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Biomedical subjects

Y G He

Publications and source records attributed to Y G He.

31 records · Page 2Linked to original sources
PubMed1992

EGF, basic FGF, and TGF beta-1 messenger RNA production in rabbit corneal epithelial cells.

The polymerase chain reaction (PCR) was used to demonstrate that rabbit corneal epithelial cells produce messenger RNAs coding for epidermal growth factor (EGF), basic fibroblast growth factor (FGFb), and transforming growth factor beta-1 (TGF beta 1) ex vivo and in primary culture. EGF, FGFb, and TGF beta 1 mRNAs were detected in central and peripheral ex vivo epithelial tissue in wounded and unwounded rabbit corneas. Southern blots of the PCR products were probed with oligonucleotides to demonstrate that the appropriately sized amplification products were specific. These results suggest that corneal epithelial cells produce growth factors that may have autocrine or paracrine effects on epithelial cells, and possibly other cells of the cornea. The functions, if any, performed by these growth factors in corneal epithelial wound healing are yet to be elucidated.

Animals↗
PubMed1992

Susceptibility of corneas from various animal species to in vitro binding and invasion by Acanthamoeba castellanii [corrected].

A crucial requirement for establishing corneal infection by the extracellular protozoal parasite, Acanthamoeba, is the ability of the parasite to bind to the corneal surface. In a series of in vitro studies, we examined the ability of Acanthamoeba castellanii [corrected] to adhere, invade, and damage normal, intact corneas of 11 mammalian and one avian species. A. castellanii [corrected] (80-90% trophozoites and 10-20% cysts) were incubated with corneas for 24 hours in vitro and examined by scanning electron microscopy (SEM). Results of several independent SEM experiments revealed that parasites not only failed to produce cytopathic effects but did not even bind to the corneal epithelium of mice, rats, cotton rats, horses, guinea pigs, cows, chickens, dogs, and rabbits. However, parasites adhered, invaded, and produced severe damage to human, pig, and Chinese hamster corneas during the 24-hour in vitro incubation period. Additional in vitro experiments quantified the binding of A. castellanii [corrected] to the corneas of selected susceptible and nonsusceptible species. In vitro binding assays revealed scant binding of parasites to mouse, rat, and rabbit (range = 5-20 parasites/7.07 mm2 corneal button). In contrast, extensive binding was observed on Chinese hamster, pig, and human corneas (range = 100-200 parasites/7.07 mm2 button). The results indicate that A. castellanii [corrected] exercises rigid host specificity at the host cell surface.

Acanthamoeba↗
PubMed1992

A pig model of Acanthamoeba keratitis: transmission via contaminated contact lenses.

A model of contact lens-induced Acanthamoeba keratitis was developed in Yucatan micropigs. Pigs fitted with parasite-laden soft contact lenses developed corneal infections that clinically and histopathologically mimicked the human counterpart. Three distinct stages of disease became apparent and were categorized as: acute, condensed infiltrate, and resolution stages. Viable parasites were isolated from corneal scrapings and smears were taken during the acute and condensed infiltrate stages. In addition, cysts could be identified deep within the stroma of histological specimens taken during the resolution stages. The characteristic dense, white ring-like infiltrates, stroma edema, keratic precipitates, and the chronic nature of the infections were similar to those observed in human Acanthamoeba keratitis. Histopathological examination of infected corneas revealed extensive neutrophilic infiltrates, stromal necrosis, and disorganization of the collagen lamellae. The strong correlation between the clinical and histopathologic features of contact lens-induced Acanthamoeba keratitis in the pig as well as the anatomical similarity of the pig eye with the human eye make the porcine model a valuable tool for investigations of the immunology, cell biology, and therapy for Acanthamoeba keratitis.

Acanthamoeba↗
PubMed1991

In vitro transfer of rabbit corneal epithelium from carriers to denuded corneas or cryolathed lenticules.

Rabbit corneal basal epithelial cells seeded onto fixed gelatin membranes or commercial collagen shields formed 3 to 5 cell layers after 1 to 3 weeks of culture at 35 degrees C in nutrient medium. The cells grew better, by comparison, in the collagen shields and eventually formed a multilayered tissue that resembled the stratified morphology of native epithelium. Transfer of multilayered cultures (prior to stratification) from these carriers in vitro to denuded corneal buttons or cryolathed lenticules resulted in complete adhesion of the grafted tissue to the underlying recipient buttons after 24- to 48-h incubations. After mechanically removing the carriers, most of the epithelial cells remained attached to the stromal surface. Our experimental findings indicated that both kinds of carriers may be suitable for epithelial transplantation, although the collagen shield is probably superior because of its better biocompatibility and physical characteristics.

Animals↗
PubMed1991

Acceptance of H-Y-disparate corneal grafts despite concomitant immunization of the recipient.

Male-specific, H-Y antigen is a widely utilized antigen system for analyzing the role of non-MHC transplantation antigens in graft rejection. In this study, we examined the role of H-Y antigen in corneal graft rejection. Orthotopic corneal grafts from male LBNF1 rats were transplanted to syngeneic female LBNF1 recipients. The male corneal grafts survived beyond 100 days on naive female recipients. In other experiments, hosts bearing clear male corneal grafts and systemically immunized with subcutaneous inoculations of male splenocytes followed by full-thickness male skin grafts failed to reject their corneal grafts, even though the male skin grafts were swiftly rejected. The inability of female hosts to reject existing male corneal grafts suggested that the cornea failed to express H-Y transplantation antigen. Further experiments, however, revealed that male-specific antigen was expressed on corneal grafts. Hosts bearing clear male grafts in the left eye rejected subsequent male skin grafts and promptly rejected male corneal grafts transplanted to the contralateral eye. Interestingly, the original male corneal grafts remained clear during the rejection of both the skin graft and the second corneal graft. The results indicate that corneal grafts representing minor histocompatibility disparities enjoy immunologic privilege in the naive host, even if the host is subsequently immunized systemically.

Animals↗
PubMed1991

The differential effects of donor versus host Langerhans cells in the rejection of MHC-matched corneal allografts.

The fate of MHC-identical, multiple minor H-disparate corneal grafts was examined in the rat. Although skin grafts exchanged between LEW and F344 rats were invariably rejected, only 26% of the corresponding corneal grafts underwent rejection. The immunologic privilege of the minor H-disparate corneal grafts was due, at least in part, to the absence of donor-derived Langerhans cells. Corneal grafts were normally devoid of donor-derived Langerhans cells; however, grafts pretreated with latex beads became infiltrated with donor-derived Langerhans cells and were rejected by 59% of the naive minor H--compatible recipients. By contrast, untreated LEW corneal grafts underwent rejection in 26% of the naive F344 hosts even though the grafts became heavily infiltrated with host-derived Langerhans cells. The immunologic privilege of minor H-disparate corneal grafts was not the result of efferent blockade or suppression of the immune response. F344 hosts bearing long-term surviving LEW corneal allografts were challenged with LEW skin grafts. In all cases, orthotopic skin grafts were rejected acutely. Moreover, all previously clear corneal grafts underwent rejection following skin graft rejection. Thus, the unique absence of donor-derived Ia+ passenger cells and the avascular graft bed conspire to provide the primary minor H-disparate corneal graft with an immunologic privilege not shared by other organ grafts.

Animals↗
PubMed1991

Growing human corneal epithelium on collagen shield and subsequent transfer to denuded cornea in vitro.

Three fundamental in vitro experiments have been done in the present report: 1) comparison of three different nutrient media on their abilities to culture and passage the human corneal epithelial cells; 2) evaluation of the ability of extracellular matrix material to promote the growth of cultured human corneal epithelium on collagen corneal shields; and 3) determination of the feasibility of the shield to serve as a carrier for the transfer of cultured cells to allogeneic, denuded corneal surface in vitro. Primary cultures of human corneal epithelium were established from explants which were obtained from limbal and peripheral corneal tissue by three different nutrient media respectively: KGM (Keratinocyte Growth Medium), SHEM (Supplemental Hormonal Epithelial Medium), and one combination of the two media (KGM/SHEM). We found the KGM/SHEM combination to be more favorable because morphology was better preserved, the proliferation rate increased five-fold over the 14 days observed time course, and we were able to subculture the tissue for at least three passages. With this combined medium, a suspension of cultured corneal epithelial cells (5 x 10(5)/ml) was seeded onto either the concave surface of collagen corneal shields or onto shields which had been coated with extracellular matrix materials (Matrigel or type IV collagen). The cells attached readily to all the coated shields (20/20) but to only a few of the uncoated shields (3/10), and formed a stratified tissue (2 to 3 layers) within seven days once the cells attached. However, the cells on the shields coated with Matrigel failed to become confluent under these conditions. The stratified tissue on type IV collagen coated shields could then be subsequently transferred to denuded human corneal stroma in organ culture by placing them together and incubating for 2-7 days. After that, histologic examinations showed that the epithelial cells had attached tightly to the recipient stromal surface, even after the removal of the collagen shield.

Biological Dressings↗
PubMed1991

Class I disparate corneal grafts enjoy afferent but not efferent blockade of the immune response.

Class I antigens are normally expressed on cells in all three layers of the cornea. In congenic rats that differ only at the single Class I locus RT1 A, central orthotopic corneal grafts were rejected 18% of the time with a mean survival time (MST) of 11.5 days. Pre-immunized recipients always rejected Class I disparate corneal grafts (100%, MST = 13.3 days). Surprisingly, the presence of donor Langerhans cells in the cornea at the time of grafting did not increase the rejection of grafts (20%, MST = 14.0 days). To determine if long term surviving grafts enjoyed immune priviledged in the form of efferent blockade, the recipients were challenged with skin grafts 4 to 6 weeks following corneal transplantation. All of the corneal grafts underwent rejection (100%, MST = 14.7 days). A number of important conclusions may be drawn from these studies. A single Class I mismatch is a weak barrier to successful engraftment of corneal grafts. However if the recipient has previously been exposed to donor antigens, a single Class I disparity is sufficient to provoke rejection of all subsequent corneal grafts. The susceptibility of long term surviving grafts to rejection induced by skin grafts indicates the orthotopic corneal grafts are antigenic but not immunogenic.

Afferent Pathways↗
PubMed1991

Promotion of murine orthotopic corneal allograft survival by systemic administration of anti-CD4 monoclonal antibody.

A mouse model of orthotopic corneal allograft rejection was used to examine the efficacy of anti-CD4 and anti-CD8 monoclonal antibodies in preventing immunologic rejection of corneal allografts. Although it is believed by many that corneal graft rejection is mediated, at least in part, by CD8-positive cytotoxic T-lymphocytes, systemic administration of anti-CD8 antibody did not reduce the rejection rate of corneal allografts that differed from the host at the entire major histocompatibility complex. By contrast, systemic administration of anti-CD4 monoclonal antibody reduced the rejection rate from 83% (untreated controls) to 33%. Fluorocytometric analysis of residual lymphoid populations showed that neither monoclonal antibody eliminated the inappropriate subset of T-cells in antibody-treated animals. In vitro cell-mediated cytotoxicity assays showed that both antibodies eliminated allospecific cytotoxic T-lymphocyte populations; however, only anti-CD4 antibody promoted graft survival. Thus, these results indicate that anti-CD4 monoclonal antibody is a powerful immunosuppressive agent for promoting corneal graft survival and that CD8-positive T-cells alone do not cause rejection of corneal allografts.

Animals↗
PubMed1991

Post-transcriptional and transcriptional control of collagen gene expression in normal and modulated rabbit corneal endothelial cells.

In a previous report, collagen synthesis did not correlate with steady-state collagen RNA levels; substantial amounts of type I collagen RNAs in endothelial cells were not translated into the respective protein. The current investigation was extended to study the level of the control mechanism in collagen gene expression in normal corneal endothelial cells or those modulated by corneal endothelium modulation factor released by polymorphonuclear leukocytes. Northern-blot analysis using cloned rabbit types I and IV cDNA probes (same species as RNA sources) demonstrated specific mRNA transcripts for collagen types I and IV in the endothelial cells, although the steady-state level of these mRNAs in modulated endothelial cells was low. The turnover rate of collagen RNAs was determined; normal cells contain very stable alpha 2(I) and alpha 2(IV) mRNAs whose half-lives exceed 24 hr. The same messages decayed rapidly in the modulated cells, where they had an apparent half-life of approximately 8 hr. Using nuclear run-off transcription, the rate of transcription in normal cells was found to be slightly lower than that in modulated cells. When the relative rate of collagen gene transcription was compared, that of alpha 2(I) was the lowest and of alpha 2(IV), the highest in both cells. The relative transcriptional rates of individual collagen chains did not account for the steady-state levels, suggesting that transcriptional regulation in corneal endothelial cells was less than was translational regulation. On the other hand, during early stages of corneal endothelial cell modulation induced by factors released by polymorphonuclear leukocytes there was a differential effect on both transcriptional rate and the steady-state level of collagen RNAs.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

In vivo and in vitro collagenolytic activity of Acanthamoeba castellanii.

Axenic cultures of Acanthamoeba castellanii contained a collagenolytic enzyme that digested collagen shields and purified collagen in vitro. Specificity of biologic activity was determined by the addition of selected enzyme inhibitors to the assays and revealed that the parasite-conditioned medium contained both collagenase and lower concentrations of other proteolytic enzymes. However, most of the collagenolytic and pathogenic activity was directly attributable to specific collagenase. Intrastromal injection of sterile, Acanthamoeba-conditioned culture medium into naive Lewis rats produced corneal lesions clinically similar to and closely resembling those found in biopsy specimens of human patients diagnosed with acanthamoebic keratitis. Histopathologic analysis revealed moderate-to-severe neutrophil infiltration, disruption of stromal lamellae, and edema. Identical pathologic sequelae were produced by intrastromal injection of purified collagenase (25 units/ml). The pathogenicity of the soluble parasite-derived product was removed by passage over affinity columns armed with antibody specific for collagenase. These results indicated that soluble parasite-derived factors were capable of producing lesions characteristic of acanthamoebic keratitis and that the pathogenicity of these factors was either directly or indirectly attributable to specific collagenase activity.

Acanthamoeba↗
PubMed1990

Corneal endothelium modulation factor released by polymorphonuclear leukocytes. Partial purification and initial characterization.

Polymorphonuclear leukocytes produce a polypeptide factor that is released into the medium. This factor is partially purified 83-fold by ammonium sulfate precipitation followed by chromatography on a DEAE-Sephadex or heparin-Sepharose column. The partially purified factor is trypsin-sensitive. This factor affects a population of rabbit corneal endothelial cells by modulating them to fibroblastlike cells and by further stimulating their growth, leading to the formation of colonies of multilayered modulated cells. There is a dose-dependent phenotypic modulation of corneal endothelial cells by the partially purified corneal endothelium modulation factor (CEMF); cell shape is changed and type I collagen synthesis is increased with greater concentrations of CEMF. Since the fully modulated endothelial cells have collagen phenotypes distinct from those of normal cells, collagen synthesized by the first-passaged cells (a mixture of normal and modulated cells) was determined by immunoblot analysis with antibodies specific against types I and IV collagens. The first-passaged cells, in the presence of CEMF, contained a large amount of type I collagen (modulated phenotype) and a dramatically reduced amount of type IV collagen (physiologic type), whereas the normal endothelial cells demonstrated strongly positive staining only with antibodies to type IV collagen. Using cloned cDNA probes, the relative quantities of the transcripts of these collagens were determined by slot-blot hybridization; the first-passaged cells contained type IV collagen RNA in an amount similar to the normal cells, but a slightly larger amount of type I mRNA. These results demonstrate a functional involvement of a protein factor released by polymorphonuclear leukocytes in modulating cell shape and collagen gene expression in corneal endothelial cells.

Animals↗