Biocompatibility of a fish scale-derived artificial cornea: Cytotoxicity, cellular adhesion and phenotype, and in vivo immunogenicity.

PURPOSE: To determine whether a fish scale-derived collagen matrix (FSCM) meets the basic criteria to serve as an artificial cornea, as determined with in vitro and in vivo tests. METHODS: Primary corneal epithelial and stromal cells were obtained from human donor corneas and used to examine the (in)direct cytotoxicity effects of the scaffold. Cytotoxicity was assessed by an MTT assay, while cellular proliferation, corneal cell phenotype and adhesion markers were assessed using an EdU-assay and immunofluorescence. For in vivo-testing, FSCMs were implanted subcutaneously in rats. Ologen(®) Collagen Matrices were used as controls. A second implant was implanted as an immunological challenge. The FSCM was implanted in a corneal pocket of seven New Zealand White rabbits, and compared to sham surgery. RESULTS: The FSCM was used as a scaffold to grow corneal epithelial and stromal cells, and displayed no cytotoxicity to these cells. Corneal epithelial cells displayed their normal phenotypical markers (CK3/12 and E-cadherin), as well as cell-matrix adhesion molecules: integrin-α6 and ß4, laminin 332, and hemi-desmosomes. Corneal stromal cells similarly expressed adhesion molecules (integrin-α6 and ß1). A subcutaneous implant of the FSCM in rats did not induce inflammation or sensitization; the response was comparable to the response against the Ologen(®) Collagen Matrix. Implantation of the FSCM in a corneal stromal pocket in rabbits led to a transparent cornea, healthy epithelium, and, on histology, hardly any infiltrating immune cells. CONCLUSION: The FSCM allows excellent cell growth, is not immunogenic and is well-tolerated in the cornea, and thus meets the basic criteria to serve as a scaffold to reconstitute the cornea.

Mutation of a Nopp140 gene dao-5 alters rDNA transcription and increases germ cell apoptosis in C. elegans.

Human diseases of impaired ribosome biogenesis resulting from disruption of rRNA biosynthesis or loss of ribosomal components are collectively described as 'ribosomopathies'. Treacher Collins syndrome (TCS), a representative human ribosomopathy with craniofacial abnormalities, is attributed to mutations in the tcof1 gene that has a homologous gene called nopp140. Previous studies demonstrated that the dao-5 (dauer and aged animal overexpression gene 5) of Caenorhabditis elegans is a member of nopp140 gene family and plays a role in nucleogenesis in the early embryo. Here, we established a C. elegans model for studying Nopp140-associated ribosomopathy. A null dao-5 mutant ok542 with a semi-infertile phenotype showed a delay in gonadogenesis, as well as a higher incidence of germline apoptosis. These phenotypes in dao-5(ok542) are likely resulted from inefficient rDNA transcription that was observed by run-on analyses and chromatin immunoprecipitation (ChIP) assays measuring the RNA Pol I occupancy on the rDNA promoter. ChIP assays further showed that the modifications of acetylated histone 4 (H4Ac) and dimethylation at the lysine 9 of histone 3 (H3K9me2) around the rDNA promoter were altered in dao-5 mutants compared with the N2 wild type. In addition, activated CEP-1 (a C. elegans p53 homolog) activity was also linked to the loss of DAO-5 in terms of the transcriptional upregulation of two CEP-1 downstream effectors, EGL-1 and CED-13. We propose that the dao-5 mutant of C. elegans can be a valuable model for studying human Nopp140-associated ribosomopathy at the cellular and molecular levels.

Medical malpractice experience of Taiwan: 2005 versus 1991.

BACKGROUND: Medical malpractice litigation has become an important issue worldwide. Although many epidemiological studies have been carried out, most studies were conducted cross-sectionally in developed countries and focused on malpractice litigation. We conducted nationwide surveys to investigate physicians' experiences associated with malpractice in 1991 and 2005, respectively. METHODS: By stratified systemic sampling, questionnaires were mailed to physicians in 1991 and 2005. Physicians were asked about the experience of medical malpractice and outcomes of malpractice. The outcomes of the malpractice were classified as resolution, settlement and lawsuit. We also collected physicians' demographic and professional characteristics. RESULTS: The prevalence of malpractice experience decreased from 44.1% in 1991 to 36.0% in 2005 (P = 0.004). The estimated annual malpractice claims decreased from 0.14 to 0.10 per physician in 1991 and 2005, respectively (P < 0.001). Physicians 45-64 years of age, obstetrician/gynaecologists and surgeons had significantly higher risk of malpractice. Compared with 1991, malpractice claims in 2005 were more likely to be brought into courts (23.1% in 2005 vs 15.7% in 1991, odds ratio (OR) = 1.48, P = 0.020). In litigation cases, malpractice events in 2005 had more than triple the risk of 1991 to be sued in both civil and criminal courts (12.4% in 2005 vs 4.1% in 1991, OR = 3.31, P < 0.001). CONCLUSION: Compared with 1991, medical malpractice experiences were decreasing in prevalence, but increasing in severity in 2005. Additional studies, especially among different legal systems, are necessary to confirm these observations.

Sera from children with active Henoch-Schönlein purpura can enhance the production of interleukin 8 by human umbilical venous endothelial cells.

OBJECTIVE: To investigate the ability of sera from children with active Henoch-Schonlein purpura (HSP) to enhance endothelial interleukin (IL) 8 production and intercellular adhesion molecule (ICAM)-1 expression. METHODS: Nine children with active HSP and nine normal healthy children were enrolled. IL8 serum levels of patients and controls at different stages were analysed. Production of IL8 and expression of ICAM-1 by human umbilical venous endothelial cells were detected (ELISA for IL8, flow cytometry for ICAM-1) and compared under various stimuli, including sera of patients at different stages, sera of controls, and medium alone. RESULTS: Serum levels of IL8 were increased at the acute stage. Levels of IL8 in supernatants from human umbilical venous endothelial cells (HUVEC) co-cultured with sera from children with active HSP were significantly higher than those from HUVEC without any treatment (p = 0.001), HUVEC treated with inactive sera (p = 0.004), and HUVEC treated with sera from healthy controls (p = 0.004). Sera from patients and from controls did not enhance the expression of ICAM-1 on HUVEC. CONCLUSIONS: Some factors may be present in sera from children with active HSP that could activate endothelial cells to produce IL8. This process may account, in part, for the mechanisms of perivascular neutrophil infiltration and leucocytosis in HSP.

Effects of mannitol or catalase on the generation of reactive oxygen species leading to DNA damage by Chromium(VI) reduction with ascorbate.

Interaction of Cr(VI) and ascorbate in vitro generates Cr(V), Cr(IV), Cr(III), carbon-based alkyl radicals, COO(*)(-), (*)OH, and ascorbate radicals and induces DNA interstrand cross-links at guanines. To determine which specific Cr species and free radicals cause DNA damage, we investigated the effects of mannitol and catalase on the formation of reactive intermediates, Cr-DNA associations, DNA polymerase-stop sites, and 8-hydroxydeoxyguanosine (8-OHdG) adducts induced by Cr(VI)/ascorbate in a Hepes buffer. EPR spectra showed that mannitol trapped reactive Cr(V), forming a stable Cr(V)-diol complex, and inhibited the radicals induced by Cr(VI)/ascorbate, whereas catalase or heat-denatured catalase enhanced the levels of Cr(V) without altering the radical signals. Mannitol markedly inhibited the retarded gel electrophoretic mobility of supercoiled plasmids and the formation of DNA polymerase-stop sites induced by Cr(VI)/ascorbate, but catalase did not. On the other hand, mannitol reduced only 32% of the Cr-DNA adducts induced by Cr(VI)/ascorbate, suggesting that Cr monoadducts (possibly DNA-Cr-mannitol adducts) are the major lesions generated in the Cr(VI)/ascorbate/mannitol/DNA solution. Native catalase but not heat-denatured catalase protected approximately 25% of the Cr-DNA adducts induced by Cr(VI)/ascorbate, suggesting that hydrogen peroxide may be involved. Mannitol could not completely inhibit the formation of 8-OHdG adducts induced by Cr(VI)/ascorbate, indicating that this DNA damage may be generated before the action of mannitol to trap Cr(V) and reactive oxygen species. Alternatively, Cr-peroxide intermediates may also lead to 8-OHdG formation to account for the incomplete prevention by mannitol. Catalase or heat-denatured catalase partially protected the formation of 8-OHdG adducts induced by Cr(VI)/ascorbate, suggesting an effect of proteins. Together, the results from this study suggest that the primary species generated during the reduction of Cr(VI) by ascorbate are hydroxyl radicals and Cr(V) species, responsible for the formation of 8-OHdG and DNA cross-links, respectively.