Ocular surface epithelial and stem cell development

Phenotypic features and developmental events involved in the genesis of the limbo-corneal and conjunctival epithelia are described. Together, these two epithelia define the ocular surface. They derive from a small cohort of optic vesicle-induced PAX6+ head ectodermal cells that remain on the surface following lens vesicle formation by the main PAX6+ cell cohort. Both epithelia are stratified, and display wet, non-keratinizing phenotypes. The most significant spatial feature of the limbo-corneal epithelium is the segregation of its supporting stem and early precursor cells to the limbus, the outer vascularized rim separating the cornea from the conjunctiva. These stem cells express ABCG2, a xenobiotic transporter present in stem cells from other organs. ABCG2 transport activity excludes the DNA dye Hoechst 33342, allowing the isolation of the ocular stem cells by flow cytometry, as a unique cohort known as a side 'side population'. Limbal stem cells do not form gap junctions and exist as metabolically isolated entities. Tracking of expression changes in Cx43, the main gap junction protein expressed in both the pre-epithelial ectoderm and in the mature central corneal epithelium, indicates that a limbal stem cell phenotype starts developing very soon after lens vesicle invagination, in advance of the appearance of any recognizable anatomical sub-epithelial limbal feature. Differences in Cx43 expression also reveal the very early nature of the divergence in limbo-corneal and conjunctival lineages. The putative involvement of several early genes, including gradients of PAX6 and differences in expression patterns for members of the Id or msh gene expression regulators are reviewed.     

GM1 gangliosidosis and Morquio B disease: An update on genetic alterations and clinical findings

GM1 gangliosidosis and Morquio B syndrome, both arising from beta-galactosidase (GLB1) deficiency, are very rare lysosomal storage diseases with an incidence of about 1:100,000-1:200,000 live births worldwide. Here we report the beta-galactosidase gene (GLB1) mutation analysis of 21 unrelated GM1 gangliosidosis patients, and of 4 Morquio B patients, of whom two are brothers. Clinical features of the patients were collected and compared with those in literature. In silico analyses were performed by standard alignments tools and by an improved version of GLB1 three-dimensional models. The analysed cohort includes remarkable cases. One patient with GM1 gangliosidosis had a triple X syndrome. One patient with juvenile GM1 gangliosidosis was homozygous for a mutation previously identified in Morquio type B. A patient with infantile GM1 gangliosidosis carried a complex GLB1 allele harbouring two genetic variants leading to p.R68W and p.R109W amino acid changes, in trans with the known p.R148C mutation. Molecular analysis showed 27 mutations, 9 of which are new: 5 missense, 3 microdeletions and a nonsense mutation. We also identified four new genetic variants with a predicted polymorphic nature that was further investigated by in silico analyses. Three-dimensional structural analysis of GLB1 homology models including the new missense mutations and the p.R68W and p.R109W amino acid changes showed that all the amino acid replacements affected the resulting protein structures in different ways, from changes in polarity to folding alterations. Genetic and clinical associations led us to undertake a critical review of the classifications of late-onset GM1 gangliosidosis and Morquio B disease.     

Reactive oxygen species are required for hyperoxia-induced Bax activation and cell death in alveolar epithelial cells

Exposure of animals to hyperoxia results in respiratory failure and death within 72 h. Histologic evaluation of the lungs of these animals demonstrates epithelial apoptosis and necrosis. Although the generation of reactive oxygen species (ROS) is widely thought to be responsible for the cell death observed following exposure to hyperoxia, it is not clear whether they act upstream of activation of the cell death pathway or whether they are generated as a result of mitochondrial membrane permeabilization and caspase activation. We hypothesized that the generation of ROS was required for hyperoxia-induced cell death upstream of Bax activation. In primary rat alveolar epithelial cells, we found that exposure to hyperoxia resulted in the generation of ROS that was completely prevented by the administration of the combined superoxide dismutase/catalase mimetic EUK-134 (Eukarion, Inc., Bedford, MA). Exposure to hyperoxia resulted in the activation of Bax at the mitochondrial membrane, cytochrome c release, and cell death. The administration of EUK-134 prevented Bax activation, cytochrome c release, and cell death. In a mouse lung epithelial cell line (MLE-12), the overexpression of Bcl-XL protected cells against hyperoxia by preventing the activation of Bax at the mitochondrial membrane. We conclude that exposure to hyperoxia results in Bax activation at the mitochondrial membrane and subsequent cytochrome c release. Bax activation at the mitochondrial membrane requires the generation of ROS and can be prevented by the overexpression of Bcl-XL.     

Assessment of serum thiol/disulfide homeostasis in multiple myeloma patients by a new method

Objectives: The etiology of multiple myeloma (MM) is not exactly known. This study investigated the role of thiol/disulfide homeostasis in the etiopathogenesis of MM.

Methods: Some 50 patients with MM (aged 39–84 years) and 50 sex-matched healthy volunteer controls (aged 50–91 years) participated in this study. Venous blood samples were collected, and levels of native thiols, total thiols, and disulfide were measured.

Results: Native and total thiol levels in the control group were determined to be higher than in the study and patient groups (P<0.001). Disulfide levels were found to be higher in the control group than in the study group and higher in newly diagnosed patients than in outpatients who were undergoing treatment (P=0.002). The ratios of thiol levels were found to be similar in both the study and control groups (P>0.05).

Discussion: The results of the study show that although there was a decrease in the levels of disulfide, native thiol, and total thiol, the balance of thiol/disulfide was maintained. This is the first study to research the homeostasis of dynamic thiol/disulfide from the perspective of the new method that was used. We hope that this study will encourage and facilitate further studies in this area.     

Analysis of an outbreak due to Chryseobacterium meningosepticum in a neonatal intensive care unit

The aim of this study was to describe the epidemiological and clinical features of an outbreak due to Chryseobacterium meningosepticum. During a 11-day period, the outbreak was observed among four newborns in a neonatal intensive care unit (NICU) in a teaching hospital. All patients yielded C. meningosepticum in their blood cultures, in addition one was colonised in the throat. Antimicrobial susceptibility assay showed complete resistance to penicillins, cephalosporins, aminoglycosides, imipenem, aztreonam, and tetracycline, sensitivity to ciprofloxacin and trimethoprim-sulfamethoxazole. All patients were empirically treated with amikacin and meropenem. The neonate who was the first to develop sepsis died before the culture result. When C. meningosepticum was identified, antimicrobial therapy was changed to a combination of ciprofloxacin, rifampicin and vancomycin, and three neonates were treated successfully. Environmental screening recovered C. meningosepticum from two venous catheter lines and one nutritional solution that was opened by health care staff and used for two neonates. Arbitrary primed polymerase chain reaction and antibiogram typing indicated that all isolates were epidemiologically related. This study demonstrates that rapid selection of appropriate antibiotics is critical for clinical cure and standard precautions should be reconsidered to limit the spread of this bacterium on the NICU in our hospital.     

Activities of Linezolid against nontuberculous mycobacteria

The activity of linezolid (Pfizer, USA) was tested by broth microdilution against 53 clinical isolates of non-tuberculous mycobacteria (NTM), including the common disease producing species Mycobacterium avium, M. intracellulare, M. fortuitum, M. chelonae and M. abscessus, obtained from western Turkey. The isolates of M. abscessus and M. intracellulare were the least susceptible, M. mucogenicum, M. gordonae and M. avium were the most susceptible to linezolid of the common species of NTM. Linezolid showed a variable sensitivity in all strains; therefore, each species and strain must be individually evaluated, and it is always advisable to perform in vitro sensitivity tests before using the drug for human therapy.     

Lipid peroxidation and antioxidant status in blood and tissue of malignant breast tumor and benign breast disease

Changes in the levels of malondialdehyde (MDA), nitrate and nitrite (as an index of nitric oxide production), lipid hydroperoxide (LOH), total antioxidant capacity (TAC), lipids (total cholesterol and triglycerides) and lipoproteins (HDL- and LDL-cholesterol) were estimated in breast cancer patients (n = 15) and benign breast disease (n = 15). Serum and tissue MDA levels were found to be decreased in breast cancer patients compared to the benign group (p < 0.05). In contrast, nitrate and nitrite levels were increased in serum and tissue of the cancer group compared to benign breast disease patients (p < 0.05). Compared to the benign group, tissue TAC levels were elevated in the breast cancer patient group (p < 0.05). Total cholesterol and HDL-cholesterol were elevated in the benign group compared with cancer patients (p < 0.05). These findings support the hypothesis that lipid peroxidation in serum and tissue of benign breast disease is greater than in breast cancer. However, the enhanced levels of nitric oxide may be in response to inflammation in patients with breast cancer. Total antioxidant status is lower in benign tissue than in cancerous tissue, probably to compensate for this elevated free radical production.     

The intrinsic apoptotic pathway is required for lipopolysaccharide-induced lung endothelial cell death

LPS has been implicated in the pathogenesis of endothelial cell death associated with Gram-negative bacterial sepsis. The binding of LPS to the TLR-4 on the surface of endothelial cells initiates the formation of a death-inducing signaling complex at the cell surface. The subsequent signaling pathways that result in apoptotic cell death remain unclear and may differ among endothelial cells in different organs. We sought to determine whether LPS and cycloheximide-induced cell death in human lung microvascular endothelial cells (HmVECs) was dependent upon activation of the intrinsic apoptotic pathway and the generation of reactive oxygen species. We found that cells overexpressing the anti-apoptotic protein Bcl-X(L) were resistant to LPS and cycloheximide-induced death and that the proapoptotic Bcl-2 protein Bid was cleaved following treatment with LPS. The importance of Bid was confirmed by protection of Bid-deficient (bid(-/-)) mice from LPS-induced lung injury. Neither HmVECs treated with the combined superoxide dismutase/catalase mimetic EUK-134 nor HmVECs depleted of mitochondrial DNA (rho(0) cells) were protected against LPS and cycloheximide-induced death. We conclude that LPS and cycloheximide-induced death in HmVECs requires the intrinsic cell death pathway, but not the generation of reactive oxygen species.