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D F Larkin

Publications and source records attributed to D F Larkin.

At least 37 records · Page 2Linked to original sources
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

Identification and characterization of cells infiltrating the graft and aqueous humour in rat corneal allograft rejection.

In a rat model of corneal transplantation, Fischer 344 (RT1(lv1)) rats received orthotopic corneal isografts or Wistar-Furth (RT1(u)) donor allografts. Rejection was observed in 25 of 26 allograft recipients, at a median time of 18 days, with all isografts surviving > 100 days. Flow cytometric analysis of aqueous humour identified cellular infiltration of the aqueous at the time of allograft rejection, in contrast to the acellular aqueous found in isografts at corresponding times following transplantation. A higher proportion of CD8+ than CD4+ cells was found at days 1-3 following rejection, whereas there was a higher proportion of CD4+ cells at days 5-8. No changes in peripheral blood T cell subsets were found at the time of rejection. Immunohistochemical analysis of cells infiltrating recipient iris and grafted cornea undertaken at days 1-2, 4 and 7-10 following onset of rejection, demonstrated inflammatory cells in the graft epithelium, stroma and aggregated on the endothelium. Large numbers of macrophages, T cells (CD4+ > CD8+ at all time points), natural killer (NK) cells and neutrophils were detected in graft tissue at days 1-2 and 4, diminishing after that time. Most infiltrating cells expressed MHC class II antigen, and a smaller number expressed IL-2R. Expression of the co-stimulatory marker B7 was identified in a few cells at day 4 in the region of the graft-host wound. The immune response in graft rejection was characterized at day 4 also by expression of intercellular adhesion molecule-1 (ICAM-1) on endothelial cells of iris and corneal vessels, demonstration of interferon-gamma on mononuclear cells in the peripheral (recipient) cornea, and tumour necrosis factor-alpha on aggregated mononuclear cells on the graft, but not recipient, endothelium. Only sparse cellular infiltrates were found in isograft controls, with inflammation located at the graft-host wound. These findings suggest that inflammatory cells reach a corneal allograft by two routes--from vessels in the peripheral recipient cornea, and from vessels in the recipient iris via the aqueous humour. Different aqueous and intragraft T cell subset proportions were seen early in rejection, although a preponderance of CD4+ cells was found in both aqueous and graft at later times.

Animals↗
PubMed1997

A method for separation and staining of flat mounts of human corneal endothelium.

A technique is described in which sheets of corneal endothelium are removed from human donor corneo-scleral discs. Celloidin solution was applied to the endothelial surface, allowed to dry, peeled off with the attached endothelial cell layer and mounted on a glass slide. Following removal of the celloidin with acetone, this endothelial cell flat mount was then stained with H&E and monoclonal antibodies to cell adhesion molecules. A pilot study of endothelial cell adhesion molecule expression in flat mount preparations of 14 corneas showed constitutive neural cell adhesion molecule (NCAM) expression, but a lower degree of focal expression of intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VACM)-1, P/E-selectin and HLA-DR.

Cell Separation↗
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↗
PubMed1995

Experimental orthotopic corneal xenotransplantation in the rat. Mechanisms of graft rejection.

Orthotopic penetrating guinea pig to rat and chicken to rat corneal xenografts were performed to examine the nature of the host response. Guinea pig to rat xenografts failed at a median of day 3 after surgery. A similar but slightly accelerated pattern of failure was seen in guinea pig xenografts performed in prevascularized recipient rat corneas. Chicken to rat xenografts failed at a median of day 2 after grafting. Rat corneal isograft controls survived indefinitely. Corneal endothelial cells were visible by silver staining on the xenografts immediately after operation, which indicates that failure was not due to loss of these cells during surgery. Histopathology and immunoperoxidase staining indicated that xenograft failure in euthymic recipients was characterized by early corneal epithelial and endothelial cell damage, granulocytic infiltration, and hemorrhage from recipient corneal and iris capillaries, followed at 7-14 days by infiltration with T cells, macrophages, and eosinophils. An accelerated pattern of graft failure was also observed in guinea pig grafts into homozygous nude rat recipients, which suggests that preformed anti-donor antibody and complement were responsible for some of the early graft damage. Flow cytometry demonstrated the presence of pre-existing natural antibodies to guinea pig and chicken lymphocytes and erythrocytes, as expected from other studies. Immunohistochemistry showed the presence of rat IgG2a, IgG1, and IgM, but not IgD deposited on grafts in immunocompetent recipients on the first postoperative day. We conclude that orthotopic corneal xenografts undergo substantial accelerated damage mediated by pre-existing antibody, followed at 7-14 days by a cell-mediated response that causes further destruction.

Animals↗
PubMed1995

The host response in experimental corneal xenotransplantation.

In addressing the worldwide shortage of human donor cornea for transplantation, animal cornea may be a substitute if mechanisms of xenogeneic (cross-species) rejection can be identified and controlled. Xenotransplantation of solid organs is followed by hyperacute rejection with minutes due to humoral graft rejection. In an experimental model corneal xenografts in rats survived for 2-3 days, depending on the phylogenetic disparity of the donor animal. Endothelial injury was the specific cause of graft failure, probably mediated by humoral rejection mechanisms. A later cell-mediated rejection response was seen. The potent humoral response is the most important feature differentiating xenograft from allograft rejection.

Animals↗
PubMed1992

Treatment of Acanthamoeba keratitis with polyhexamethylene biguanide.

Polyhexamethylene biguanide (PHMB) is a polymeric biguanide disinfectant that has not previously been used in the treatment of infection. Six patients with confirmed Acanthamoeba keratitis were treated with PHMB 0.02%. All patients had uncontrolled keratitis refractory to therapy with multiple conventional antiamebic agents. The rationale for use and the dose of PHMB was determined by in vitro sensitivity testing of the Acanthamoeba corneal isolates to the drugs available for use. Trophozoite forms were sensitive to most agents. Only PHMB was cysticidal at low concentrations in all cases. Sensitivity to the other drugs, including propamidine, showed wide variation. In 5 of 6 cases, complete resolution of inflammation followed the introduction of PHMB. Toxicity to the ocular surface was not evident with PHMB, unlike propamidine or neomycin. The reasons for the treatment failure in one case, despite cyst sensitivity to both PHMB and propamidine, are not clear. PHMB is a promising new treatment for this infection.

Acanthamoeba↗
PubMed1991

Quantitative alterations of the commensal eye bacteria in contact lens wear.

A study was performed on the commensal external eye flora in 34 long-term contact lens wearers and a matched control group, in order to identify the effect of lens wear. Samples were taken from the lid margin and conjunctiva by semi-quantitative, and tear film by quantitative methods. Cultured bacteria were identified with particular attention to coagulase-negative staphylococci. No qualitative alteration in the commensal bacteria was found, although lens wearers were found to have a significantly higher number of species at all sites than controls. Significant quantitative changes were identified on the lid margin, with particularly high counts in some lens wearers, and tear film. Increased numbers of bacteria obtained from the conjunctiva were not statistically significant. Quantitative changes in the tear film are thought to be secondary to changes at the lid margin, for which no explanation is apparent.

Adult↗
PubMed1991

In vitro corneal pathogenicity of Acanthamoeba.

The comparative cytopathic effects of a keratitis and an environmental isolate of Acanthamoeba were studied on confluent monolayers of human and rabbit corneal cells grown in culture. The presence of cells in culture induced excystment of amoebae to the active trophozoite form. Total destruction of cell monolayers was observed to be similar for both isolates, and dependent on incubation time and amoebic concentration. The relevance of these findings to human and experimental Acanthamoeba keratitis is discussed.

Acanthamoeba↗
PubMed1991

Experimental Acanthamoeba keratitis: II. Immunohistochemical evaluation.

In a Wistar rat experimental model of Acanthamoeba keratitis immunohistochemical techniques were used to analyse the host cellular response. The inflammatory cell profile was observed to change at intervals. In tissue sections the cellular response consisted of neutrophils on the first day but predominantly macrophages on the following days. Some T lymphocytes but no B lymphocytes were observed.

Acanthamoeba↗
PubMed1990

External eye flora as a nutrient source for Acanthamoeba.

Certain bacteria cause excystment of Acanthamoeba from cyst to trophozoite form and are then ingested by migrating trophozoites. We studied the response of Acanthamoeba cysts to inoculation on agar seeded with three types of commensal eye bacteria and Escherichia coli. Amoebae excysted on all bacteria tested, and the migration rate of Acanthamoeba trophozoites on each was compared. Acanthamoeba migrated with equal speed on E. coli and Staphylococcus epidermidis. Migration was observed, but was more slow on Micrococcus and Corynebacterium. Commensal bacteria on the eyelids, conjunctiva and tear film may have a role in pathogenesis of Acanthamoeba keratitis.

Acanthamoeba↗
PubMed1990

Acanthamoeba adherence to contact lenses and removal by cleaning agents.

The quantitative adherence of a keratitis isolate of Acanthamoeba polyphaga to low- and high-water content non-ionic soft contact lenses and one type of rigid gas-permeable lens was investigated. Adherence of trophozoite and cyst forms of the organism was observed in vitro, and adherent amoebae counted by a plaque assay method following detachment. Trophozoites adhered to all lens types with adherence being statistically significantly greater to high water content soft lenses. Cyst attachment occurred only to the soft lenses but not to gas-permeable lenses, and was significantly higher for the high water content lenses. Attachment of cysts was significantly lower than that of trophozoites to each lens tested. Recommended cleaning procedures using two commercial solutions removed all adherent trophozoites and cysts from lenses. These studies demonstrate (i) that lenses may act as a vector in Acanthamoeba keratitis, particularly for high-water content lenses and trophozoite amoebae, (ii) that lens cleaning agents may prevent keratitis by removing adherent Acanthamoeba.

Acanthamoeba↗
PubMed1990

Laboratory investigation of Acanthamoeba keratitis.

Following the diagnosis of Acanthamoeba keratitis in a contact lens wearer, the antimicrobial susceptibility of the clinical isolate and the environmental source of the infection were investigated. Contrary to previous reports, in vitro antimicrobial testing showed that the infecting strain was inherently resistant to propamidine isethionate. Restriction endonuclease digestion analysis of Acanthamoeba whole-cell DNA of strains isolated from the patient's cornea, contact lens storage container, saline rinsing solution, and kitchen cold-water tap showed that the isolates were identical. This implicates, for the first time, domestic tap water as the source of Acanthamoeba sp. in this infection. It is therefore recommended that the use of homemade saline solutions and the rinsing of contact lenses in tap water be strongly discouraged.

Acanthamoeba↗
PubMed1990

Contamination of contact lens storage cases by Acanthamoeba and bacteria.

In order to identify possible risk factors for microbial keratitis the storage cases for contact lenses of 102 asymptomatic lens wearers were tested for contamination by bacteria and free-living amoebae. Of this group 43 had significant counts of viable bacteria and only 40 had negligible counts. Seven had contamination by acanthamoebae, of whom six also had significant bacterial counts. These results were categorised according to the type of contact lens worn and the lens disinfection method. The high rates of contamination by apathogenic and pathogenic organisms, in particular Acanthamoeba, and the probable support by contaminating bacteria of Acanthamoeba, are discussed.

Acanthamoeba↗