fluo-4AM), and lipid peroxidation (BODIPY). Cellular alterations were observed in response to all hepatotoxicants tested. The most sensitive parameter was TMRM, with high sensitivity at a low dose, next was BODIPY, followed by Fluo-4AM. HCA data from HepG2 cells and hPHs were generally concordant, although some inconsistencies were noted. Both hepatocyte types showed mild or severe mitochondrial impairment and lipid peroxidation in response to several hepatotoxicants. The results demonstrate that the application of HCA to in vitro hepatotoxicity testing enables more efficient hazard identification, and further, they suggest that certain parameters could serve as sensitive endpoints for predicting the hepatotoxic potential of chemical compounds.
Animals ; Calcium ; Cytosol ; Fluorescence ; Hep G2 Cells ; Hepatocytes ; Humans ; In Vitro Techniques ; Lipid Peroxidation ; Liver ; Membrane Potential, Mitochondrial

BACKGROUND AND OBJECTIVES: Occlusion of semicircular canal has been used for otoneurologic and skull base surgery with preservation of postoperative hearing. However, research on healing process of the occluded bony semicircular canal is scarce. MATERIALS AND METHODS: We transected and occluded the lateral semicircular canal of guinea pigs using a surgical drill. Histopathologic changes of the temporal bones were observed up to the eighth postoperative week. RESULTS: The temporal bone specimens showed that ostcogenic cells proliferated from the perilymphatic fibrous mesh formed a new bone in the occluded portion of the lateral semicircular canal. Periosteal cells of the endosteum produced a compact bone layer lining the bony semicircular canal lumen. Periosteal layer produced a woven bone at the surgical defect of the lateral semicircular canal. CONCLUSION: This study shows that the perilymphatic fibrous mesh, the endosteum and the periosteal layer participated in osteogenesis in healing of the bony lateral semicircular canal after transection and occlusion.
Animals ; Ear, Inner* ; Guinea Pigs ; Hearing ; Osteogenesis ; Semicircular Canals* ; Skull Base ; Temporal Bone

Cytokine has been used as an immune stimulator and administered to patients for a treatment of cancer. Interleukin-12 (IL-12) is a potent cytokine which acts through a variety of functions including interferon-gamma production and cytotoxic T-cell activation. Considering the toxicity of high dose systemic IL-12 administration into human, local administration of low dose IL-12 can be a more efficient strategy. In ex vivo therapy, human dermal fibroblast has been considered as a useful vehicle for transferring genes, Here we show that human dermal fibroblast transduced with retrovirus containing IL-12 gene can be manipulated to produce reasonable amount of IL-12 protein. Human dermal fibroblast was isolated from freshly harvested skin specimens by collagenase digestion, grown in primary cultures, and transduced with a retroviral vector containing genes for human IL-12 and a selectable marker Neo(R). Following selection in G418, IL-12 producing fibroblasts were tested for secreted IL-12 level by ELISA. Six specimens of human skin were processed to obtain fibroblasts. ELISA results show that 40-150 units of IL-12 was produced for 24 h from 1x10(6) cells of transduced and selected fibroblast cultures. The primary cultures were maintained for up to nine passages about 108 days. The mean +/- overall time for obtaining enough number of cells was 49 +/- 2 days. The fibroblasts continued to produce IL-12 in culture for 90 days. These preliminary results can be used for the design of ex vivo gene therapy clinical trial using human dermal fibroblast.
Collagenases ; Digestion ; Enzyme-Linked Immunosorbent Assay ; Fibroblasts* ; Genes, Neoplasm* ; Genetic Therapy ; Humans* ; Interferon-gamma ; Interleukin-12* ; Retroviridae ; Skin ; T-Lymphocytes ; Zidovudine

Coccidiosis in chickens is an intestinal parasitic disease caused by protozoan parasites named Eimeria spp. In some Eimeria infections, intestinal lymphocytes are known to highly express chicken NK-lysin (cNK-lysin), an antimicrobial peptide with anticoccidial activity. Therefore, this study aims to investigate the expression of cNK-lysin in E. necatrix-infected chickens and its role in E. necatrix infection. The expression of cNK-lysin transcript was significantly increased in E. necatrix sporozoites-treated lymphocytes. In E. necatrix infection, cNK-lysin transcript was induced in intestinal lymphocytes but not in the spleen. The recombinant cNK-lysin exhibited anticoccidial activity against E. necatrix sporozoites as well as immunomodulatory activity on macrophages by inducing proinflammatory cytokines. These results indicated that E. necatrix infection induces high local expression of cNK-lysin and the secreted cNK-lysin helps protect coccidiosis.

PURPOSE: We studied the E2A-PBX1 positivity in t(1;19)-positive childhood acute lymphoblastic leukemia (ALL) patients during chemotherapy and follow-up period to evaluate the clinical implications of minimal residual disease (MRD) and the effect of delayed intensification chemotherapy on long-term survival. METHOD: Fifty-six bone marrow or peripheral blood samples collected retrospectively or prospectively before or during chemotherapy from 14 childhood ALL patients who had t(1;19) or E2A-PBX1 transcript at initial diagnosis were studied for the presence of E2A-PBX1 by RT- PCR. All patients received delayed intensification chemotherapy regardless of standard prognostic grouping for childhood ALL to evaluate its effect on the improvement of long-term survival. RESULTS: There were 11 t(1;19)-positive cases documented by karyotyping and 3 E2A-PBX1 transcript-positive cases amplified by PCR from the initial bone marrow samples. There were 11 cases of FAB L1 and 3 cases of L2. Immunophenotypic classification revealed 10 cases of group V, 2 cases of group IV, and 1 case of group III. Among 11 cases with documented karyotype, 9 cases (81.8%) had der(19)t(1;19) and the other 2 had balanced t(1;19). Fifty-six samples collected at 2 to 7 different time points of 14 patients revealed 4 cases of MRD immediately after completion of induction chemotherapy despite hematologic complete remission. Three of these cases relapsed eventually, and 1 was lost to follow-up. Among 12 cases who received adequate delayed intensificiation chemotherapy, 10 are alive in complete remission. CONCLUSION: MRD detection by RT-PCR amplification of E2A-PBX1 transcript was feasible, and the sample after completion of induction chemotherapy was most informative for the prediction of long-term survival and relapse. The presence of MRD after completion of induction chemotherapy conferred poor prognosis. The addition of delayed intensification chemotherapy to standard chemotherapy regimen could abolish the adverse effect of t(1;19) in childhood ALL patients.
Bone Marrow ; Classification ; Diagnosis ; Drug Therapy* ; Follow-Up Studies ; Humans ; Induction Chemotherapy ; Karyotype ; Karyotyping ; Lost to Follow-Up ; Neoplasm, Residual ; Polymerase Chain Reaction ; Precursor Cell Lymphoblastic Leukemia-Lymphoma* ; Prognosis ; Prospective Studies ; Recurrence ; Retrospective Studies

PURPOSE: The diagnostic criteria of the biphenotypic acute leukemia (BAL) are based on the number and degree of the specificity of the immunological markers expressed on the leukemic blasts. The newer diagnostic criteria proposed by The European Group for the Immunological Classification of Leukaemias (EGIL) is now well accepted. We have recently evaluated our BAL patients using the EGIL criteria and analyzed the clinical characteristics, treatment and clinical outcome. METHODS: Fourteen children diagnosed with BAL among 193 childhood acute leukemia patients in our hospital from January 1995 to December 2001 were retrospectively reviewed. RESULTS: The incidence of BAL was 7.3% (14 out of 193 cases). Of these 14 patients, 12 were de novo and 2 were secondary. In the de novo group, the immunological marker studies showed myeloid/B-lymphoid phenotype in 6 (50%), myeloid/T-lymphoid in 3 and B-lymphoid/T-lymphoid in 1. In addition, two patients showed trilineage differentiation. Cytogenetic analysis revealed various abnormal karyotypes in the majority of the cases, showing normal karyotype only in 3 cases. Among the karyotype abnormalities, two were t (9; 22) and one was t (4; 11). Chemotherapy was mostly based on the induction regimen of acute lymphoblastic leukemia therapy (12 of 14 cases). One patient was treated with acute myeloid leukemia regimen and one patient received combination of both acute lymphoid and myeloid regimen. Induction of complete remission was achieved in 100% of the patients and the median duration of induction therapy to complete remission was 33 days. Five of the 12 de novo patients died during the median follow-up of 16.5 months (4 months to 37.5 months) and the 2 year survival rate was only 52%. CONCLUSION: The incidence of BAL in children is relatively rare and the most common immunophenotypic expression consists of myeloid and B-lymphoid coexpression. Most of the cases showed chromosomal abnormalities. The outcome of BAL treated mostly with the traditional acute lymphoblastic leukemia induc without stem cell transplantation should be sought especially in those expressing poor prognostic cytogenetic abnormalities or strong myeloid marker expression.
Abnormal Karyotype ; Child ; Chromosome Aberrations ; Classification ; Cytogenetic Analysis ; Drug Therapy ; Follow-Up Studies ; Humans ; Incidence ; Karyotype ; Leukemia ; Leukemia, Biphenotypic, Acute* ; Leukemia, Myeloid, Acute ; Phenotype ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; Retrospective Studies ; Sensitivity and Specificity ; Stem Cell Transplantation ; Survival Rate

OBJECTIVE: The purpose of this study was to investigate the relationship between the mandibular dental and basal arch forms in subjects with normal occlusion and compare them with those of Class III malocclusion using cone-beam computed tomography (CBCT). METHODS: CBCT images of 32 normal occlusion (19 males, 13 females; 24.3 years) and 33 Class III malocclusion subjects (20 males, 13 females, 22.2 years) were selected. Facial axis and root center points were identified from the left to right mandibular first molars. Distances between the facial axis and root center points for each tooth were calculated, and 4 linear and 2 ratio variables were measured and calculated for each arch form. The variables were compared between groups by independent t-test. Pearson correlation coefficient was applied to assess the relationships between dental and basal variables within each group. RESULTS: The mandibular dental and basal intercanine widths were significantly greater in the Class III group than in normal occlusion subjects (p < 0.05). The dental and basal intercanine widths as well as the dental and basal intermolar widths were strongly correlated in normal occlusion and moderately correlated in Class III malocclusion. CONCLUSIONS: The dental arch form demon strated a strong positive correlation with the basal arch form in the normal occlusion group and moderate correlation in the Class III malocclusion group. These results might be helpful for clinicians to have a better understanding of the importance of basal arch form in the alveolar bone.
Axis, Cervical Vertebra ; Cone-Beam Computed Tomography ; Dental Arch ; Female ; Humans ; Male ; Malocclusion ; Molar ; Tooth

The purpose of our experiment is to examine the level of anti-Haemophilus influenza polysaccharide antibody titer in the Korean population. Using ELISA, the level of Hib-PS antibodies in 384 infants and children who were all free from Hib invasive diseases, was tested. And the blood of 50 mothers within 24 hours of delivery and cord blood from their respective full-term neonates was also tested. The transport of Hib-PS IgG and IgG subclasses in paired sera from mothers and neonates was also measured. The titer of Hib-PS IgG varies with age. At birth the mean optical density of cord blood was 1.028; however, it declined to 0.609 up to 6 months and further decline was noted up to 2 years to 0.488. Then the mean O.D. remained around 0.5 from 3 to 14 years of age. The mean O.D. of Hib-PS IgG in the mothers blood was 0.856. The ratio of mean O.D. of anti-Hib PS IgG antibody in the cord blood to that in the maternal blood was 1.20. The mean optical densities of IgG subclasses were: 1.18 for anti-Hib PS IgG1, 1.07 for anti-Hib PS IgG2, 1.01 for anti-Hib PS IgG3, and 1.09 for anti-Hib PS IgG4. The sera from Korean children of almost all age groups reacted to Hib-PS antigen on ELISA. Also the active transport of anti-Hib PS IgG antibody through placenta was observed. Among four IgG subclasses, only IgG1 transport had significant experimental meaning.
Adolescent ; Adult ; Antibodies, Bacterial/*immunology ; Antigens, Bacterial/*immunology ; Bacterial Capsules ; Child ; Child, Preschool ; Enzyme-Linked Immunosorbent Assay ; Female ; Fetal Blood/immunology ; Haemophilus Vaccines/*immunology ; Haemophilus influenzae/*immunology ; Humans ; Immunoglobulin G/classification/immunology ; Infant ; Infant, Newborn ; Korea ; Male ; Maternal-Fetal Exchange ; Polysaccharides, Bacterial/*immunology ; Pregnancy

BACKGROUND: Lymphocytes seen during the chemotherapy of childhood ALL are not fully understood regarding their clinical significance. The lymphoid aggregates found during the complete remission period are more confusing. We investigated the characteristics of lymphoid aggregates and the clinial course of children with these in the marrow during the chemotherapy of childhood ALL. This is the first study about this subject. METHODS: From January 1996 to April 1998, 210 bone marrow specimens were diagnosed as complete remission status of ALL and among them, ten patients (4.8%) showed lymphoid aggreagates on the marrow clot sections at the time of complete remission. We reviewed bone marrow specimens, performed immunohistochemical stains for CD3, CD 10 and CD79a and investigated the clinical course. RESULTS: The ten cases were composed of nine ALL, L1 and one ALL, L2. All of them were treated under guidance of the CCG (children's cancer group) protocol. Fourteen lymphoid aggregates from ten cases were found. They showed mean number of 1.4 per clot section, mean diameter of 132 micrometer, regular (36%) or irregular (64%) margin and composition of mature lymphocytes (21%), immature lymphocytes (29%) or mixed pattern (50%). The mean interval between the diagnosis and the emergence of lymphoid agregates was 29 months (2~55 months). One patient in the course of consolidation chemotherapy expired due to upper gastrointestinal bleeding and other nine cases are still in the continuous complete remission state. The lymphoid cells consisting of lymphoid aggregates showed positive reaction only for CD79a and negative reactions for CD3 and CD10. CONCLUSION: Lymphpoid aggregates found at the time of complete remission are collections of regenerating B-lymphocytes and they are not residual leukemic blasts, and show no effect on the complete remission state.
B-Lymphocytes ; Bone Marrow* ; Child ; Coloring Agents ; Consolidation Chemotherapy ; Diagnosis ; Drug Therapy ; Hemorrhage ; Humans ; Lymphocytes ; Precursor Cell Lymphoblastic Leukemia-Lymphoma

OBJECTIVE: Activating mutations in the fibroblast growth factor receptor-2 (FGFR2) have been shown to cause syndromic craniosynostosis such as Apert and Crouzon syndromes. The purpose of this pilot study was to investigate the resultant phenotypes induced by the two distinctive bone-targeted gene constructs of FGFR2, Pro253Arg and Cys278Phe, corresponding to human Apert and Crouzon syndromes respectively. METHODS: Wild type and a transgenic mouse model with normal FGFR2 were used as controls to examine the validity of the microinjection. Micro-CT and morphometric analysis on the skull revealed the following results. RESULTS: Both Apert and Crouzon mutants of FGFR2 induced fusion of calvarial sutures and anteroposteriorly constricted facial dimension, with anterior crossbite present only in Apert mice. Apert mice differed from Crouzon mice and transgenic mice with normal FGFR2 in the anterior cranial base flexure and calvarial flexure angle which implies a possible difference in the pathogenesis of the two mutations. In contrast, the transgenic mice with normal FGFR2 displayed normal craniofacial phenotype. CONCLUSION: Apert and Crouzon mutations appear to lead to genotype-specific phenotypes, possibly causing the distinctive sites and sequence of synostosis in the calvaria and cranial base. The exact function of the altered FGFR2 at each suture needs further investigation.
Acrocephalosyndactylia ; Animals ; Craniofacial Dysostosis* ; Craniosynostoses ; Fibroblast Growth Factors ; Humans ; Malocclusion ; Mice ; Mice, Transgenic ; Microinjections ; Phenotype ; Pilot Projects ; Skull ; Skull Base ; Sutures ; Synostosis