Biomedical subjects

H B Oral

Publications and source records attributed to H B Oral.

7 recordsLinked to original sources

PubMed2000

Tularemia in Bursa, Turkey: 205 cases in ten years.

Tularemia is a zoonotic disease caused by the coccobacillus F. tularensis. Small epidemics and sporadic cases were seen around Bursa since November 1988. In this study, a total of 205 cases of tularemia were observed. All the cases were diagnosed on clinical, bacteriological and serological grounds. The epidemics were thought to be waterborne. The majority of the patients were young and female. In most of the cases the disease presented itself in oropharyngeal form (83%). Analysing sera from the patients with microagglutination method demonstrated that titers were > or = 1:160 in approximately 85% of the cases, including the ones in subclinical form. Five of ten patients from who the bacteria was isolated were seronegative. Streptomycin was given to the most of the patients by combining with tetracycline, doxycycline or chloramphenicol. The early administration of these antibiotics (before the third week of disease) was found to be much more effective to resolve the infection. As a result, the main mode of transmission of F. tularensis is waterborne in our region. In our region, tularemia should be considered in differential diagnosis for the cases with fever, tonsillopharyngitis and cervical lymphadenopathy to make an early diagnosis and to design relevant treatment.

Adolescent↗

PubMed1999

A method for determining the cytoprotective effect of catalase in transiently transfected cell lines and in corneal tissue.

Both when developing gene constructs for therapeutic purposes and when testing the biological function of proteins, it would be convenient to use cells or tissues that have been transiently transfected with the gene of interest. However, determining the protective effects of transient gene expression is complicated by a low transfection efficiency, resulting in only a minority of the cells expressing the introduced gene and consequently a reduced sensitivity of assays measuring the death of transfected cells. In this study we have developed a convenient technique for determining cell death in transiently transfected vascular endothelial cell monolayers and in corneal tissue. Vascular endothelial cells were cotransfected with human catalase cDNA and the lacZ gene encoding beta-galactosidase, under conditions in which cells expressing beta-galactosidase also expressed catalase. By assaying release of beta-galactosidase upon cell death, it was possible to show that catalase transfection led to significant protection against the cytotoxic effect of increasing concentrations of hydrogen peroxide. The assay was adapted to demonstrate the protective effects of catalase transfection on hydrogen peroxide-mediated injury of intact corneal endothelium under ex vivo culture conditions. This assay should also be useful for characterizing the cytoprotective effects of other genes in transient transfection systems.

Animals↗

PubMed1998

Lipoadenofection-mediated gene delivery to the corneal endothelium: prospects for modulating graft rejection.

BACKGROUND: Gene transfer to the corneal endothelium has potential for the prevention or reversal of corneal allograft rejection. Previous work has examined adenoviral vectors for gene transfer to endothelium. These have a number of theoretical and practical disadvantages, both for experimental and clinical applications. We have therefore used lipoadenofection, in which plasmid DNA is delivered using a combination of liposomes and adenovirus, to transfer marker genes to the cornea. METHODS: Corneas were obtained from New Zealand White rabbits and cultured ex vivo using standard conditions. The corneas were transfected using either lipofection or lipoadenofection with plasmids encoding marker genes. The efficiency of gene transfer and the location and kinetics of gene expression were determined. We also investigated the delivery of a gene construct containing an inducible promoter that is activated by tumor necrosis factor (TNF), to determine whether expression of the relevant genes could be controlled by exogenous factors such as cytokines. RESULTS: This study shows that gene expression is limited to the endothelium and that expression is transient. Furthermore, we have shown that expression of a gene controlled by an inducible promoter only occurs when TNF is present. CONCLUSIONS: These data indicate that lipofection is an efficient method to transfer therapeutic genes to the corneal epithelium, and that it can be used to transfer constructs that utilize an inducible promoter controlled by TNF. As TNF is present in the aqueous humor during allograft rejection, and this is in contact with the corneal endothelium, this has the potential to restrict expression of a therapeutic gene to rejection episodes in the cornea.

Animals↗

PubMed1998

A sensitive fluorometric assay for determining hydrogen peroxide-mediated sublethal and lethal endothelial cell injury.

A rapid and sensitive quantitative fluorometric assay was developed to measure the response of endothelial cells to hydrogen peroxide (H2O2). The response of an endothelial cell-derived cell line, EA-hy-926, or human umbilical vein endothelial cells to H2O2 was determined using calcein-AM, a dye which becomes fluorescent upon cleavage by intracellular esterase(s). The ability of the cells to take up and convert calcein-AM was measured directly in the wells of 96-well flat-bottomed tissue culture plates with cell monolayers using a computerised microplate fluorimeter, or in cell suspensions using flow cytometry. The results obtained by these techniques were compared with each other and with a standard 51Cr release cytotoxicity assay. We found that calcein-AM is a highly sensitive probe for measuring H2O2-mediated cell injury, as it can not only detect the irreversible cytotoxicity measured by 51Cr release assay, but can also distinguish sublethal and reversible injury seen at low H2O2 concentrations.

Cell Line↗

PubMed1997

Ex vivo adenovirus-mediated gene transfer and immunomodulatory protein production in human cornea.

One attractive strategy to prevent or control allograft rejection is to genetically modify the donor tissue before transplantation. In this study, we have examined the feasibility of gene transfer to human corneal endothelium, using a number of recombinant adenovirus constructs. Ex vivo infection of human corneas with adenoviral vectors containing lacZ, under transcriptional control of either cytomegalovirus (CMV) or Rous sarcoma virus (RSV) promoters, provided high-level gene expression, which was largely restricted to endothelium. Expression of the reporter gene persisted at relatively high levels for up to 7 days, followed by a decline to indetectable levels by 28 days. RT-PCR analysis of lacZ transcription showed a similar picture with a short period (3-7 days) of RNA transcription after infection. In contrast, adenoviral DNA persisted for at least 56 days. Subsequently, we examined the expression of a potential therapeutic gene, CTLA-4 Ig fusion protein. Following infection of human corneas with adenoviral vectors encoding CTLA-4 Ig protein, high levels of the fusion protein were detected in corneal culture supernatants for up to 28 days. This protein was functionally active, as determined by binding to B7.1 (CD80)-expressing transfectants. This study suggests that genetic alteration of donor cornea before transplantation is a feasible approach for preventing or controlling allograft rejection. Similar gene-based strategies might also be feasible to prevent rejection of other transplanted tissues or organs.

Abatacept↗

PubMed1997

Gene transfer to ex vivo stored corneas.

PURPOSE: We examined the efficiency and kinetics of recombinant adenovirus vector-mediated gene transfer to rat and rabbit cornea in culture ex vivo. METHODS: A recombinant replication-defective adenovirus was used to transfer a lacZ marker gene to whole rat and rabbit corneas in culture. Histochemistry was used to localise transgene expression and a colorimetric assay to quantify recombinant protein expression. RESULTS: After infection with recombinant virus and culture for 3 days, high-efficiency gene transfer was found, with expression in most endothelial cells of both species. Minimal expression was found in other corneal cell types. On histochemistry, longer duration of expression was found in rat than in rabbit endothelium. In both rat and rabbit cornea, highest levels of recombinant protein were found at days 3-7 after incubation with virus, decreasing to low or undetectable levels at 21 days. CONCLUSION: Adenovirus vectors allow high-efficiency transgene expression in cornea, largely restricted to the endothelial cells of ex vivo cultured cornea. Kinetics of expression differ according to the species of cornea studied, a factor that must be considered if this vector is used in further studies.

Adenoviridae↗

PubMed1996

Adenovirus-mediated gene delivery to the corneal endothelium.

Genetic manipulation of donor cornea prior to transplantation has the potential to modulate the allogeneic response, as well as the endothelial cell function. This study examined the feasibility of gene transfer to corneal endothelial cells using replication-defective recombinant adenoviral vectors. Adult rabbits corneas were infected with recombinant adenovirus RAd35, containing the Escherichia coli beta-galactosidase (lacZ) gene. Localization of gene transfer was assessed by histochemical staining for beta-galactosidase and recombinant protein production was quantified by a soluble assay. In initial experiments, the efficiency of gene transfer and kinetics of expression were studied ex vivo, using organ culture of transfected corneas. Following coculture of whole corneal fragments with RAd35, high levels of gene expression were evident on days 107, diminishing after that time. Gene transfer was found to be almost entirely restricted to corneal endothelial cells, with scattered expression in epithelial cells. Following these ex vivo studies, genetically modified corneas were transplanted as orthotopic allografts in rabbits. Similar kinetics of gene expression were seen after transplantation as in the ex vivo experiment, with maximal levels of gene expression in endothelial cells on days 1-4 after grafting. Corneal function following transplantation was not affected by the gene transfer, with the corneas attaining clarity within 1 day of grafting, and thereafter showing the expected thinning on ultrasonic pachymetry. In the absence of any immunosuppression, no inflammation was evident in graft recipient eyes, with the exception of allograft rejection in 1 animal 23 days after grafting. In this study we show that gene transfer to nonreplicating corneal endothelial cells is feasible using recombinant adenovirus vectors, and so may have potential application in the setting of corneal transplantation.

Adenoviridae↗