Phenotypic analyses of limbal epithelial cell cultures derived from donor corneoscleral rims.

Grafted cultures of limbal epithelial cells aid repair of the corneal epithelium, but their phenotype is unclear. In this study, the phenotype of cultures that were similar in age to those used clinically were analysed. Limbal epithelial cells were isolated from donor corneoscleral rims and grown in various media, including those designed for keratinocytes.

Gut mucosal injury is attenuated by recombinant bactericidal/permeability-increasing protein in hind limb ischemia-reperfusion injury.

Lower limb ischemia-reperfusion injury (IRI) is associated with increased gut permeability to endotoxin, which not only directly damages enterocytes but also stimulates a systemic inflammatory response syndrome (SIRS), compounding gut injury. Recombinant bactericidal/permeability-increasing protein (rBPI21) is a novel anti-endotoxin therapy with proven benefit in sepsis. Its potential role in modulating remote gut injury in hind limb IRI was studied.

Recombinant bactericidal/permeability-increasing protein attenuates the systemic inflammatory response syndrome in lower limb ischemia-reperfusion injury.

Objectives: Hind limb ischemia-reperfusion (I/R) injury increases gut permeability, and resultant endotoxemia is associated with an amplified systemic inflammatory response syndrome leading to multiple organ dysfunction syndrome. We studied the potential role of recombinant bactericidal/permeability-increasing protein (rBPI(21) ), a novel antiendotoxin therapy, in modulating endotoxin-enhanced systemic inflammatory response syndrome in hind limb I/R injury.

Methods: In this prospective, randomized, controlled, experimental animal study, 48 male Wistar rats, weighing 300 to 350 g, were randomized to a control group (sham) and five groups undergoing 3 hours bilateral hind limb ischemia with 2 hours reperfusion (I/R) (n = 8 per group).

A novel approach to studying the migratory morphology of embryonic mesenchymal cells.

A polarized morphology, defined by extension of an anterior pseudopod, is essential for the amoeboid migration of embryonic mesenchymal cells. Leukocytes adopt a similar morphology immediately following suspension in simple buffers containing chemotactic factors. Polarization in suspension therefore provides a rapid and sensitive screening assay for putative regulators of leukocyte migration.

Uncovering functionally relevant signaling pathways using microarray-based expression profiling.

The introduction of microarray technology to the scientific and medical communities has fundamentally altered the way in which we now address basic biomedical questions. Microarrays technology facilitates a more complete and inclusive experimental approach where alterations in the transcript level of entire genomes can be simultaneously assayed in response to a variety of stimuli. Conceptually different approaches to the development of microarray technology have resulted in the generation of two different array formats: oligonucleotide arrays and cDNA arrays.

Measuring a cell's response to stress: the p53 pathway.

The characterization of complex cellular responses to diverse stimuli can be studied by the use of emerging chip-based technologies.

Isolation and mapping of a human septin gene to a region on chromosome 17q, commonly deleted in sporadic epithelial ovarian tumors.

Allele losses from chromosome 17 are common in sporadic ovarian tumors. Previously, we reported high rates of LOH (up to 70%) from 17q25 at the marker THH59 in a bank of malignant ovarian tumors. We have extended this study to 70 tumors with 17 markers from the long arm of chromosome 17.

Inhibition of C5a-induced neutrophil chemotaxis and macrophage cytokine production in vitro by a new C5a receptor antagonist.

A cyclic peptide, Phe-[Orn-Pro-D-Cyclohexylalanine-Trp-Arg] (F-[OPdChaWR]), was recently shown in vitro to antagonise the binding of C5a to its receptor (CD88) on human polymorphonuclear leukocytes (PMNs) and in vivo to inhibit the neutropenia associated with septic shock induced by lipopolysaccharide (LPS) in rats. The aim of this study was to investigate whether F-[OPdChaWR] inhibits C5a-mediated chemotaxis of human PMNs using a modified Boyden chamber and C5a-stimulated release of cytokines from human monocytes in vitro. Approximately 50% of the chemotactic activity induced by 10 nM C5a was inhibited by 76 nM F-[OPdChaWR].

Reperfusion injury is greater with delayed restoration of venous outflow in concurrent arterial and venous limb injury.

Background: Complex limb trauma often involves combined arterial and venous injury, and the resultant ischaemia-reperfusion injury (IRI) causes both local and remote organ injury. This study assessed the influence of the timing of restoration of venous drainage on IRI.

Methods: Male New Zealand white rabbits (n = 36) were randomized into six groups: sham operation (group 1) and unilateral hind limb arterial and venous occlusion for 1 h followed by no reflow for 2 h (group 2), arterial and venous reflow for 2 h (group 3), arterial reflow alone for 2 h (group 4), arterial reflow alone for 1 h followed by arterial and venous (delayed) reflow for a further 1 h (group 5), and pretreatment with an enteral combination antioxidant before occlusion of both artery and vein and delayed venous reflow (group 6).

Imaging of renal medullary interstitial cells in situ by confocal fluorescence microscopy.

Renal medullary interstitial cells are a prevalent and characteristic feature of the inner medulla of the kidney, but the physiological significance of this is unclear. We have developed a method for imaging renal medullary interstitial cells in situ by loading the cells with fluorescent dyes and monitoring their distribution using confocal microscopy. The pH-sensitive probe 2'7'-bis-(carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester was used as a marker of cytoplasmic volume and therefore of cell morphology.

Induction of GADD45 and JNK/SAPK-dependent apoptosis following inducible expression of BRCA1.

The breast cancer susceptibility gene BRCA1 encodes a protein implicated in the cellular response to DNA damage, with postulated roles in homologous recombination as well as transcriptional regulation. To identify downstream target genes, we established cell lines with tightly regulated inducible expression of BRCA1. High-density oligonucleotide arrays were used to analyze gene expression profiles at various times following BRCA1 induction.

Increased expression of the RIalpha subunit of the cAMP-dependent protein kinase A is associated with advanced stage ovarian cancer.

The primary element in the cAMP signal transduction pathway is the cAMP-dependent protein kinase (PKA). Expression of the RIalpha subunit of type I PKA is elevated in a variety of human tumours and cancer cell lines. The purpose of this study was to assess the prognostic importance of RIalpha expression in patients with ovarian cancer.

Inactivating mutations in an SH2 domain-encoding gene in X-linked lymphoproliferative syndrome.

X-linked lymphoproliferative syndrome (XLP) is an inherited immunodeficiency characterized by increased susceptibility to Epstein-Barr virus (EBV). In affected males, primary EBV infection leads to the uncontrolled proliferation of virus-containing B cells and reactive cytotoxic T cells, often culminating in the development of high-grade lymphoma. The XLP gene has been mapped to chromosome band Xq25 through linkage analysis and the discovery of patients harboring large constitutional genomic deletions.

Inhibition of cellular proliferation by the Wilms tumor suppressor WT1 requires association with the inducible chaperone Hsp70.

The Wilms tumor suppressor WT1 encodes a zinc finger transcription factor that is expressed in glomerular podocytes during a narrow window in kidney development. By immunoprecipitation and protein microsequencing analysis, we have identified a major cellular protein associated with endogenous WT1 to be the inducible chaperone Hsp70. WT1 and Hsp70 are physically associated in embryonic rat kidney cells, in primary Wilms tumor specimens and in cultured cells with inducible expression of WT1.

Eosinophilic interleukin 5 (IL-5) transgenic mice: eosinophil activity and impaired clearance of Schistosoma mansoni.

Eosinophilia is a feature common to many invasive helminth infections and eosinophils are often considered to be effector cells in immunity to helminths. This study examined the possible influence of constitutive eosinophilia on the clearance of Schistosoma mansoni infections in mice. Eosinophils from interleukin-5 transgenic mice exhibit normal ultrastructure and function with regard to phagocytosis and killing of bacteria and responses to chemotactic stimuli.

Heterozygous ATM mutations do not contribute to early onset of breast cancer.

Ataxia telangiectasia (AT) is a recessive syndrome, including cerebellar degeneration, immunologic defects and cancer predisposition, attributed to mutations in the recently isolated ATM (ataxia telangiectasia, mutated) gene. AT is diagnosed in 1/40,000 to 1/100,000 live births, with carriers calculated to comprise approximately 1% of the population. Studies of AT families have suggested that female relatives presumed to be carriers have a 5 to 8-fold increased risk for developing breast cancer, raising the possibility that germline ATM mutations may account for approximately 5% of all breast cancer cases.

Prevalence of germ-line mutations in p16, p19ARF, and CDK4 in familial melanoma: analysis of a clinic-based population.

Five to ten percent of individuals with melanoma have another affected family member, suggesting familial predisposition. Germ-line mutations in the cyclin-dependent kinase (CDK) inhibitor p16 have been reported in a subset of melanoma pedigrees, but their prevalence is unknown in more common cases of familial melanoma that do not involve large families with multiple affected members. We screened for germ-line mutations in p16 and in two other candidate melanoma genes, p19ARF and CDK4, in 33 consecutive patients treated for melanoma; these patients had at least one affected first or second degree relative (28 independent families).

Physical map and cosmid contig encompassing a new interstitial deletion of the X-linked lymphoproliferative syndrome region.

The X-linked lymphoproliferative syndrome (XLP) is an inherited immuno-deficiency to Epstein-Barr virus infection that has been mapped to chromosome Xq25. Molecular analysis of XLP patients from ten different families identified a small interstitial constitutional deletion in 1 patient (XLP-D). This deletion, initially defined by a single marker, DF83, known to map to interval Xq24-q26.

Effects of electroporation on the tubulin cytoskeleton and directed migration of corneal fibroblasts cultured within collagen matrices.

Electroporation provides a useful method for loading fibroblasts with fluorescent probes for the cytoskeleton, but the possible deleterious effects of this loading technique on cell motility are unknown. We have used conventional and confocal microscopy of living cells and immunohistochemistry to examine the migration and cytoskeleton of chick embryo corneal fibroblasts electroporated while cultured within collagen gels. Fibroblasts cultured in collagen (1 mg/ml) are successfully electroloaded (0.

Chemotaxis of polymorphonuclear leukocytes towards human pre-ovulatory follicular fluid and serum using a 'sparse-pore' polycarbonate filtration membrane.

The chemotactic responses of polymorphonuclear leukocytes (PMN) towards pre-ovulatory follicular fluid and serum from patients participating in an in vitro fertilization program were tested in a modified Boyden chamber using a 'sparse-pore' polycarbonate filtration membrane method. The chemotactic activities of follicular fluids were generally low, but were significantly higher in conceptual cycles than in non-conceptual cycles. The chemotactic activities of sera were generally high, but were significantly lower in conceptual cycles.