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Biomedical subjects

D F Larkin

Publications and source records attributed to D F Larkin.

At least 19 recordsLinked to original sources
PubMed1999

Activated polyamidoamine dendrimers, a non-viral vector for gene transfer to the corneal endothelium.

We investigated the efficiency of activated polyamidoamine dendrimers, a new class of nonviral vectors, to transfect rabbit and human corneas in ex vivo culture. In addition to assessing the expression of a marker gene we have demonstrated that this approach can be used to induce the production of TNF receptor fusion protein (TNFR-Ig), a protein with therapeutic potential. Whole thickness rabbit or human corneas were transfected ex vivo with complexes consisting of dendrimers and plasmids containing lacZ or TNFR-Ig genes. Following optimisation 6-10% of the corneal endothelial cells expressed the marker gene. Expression was restricted to the endothelium and was maximal after transfection with 18:1 (w/w) activated dendrimer:plasmid DNA ratio and culture for 3 days. The supernatant of corneas transfected with TNFR-Ig plasmid contained TNFR-Ig protein which was able to inhibit TNF-mediated cytotoxicity in a bioassay. We have therefore shown that activated dendrimers are an efficient nonviral vector capable of transducing corneal endothelial cells ex vivo. They may have applications in gene-based approaches aimed at prevention of corneal allograft rejection or in treatment of other disorders of corneal endothelium.

Animals↗
PubMed1999

A new model of orthotopic penetrating corneal transplantation in the sheep: graft survival, phenotypes of graft-infiltrating cells and local cytokine production.

BACKGROUND: Orthotopic penetrating keratoplasty in the sheep was developed as an outbred preclinical model to allow correlation of the cellular infiltrate during graft rejection with local production of cytokine mRNA. METHODS: Penetrating corneal autografts and allografts were performed in Merino sheep. Graft outcome was followed at the slit-lamp. Corneal infiltrates were examined by immunoperoxidase staining on postmortem specimens. Cytokine mRNA was detected by polymerase chain reaction. RESULTS: Corneal autografts survived indefinitely. Allografts became vascularized and underwent rejection at a median of 20 days postgraft. Both endothelial and epithelial rejection lines were observed. Immunohistochemical staining of rejecting grafts showed up-regulation of major histocompatibility complex class I molecules on corneal graft epithelium, damaged or absent graft endothelium and a marked, predominantly mononuclear cell infiltrate. CD4-positive T cells were observed in the graft within 2 days of the onset of rejection, followed several days later by CD8-positive T cells. Messenger RNA transcripts for interleukin (IL)-2, tumour necrosis factor (TNF)-alpha and IL-10 (but not for interferon (IFN)-gamma or IL-4) were found in autografted corneas. Proportionately, more allografts than autografts contained transcripts for IL-2 and TNF-alpha, and IFN-gamma was detected in three of four allografts. CONCLUSIONS: Corneal graft rejection in the sheep is macroscopically and histologically similar to human corneal graft rejection. Allografts become infiltrated by both CD4- and CD8-positive T cells and local production of pro-inflammatory cytokines occurs during graft rejection.

Animals↗
PubMed1998

Lipid-mediated enhancement of transfection by a nonviral integrin-targeting vector.

Nonviral vectors consisting of integrin-targeting peptide/DNA (ID) complexes have the potential for widespread application in gene therapy. The transfection efficiency of this vector, however, has been limited by endosomal degradation. We now report that lipofectin (L) incorporated into the ID complexes enhances integrin-mediated transfection, increasing luciferase expression by more than 100-fold. The transfection efficiency of Lipofectin/Integrin-binding peptide/DNA (LID) complexes, assessed by beta-galactosidase reporter gene expression and X-gal staining, was improved from 1% to 10% to over 50% for three different cell lines, and from 0% to approximately 25% in corneal endothelium in vitro. Transfection complexes have been optimized with respect to their transfection efficiency and we have investigated their structure, function, and mode of transfection. Both ID and LID complexes formed particles, unlike the fibrous network formed by lipofectin/DNA complexes (LD). Integrin-mediated transfection by LID complexes was demonstrated by the substantially lower transfection efficiency of LKD complexes in which the integrin-biding peptide was substituted for K16 (K). Furthermore, the transfection efficiency of complexes was shown to be dependent on the amount of integrin-targeting ligand in the complex. Finally, a 34% reduction in integrin-mediated transfection efficiency by LID complexes was achieved with a competing monoclonal antibody. The role of lipofectin in LID complexes appears, therefore, to be that of a co-factor, enhancing the efficiency of integrin-mediated transfection. The mechanism of enhancement is likely to involve a reduction in the extent of endosomal degradation of DNA.

Amino Acid Sequence↗
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

Retreatment for significant regression after excimer laser photorefractive keratectomy. A prospective, randomized, masked trial.

PURPOSE: Regression, a gradual partial or complete return to the myopic state, remains a common complication of excimer laser photorefractive keratectomy (PRK) and limits the predictability of refractive outcome, especially in high myopia. An estimated 10% to 20% of patients, therefore, request a repeat PRK procedure. This study was designed to provide patient selection criteria and guidelines for successful retreatment. METHODS: One hundred six patients who had regressed were randomized to 1 of 4 retreatment groups comprising (1) those with minimal haze after their first PRK who received an exact retreatment; (2) those with minimal haze treated with a 50% deliberate overcorrection; (3) those with significant haze (> or = 2+ haze) given an exact retreatment; and (4) those with significant haze who received a 50% overcorrection. A Visx 20/20 laser was used in each case. Mean follow-up after retreatment was 12 months (range, 6-18 months). RESULTS: Deliberate overcorrection (groups 2 and 4) resulted in a statistically significantly better refractive outcome (P = 0.026 at 6 months). Analysis of variance showed that significant haze after the first PRK was the most important predictor of a poor outcome after retreatment, other factors being high original myopia, marked regression, and loss of best-corrected visual acuity. CONCLUSIONS: A retreatment PRK procedure for significant regression will reduce residual myopia significantly in the majority of patients, and a deliberate overcorrection (50%) reduces the chance of further regression. However, patients with high myopia who have regressed beyond approximately -3.50 diopters originally and who show significant anterior stromal haze (> 2+) should be retreated only with great caution, because of the risk of further regression, haze, and loss of visual acuity.

Adult↗
PubMed1998

Distribution of integrins alpha v beta 5, alpha v beta 3 and alpha v in normal human cornea: possible implications in clinical and therapeutic adenoviral infection.

PURPOSE: Integrins are heterodimeric cell surface molecules involved in cell-cell and cell-matrix interactions. Adenoviral entry into human cells has been shown to be dependent on integrins alpha v beta 5 and alpha v beta 3 that promote viral internalisation. We studied the distribution of integrins alpha v beta 5, alpha v beta 3 and the alpha v chain in normal human cornea to investigate possible mechanisms of adenoviral entry to specific corneal cell types. METHODS: We used immunohistochemistry with monoclonal antibodies to study the distribution of alpha v beta 5, alpha v beta 3 and alpha v in normal human corneas maintained for up to 4 days in corneal storage medium (Optisol) at 4 degrees C (n = 9). RESULTS: Both alpha v beta 5 and alpha v were present to a variable extent on the corneal epithelium and corneal endothelium of most specimens. In some specimens staining of both alpha v beta 5 and alpha v in the epithelium was graded, with more basal than superficial staining, alpha v beta 3 was not detectable in either the corneal epithelium or the corneal endothelium in those specimens tested. CONCLUSIONS: The integrin alpha v beta 5 is present on both epithelium and endothelium in the normal human cornea. The role of alpha v integrins in clinical infection and in adenoviral entry for gene transfer is discussed.

Adenovirus Infections, Human↗
PubMed1997

Infiltrating inflammatory cell phenotypes and apoptosis in rejected human corneal allografts.

PURPOSE: The aim of this study was to survey the histopathological and immunohistochemical features of rejected human corneal allografts. METHODS: Following graft failure in each case due to rejection, paraffin-embedded specimens of 17 corneal transplants which had been replaced were examined by light microscopy and immunohistochemistry. Specimens were either first (n = 9), second (n = 4) or third (n = 4) grafts and were removed at varying intervals from 4 weeks following documented rejection. RESULTS: Those grafts which were removed earliest following onset of rejection had the most intense graft inflammatory infiltrates. Immunohistochemical staining showed a high proportion of graft stroma-infiltrating cells expressing leucocyte common antigen, and many of these cells also bore T cell or macrophage markers. Leucocyte-keratocyte apposition and regional loss of keratocytes were observed in all rejection specimens, but not in non-rejected control grafts. In situ end-labelling of DNA double-strand breaks and morphological features identified keratocyte apoptosis in 5 of 12 specimens examined for this phenomenon. Corneal endothelial cells were absent in 7 specimens and present in reduced numbers in the remaining 10 specimens. CONCLUSIONS: Endothelial cell monolayer attenuation and keratocyte loss are consistent findings in grafts removed subsequent to clinically observed endothelial rejection. Death of donor corneal cells is mediated, at least in part, by apoptosis. The stromal inflammatory infiltrate consists mainly of T lymphocytes and macrophages, which may be responsible for induction of keratocyte apoptosis.

Adult↗
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↗