An isotype-specific trans-acting factor is defective in a mutant B cell line that expresses HLA-DQ, but not -DR or -DP.

The B lymphoblastoid cell line clone 13 (a subclone of the mutant cell line P3JHR-1) has been found to express high levels of HLA-DQ; by contrast, HLA-DR and -DP antigens are not expressed and cannot be induced by interferon gamma. Northern blot analysis using gene-specific probes indicated that the lack of surface expression of the DR and DP antigens is due to a marked decrease in the levels of steady-state RNA for both the alpha and beta chains. Southern blots demonstrated that none of the transcriptionally repressed genes are grossly deleted. Preparations of interspecific transient heterokaryons between clone 13 and the class II antigen-positive murine B cell lymphoma, A20, resulted in reactivation of the DRA gene and surface expression of both the DR and DP molecules. The efficiency of the DRA promoter relative to the DQB promoter is markedly and specifically diminished in clone 13 (P3JHR-1) as compared with the parental cell line, Jijoye, as assayed both by transient expression of appropriate chloramphenicol acetyltransferase gene (CAT) constructs and by in vitro transcription analysis. These data clearly demonstrate the existence of an isotype-specific trans-acting factor, and provide direct evidence that the highly homologous class II genes have distinct regulatory mechanisms.

A novel cysteine-rich sequence-specific DNA-binding protein interacts with the conserved X-box motif of the human major histocompatibility complex class II genes via a repeated Cys-His domain and functions as a transcriptional repressor.

The class II major histocompatibility complex (MHC) molecules function in the presentation of processed peptides to helper T cells. As most mammalian cells can endocytose and process foreign antigen, the critical determinant of an antigen-presenting cell is its ability to express class II MHC molecules. Expression of these molecules is usually restricted to cells of the immune system and dysregulated expression is hypothesized to contribute to the pathogenesis of a severe combined immunodeficiency syndrome and certain autoimmune diseases. Human complementary DNA clones encoding a newly identified, cysteine-rich transcription factor, NF-X1, which binds to the conserved X-box motif of class II MHC genes, were obtained, and the primary amino acid sequence deduced. The major open reading frame encodes a polypeptide of 1,104 amino acids with a symmetrical organization. A central cysteine-rich portion encodes the DNA-binding domain, and is subdivided into seven repeated motifs. This motif is similar to but distinct from the LIM domain and the RING finger family, and is reminiscent of known metal-binding regions. The unique arrangement of cysteines indicates that the consensus sequence CX3CXL-XCGX1-5HXCX3CHXGXC represents a novel cysteine-rich motif. Two lines of evidence indicate that the polypeptide encodes a potent and biologically relevant repressor of HLA-DRA transcription: (a) overexpression of NF-X1 from a retroviral construct strongly decreases transcription from the HLA-DRA promoter; and (b) the NF-X1 transcript is markedly induced late after induction with interferon gamma (IFN-gamma), coinciding with postinduction attenuation of HLA-DRA transcription. The NF-X1 protein may therefore play an important role in regulating the duration of an inflammatory response by limiting the period in which class II MHC molecules are induced by IFN-gamma.

Functional roles of the transcription factor Oct-2A and the high mobility group protein I/Y in HLA-DRA gene expression.

The class II major histocompatibility complex gene HLA-DRA is expressed in B cells, activated T lymphocytes, and in antigen-presenting cells. In addition, HLA-DRA gene expression is inducible in a variety of cell types by interferon-gamma (IFN-gamma). Here we show that the lymphoid-specific transcription factor Oct-2A plays a critical role in HLA-DRA gene expression in class II-positive B cell lines, and that the high mobility group protein (HMG) I/Y binds to multiple sites within the DRA promoter, including the Oct-2A binding site. Coexpression of HMG I/Y and Oct-2 in cell lines lacking Oct-2 results in high levels of HLA-DRA gene expression, and in vitro DNA-binding studies reveal that HMG I/Y stimulates Oct-2A binding to the HLA-DRA promoter. Thus, Oct-2A and HMG I/Y may synergize to activate HLA-DRA expression in B cells. By contrast, Oct-2A is not involved in the IFN-gamma induction of the HLA-DRA gene in HeLa cells, but antisense HMG I/Y dramatically decreases the level of induction. We conclude that distinct sets of transcription factors are involved in the two modes of HLA-DRA expression, and that HMG I/Y may be important for B cell-specific expression, and is essential for IFN-gamma induction.

Aqueous humor alloreactive cell phenotypes, cytokines and chemokines in corneal allograft rejection.

As biopsies are not taken at the time of human corneal allograft rejection, most information on the early cellular changes in rejection is from animal models. We examined the phenotype of alloreactive cells present in the human anterior chamber during corneal graft rejection by flow cytometry and quantified aqueous humor levels of cytokines and chemokines using cytometric bead array. Aqueous and peripheral blood samples were taken from patients with graft endothelial rejection (n = 11) and from control patients undergoing cataract surgery (n = 8). CD45(+)CD4(+), CD45(+)CD8(+) and CD45(+)CD14(+) cells were found in aqueous during rejection; no CD45(+) cells were seen in control samples. Higher proportions of CD45(+) cells found in aqueous during rejection were CD14(+), denoting monocyte/macrophage lineage, than were CD4(+) or CD8(+). Large elevations were seen in aqueous levels of IL-6, MCP-1 and IP-10 during rejection compared with controls; smaller but still statistically significant increases were seen in MIP-1alpha and eotaxin. The role of CD14(+) cells in allorejection is unclear as is the potential of these chemokines and their receptors as therapeutic targets. Aqueous humor samples offer a unique opportunity to analyze components of the allogeneic response in direct contact with donor tissue but without artifacts inherent in examination of tissue.

Biology and genetics of atopic disease.

Several immunological disorders including allergic rhinitis, bronchial asthma, atopic dermatitis, food allergies, urticaria, nonhereditary angioedema, systemic anaphylaxis, and allergic conjunctivitis are associated with a positive family history, and share a positive response in the Prausnitz-Kuster (wheal and flare) reaction. Studies have shown that 20-30% of the population has a strong genetic predisposition for this condition, termed atopy, whose hallmark is a greatly elevated serum IgE concentration. A great deal is known about the cellular interactions that mediate the sensitization, immediate and late-phase reactions that follow encounters with allergen, as well as about the cell surface and signaling events that result in mediator release from inflammatory cells. Less is known of the genes that confer genetic predisposition for atopy; however, a worldwide effort to identify atopy genes is making significant progress.

The architectural transcription factor high mobility group I(Y) participates in photoreceptor-specific gene expression.

The nonhistone chromosomal proteins high mobility group I(Y) [HMG I(Y)] have been shown to function as architectural transcription factors facilitating enhanceosome formation on a variety of mammalian promoters. Specifically, they have been shown to act as a "molecular glue" mediating protein-protein and protein-DNA contacts within the enhanceosome complex. HMG I(Y) proteins are expressed at high levels in embryonic and transformed cells and have been implicated in transcriptional regulation in these cells. Terminally differentiated cells, however, have been reported to express only minimal, if any, HMG I(Y). In contrast to these observations, we show here that adult mouse retinal photoreceptors, which are terminally differentiated cells, express high levels of these proteins. Using retinoblastoma cells as an approximate model, we further demonstrate in transiently transfected cells that inhibition of HMG I(Y) expression and mutation of HMG I(Y) binding sites significantly reduce rhodopsin promoter activity. DNase I footprint analysis indicates that HMG I protein interacts with a discrete site within the rhodopsin proximal promoter. This site overlaps with the binding site for Crx, a paired-like homeodomain transcription factor that is essential for photoreceptor functioning and that when mutated causes several forms of human photoreceptor degeneration. Both biochemical and functional experiments demonstrate that HMG I(Y) physically associate with Crx and that their interaction with DNA is required for high-level transcription of the rhodopsin gene. These data provide the first demonstration that HMG I(Y) can be important for gene activation in terminally differentiated cells.

Interferon-gamma induces transcription and differential expression of MHC genes in rat insulinoma cell line RINm5F.

We have reported that enhanced levels of class I major histocompatibility complex (MHC) antigen are expressed throughout the islets of prediabetic and newly diabetic BB rats and that the endocrine cells of the islet remained class II negative. In this study we investigated the molecular biology of lymphokine-induced expression of the class I and II MHC genes in subclones of the rat insulinoma cell line RINm5F. Treatment of a particular subclone of RINm5F cells (which are normally class II negative, class I low expressors) with crude lymphokine preparation or various doses of recombinant interferon-gamma resulted in enhancement of MHC class I antigen expression but no detectable induction of class II antigen expression. This enhancement of class I antigen expression was a dose-dependent phenomenon and was preceded by a dose-dependent increase in class I-specific RNA. Both class I and II genes were induced at the transcriptional level, as determined by Northern blotting and in vitro nuclear transcription assays, but exhibited strikingly different induction kinetics. Supernatants from concanavalin A-stimulated splenocytes had a similar class I-restricted inductive effect on MHC gene expression. This subclone of RINm5F cells, which exhibits a class I lymphokine response-positive, class II response-negative phenotype, 1) mimics the behavior of beta-cells in the prediabetic and newly diabetic pancreas and 2) represents a valuable system for probing the similarities and differences in the lymphokine-mediated induction pathways for class I and II MHC genes.

Association of susceptibility to spontaneous diabetes in rat with genes of major histocompatibility complex.

This study was designed to map the diabetes susceptibility gene(s) associated with the rat major histocompatibility complex (MHC) RT1. We have crossed spontaneously diabetic male rats bearing the recombinant RT1r8 haplotype with female rats of the AC1.1r4 congenic strain. Three diabetic rats were determined to be homozygous for the r4 haplotype by serotyping. The absence of recombination within the MHC was confirmed by inspection of restriction-fragment-length patterns of the diabetic animals and the parental strains. In conjunction with previous breeding studies, this study maps the diabetes susceptibility gene to the right of the RT1-A locus and to the left of the RT1-C locus. A low incidence of diabetes in the F2 (4.5%) emphasizes the multifactorial nature of the susceptibility. The presence of depressed responsiveness of peripheral blood lymphocytes to concanavalin A stimulation increases the prevalence of the overt disease. An unusual feature of the diabetic syndrome in this study is the sparse or absent pancreatic lymphocytic inflammatory response, with true insulitis being a rare finding.

IDDM in BB rats. Enhanced MHC class I heavy-chain gene expression in pancreatic islets.

Modulation in major histocompatibility complex (MHC) gene expression correlates with the inflammatory reactions that occur during graft rejection and autoimmune disease. We analyzed the expression of class I and II MHC genes in the pancreatic islets of prediabetic and newly diabetic BB rats by immunohistochemistry of tissue sections and Northern blotting of RNA extracted from isolated islets. We show that enhanced levels of MHC class I heavy-chain RNA are present in pancreatic islets before overt inflammation and the onset of insulin-dependent diabetes mellitus (IDDM) in the spontaneously diabetic BB rat. Immunohistochemical analysis revealed enhanced class I antigen expression throughout the pancreatic islets of newly diabetic animals but no induction of class II antigen on endocrine cells within the islet. Varying degrees of inflammatory infiltrate were observed in the sections exhibiting enhanced class I antigen expression or in nearby serial sections. Southern blot analysis revealed no restriction-fragment-length polymorphism or amplification of the endogenous class I heavy-chain genes compared with those of seroidentical disease-resistant Wistar-Furth rats. I-A alpha and I-E alpha hybridizing RNA appeared de novo before overt diabetes, although concomitantly with T-lymphocyte-receptor beta-chain and interferon-gamma gene hybridizing RNA and after MHC class I heavy-chain RNA enhancement was observed. These data indicate the possibility that enhanced class I heavy-chain gene expression plays a role in the progression of IDDM.

Immunologic and genetic studies of diabetes in the BB rat.

The spontaneous development of diabetes in the Bio-Breeding (BB) rat is an excellent model of human insulin-dependent diabetes mellitus (IDDM). Disease expression is dependent on several genetically determined abnormalities, including specific major histocompatibility complex (MHC) genes. At least one MHC class II locus of the U haplotype is a necessary, but not sufficient, condition for disease expression. The immune system of BB rats is markedly abnormal. There is a striking reduction in the number and function of mature cytotoxic/suppressor T cells, a poor proliferative response to mitogens and in mixed lymphocyte culture, poor interleukin-2 production, and a reduced ability to reject skin allografts. While these immune system abnormalities are closely related to the development of diabetes, the immune recognition and effector mechanisms resulting in islet cell destruction are still poorly understood. The hypothesis that MHC class II induction on pancreatic beta cells serves to target these lymphokines, natural killer (NK) cells, macrophages, etc.) have been implicated in islet cell killing. The incidence of IDDM is reduced by immunosuppressive therapy in both rats and humans, further supporting the role of immune mechanisms in this disease.

Retinal transplantation: progress and problems in clinical application.

There is currently no real treatment for blinding disorders that stem from the degeneration of cells in the retina and affect at least 50 million individuals worldwide. The excitement that accompanied the first studies showing the potential of retinal cell transplantation to alleviate the progress of blindness in such diseases as retinitis pigmentosa and age-related macular degeneration has lost some of its momentum, as attempts to apply research to the clinic have failed so far to provide effective treatments. What these studies have shown, however, is not that the approach is flawed but rather that the steps that need to be taken to achieve a viable, clinical treatment are many. This review summarizes the course of retinal transplant studies and points to obstacles that still need to be overcome to improve graft survival and efficacy and to develop a protocol that is effective in a clinical setting. Emphasis is given particularly to the consequences of introducing transplants to sites that have been considered immunologically privileged and to the role of the major histocompatibility complex classes I and II molecules in graft survival and rejection.

High mobility group I/Y protein functions as a specific cofactor for Oct-2A: mapping of interaction domains.

The octamer motif (ATTTGCAT) present in several eukaryotic promoters and enhancers is now known to influence the transcription of several genes by interacting with members of a broad family of homeodomain proteins. The promoter of the human class II MHC gene HLA-DRA contains a conserved octamer element that can bind (among other proteins) the transcription factor Oct-2A and the high mobility group proteins (HMG) I/Y. We have previously determined that HMG I(Y) and Oct-2A cooperatively activate HLA-DRA gene expression, most likely due to the ability of HMG I(Y) to selectively recruit Oct-2A to the octamer motif. In this report, we present results of our investigations of the mechanisms of cooperative transactivation of HLA-DRA transcription by Oct-2A and HMG I(Y). We show that both the amino- and the carboxy-terminal domains of Oct-2A are required for HLA-DRA transactivation. Experiments using domain-swap chimeras of the Oct-1 and Oct-2A polypeptides indicate that cooperative activation of the DRA gene by HMG I(Y) and Oct-2A requires the carboxy-terminal domain (CTD) of Oct-2A. However, HMG I(Y) physically interacts with the conserved POU domains of both Oct-1 and Oct-2A. We therefore postulate that the nature of the CTD attached to the POU homeodomain influences the outcome of interaction with HMG I(Y). These studies support the view that HMG I(Y) is an important cofactor for HLA-DRA gene activation by Oct-2A and provide insights into its mechanism of action.

Cis-element dependence and occupancy of the human invariant chain promoter in CIITA-dependent and -independent transcription.

The major histocompatibility complex (MHC)-associated invariant chain (Ii) associates with the class II alpha/beta heterodimer during its biosynthesis, inhibiting association of endogenous peptides with the peptide-binding cleft. It is therefore not surprising that there are significant similarities in regulatory mechanisms controlling the expression of the structural class II MHC and Ii genes. One important similarity is that both classes of genes can be expressed via CIITA-dependent or -independent mechanisms. In this report, we have dissected CIITA-dependent and -independent transcription of the Ii gene using an isogenic B-LCL cell pair (Jijoye and clone-13) which do or do not express the class II MHC transactivator (CIITA), respectively. Experiments using mutant or deletion constructs of the Ii gene promoter indicate that while both the X-box and li-kappaB1 elements are critical for CIITA-dependent transcription in B lymphocytes, the Ii-kappaBI element is of greater importance for CIITA-independent Ii gene transcription, with the X-box playing a secondary role. Despite these clear differences in cis-element dependence of CIITA-dependent and -independent Ii transcription, there are only subtle differences in the occupancy of these elements in vivo as assessed by genomic footprinting. These differences are restricted to occupancy of the X-box and Y-box, with which the RF-X and NF-Y complexes interact in Ii-positive cells. This difference in the occupancy of the X-box and Y-box in this cell pair indicates that while protein/protein interactions between CIITA and promoter-bound factors stabilize promoter occupancy, these interactions are not absolutely required for occupancy and transcription of the invariant chain gene.

Identification of a stimulus-dependent DNase I hypersensitive site between the Ialpha and Calpha exons during immunoglobulin heavy chain class switch recombination.

The complete humoral response to foreign antigen depends upon two distinct recombination events within the heavy chain locus of immunoglobulin. The first recombination event takes place in what will become the antigen combining site of the antibody molecule, encoded by V, D and J segments. The second recombination event involves the looping-out of large spans of DNA which separate the various clusters of heavy chain exons which define the different immunoglobulin isotypes, or classes. While a great deal has been learned about the nature of the VDJ recombinase, very little is known about the nature of the class-switch recombinase. Using a cell system where class-switch recombination occurs primarily to the IgA locus, we have looked for stimulus-dependent changes in the chromatin structure of the IgA locus which might result from interactions between components of the recombinase and cis-elements within the region. We present evidence that strongly suggests that the class-switch recombinase interacts between the Ialpha and Calpha exons of IgA, just upstream of the highly reiterated DR1 and DR2 elements. However, although multiple potential SMAD-4 sites are located precisely within the DNase I hypersensitive site and 160 bp upstream of that site, we failed to detect any evidence of DNA/protein interactions near the hypersensitive site. Moreover, recombinant SMAD-3/4 proteins fail to interact with these sites with appreciable affinity in vitro. These data suggest that some other structural alteration at this site (e.g. RNA/DNA hybrid) may mediate the nuclease sensitivity.

A novel downstream positive regulatory element mediating transcription of the human high mobility group (HMG) I-C gene.

The high mobility group (HMG) I proteins are small, non-histone chromosomal proteins that promote gene activation during development and within rapidly dividing cells. They do so by facilitating enhanceosome formation on inducible genes, via both protein/DNA and protein/protein interactions. The HMG I-C gene is tightly regulated, normally being expressed exclusively during embryonic development. However, HMG I-C expression is also observed frequently in a number of tumor types, and this expression has been shown to contribute to the malignant transformation process. With the aim of dissecting pathways that lead to aberrant expression of HMG I-C in tumor cells, we have analyzed HMG I-C gene regulation in the human hepatoma cell line PLC/PRF/5. One of the two HMG I-C transcripts detected in this cell line originates from a novel downstream initiation site at nucleotide -161 relative to the first methionine. Transcription from the downstream initiation site is mediated by a PRE located between nt -222 and -217. We show here that the Sp1 and Sp3 transcription factors interact with the PRE and transactivate the HMG I-C promoter in a cooperative fashion. This study provides the first characterization of this downstream HMG I-C promoter.

The HMG I proteins: dynamic roles in gene activation, development, and tumorigenesis.

The high mobility group I, Y, and I-C proteins are low-molecular-weight, nonhistone chromosomal proteins that play a general role modulating gene expression during development and the immune response. Consistent with their role in early development, all three proteins are expressed at high levels during embryogenesis, and their expression is markedly diminished in differentiated cells. Exceptions to the general repression of these genes in adult tissues involve (1) A burst of synthesis of the HMG I protein during the immune response (during lymphocyte activation and preceding cytokine/adhesion molecule gene expression), (2) A constitutive expression of the HMG I and Y proteins in photoreceptor cells, and (3) Derepression of HMG I, Y, and often I-C expression in neoplastic cells. Work from several laboratories has now uncovered how these proteins participate in gene activation: (1) By altering the chromatin structure around an inducible gene-and thus influencing accessibility of the locus to regulatory proteins-(2) By facilitating the loading of transcription factors onto the promoters, and (3) By bridging adjacent transcription factors on a promoter via protein/protein interactions. Despite the similar structures and biochemical properties of the three proteins, the work has also provided clues to a division of labor between these proteins. HMG I and Y have demonstrable roles in enhanceosome formation, whereas HMG I-C has a specific role in adipogenesis. C-terminal truncations of HMG I-C and wild-type HMG Y appear to function in a manner analogous to oncogenes, as assessed by cellular transforation assays and transgenic mice. Future work should clearly define the similarities and differences in the biological roles of the three proteins, and should evolve to include attempts at pharmaceutical intervention in disease, based upon structural information concerning HMG I interactions with DNA and with regulatory proteins.

Prevention of acute allergic conjunctivitis and late-phase inflammation with immunostimulatory DNA sequences.

PURPOSE: To evaluate the therapeutic potential of immunostimulatory sequence oligodeoxynucleotides (ISS-ODN) administration in ocular allergy, using a mouse model of ragweed-specific conjunctivitis. METHODS: A murine model of allergic conjunctivitis involving SWR/J mice sensitized and challenged with short ragweed was used to test immunostimulatory DNA sequences for therapeutic potential. ISS-ODN or control ODN (0.1 mg/mouse) was administered intraperitoneally or topically to the conjunctiva 3 days before final allergen challenge. Multiple parameters of clinical symptoms evident during the acute-phase reaction and the cellular components of the late-phase reaction were evaluated in both groups of mice. RESULTS: All parameters of clinical symptoms were markedly inhibited after intraperitoneal injection of ISS-ODN, whereas topical application to the conjunctiva did not inhibit clinical symptoms significantly. Remarkably, a single topical treatment with ISS-ODN (as well as by intraperitoneal injection) completely inhibited both eosinophilia and neutrophilia in the late-phase reaction. CONCLUSIONS: Systemic or conjunctival administration of ISS-ODN was shown to significantly inhibit allergic responses in this mouse model. This indicates that ISS-ODN may be an effective form of immunotherapy for this class of allergic disease.

Differential chemokine gene expression in corneal transplant rejection.

PURPOSE: To evaluate the differential gene expression of chemokines after corneal transplantation and to determine the chemokines associated with allograft rejection. METHODS: Orthotopic mouse corneal transplantation was performed in two fully mismatched-strain combinations using C57BL/6 (H-2b) and BALB/c (H-2d) mice as recipients and BALB/c and C57BL/6 mice as donors. Normal nonsurgical eyes served as negative control specimens and syngeneic transplants (isografts) as control specimens for the alloimmune response. Chemokine gene expression in accepted and rejected allografts and appropriate control specimens was determined by a multiprobe RNase protection assay system. RESULTS: In eyes with rejected allografts, there was overexpression of regulated on activation normal T-cell expressed and secreted (RANTES), macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, MIP-2, and monocyte chemotactic protein (MCP)-1 in both C57BL/6 and BALB/c recipients. In addition, C57BL/6 eyes with rejected allografts expressed very high levels of interferon-gamma-inducible protein of 10 kDa (IP-10) mRNA, in contrast to BALB/c eyes with rejected allografts, in which IP-10 expression remained very low. In contrast, lymphotactin gene expression increased only slightly in rejected allografts, and eotaxin mRNA, which was also detected in normal eyes, remained unchanged among isograft and allograft groups. T-cell activation gene (TCA)-3 mRNA was not detected in any of the assayed eyes. CONCLUSIONS: Increased expression of mRNA for select chemokines of the CXC (alpha) and CC (beta) families is associated with corneal allograft rejection. Significantly elevated IP-10 gene expression in high-rejector C57BL/6, but not in low-rejector BALB/c, hosts suggests that differential activation of chemokines may be related to differences in alloimmune reactivity observed among different murine strains.

The effect of perioperative allergic conjunctivitis on corneal lymphangiogenesis after corneal transplantation.

INTRODUCTION: Perioperative allergic conjunctivitis accelerates the speed of corneal allograft rejection. This study examines the effect of allergic conjunctivitis, with and without dexamethasone treatment, on the early inflammatory response and lymphangiogenesis in the host cornea following corneal transplantation. METHODS: Allogeneic fully MHC-mismatched C57Bl/6 strain donor corneas were transplanted into naive A/J mice and into A/J mice with active allergic conjunctivitis. Further groups of allograft recipients with allergic conjunctivitis were treated post-operatively with twice daily topical dexamethasone 0.1% or phosphate-buffered saline. Mice were killed on days 2 and 6 and corneas were examined by (i) fluorescent immunohistochemistry of frozen sections using anti-CD11b, anti-F4/80 and anti-Gr-1 antibodies, or (ii) whole-mount staining with anti-LYVE-1 antibody. Lymphatic ingrowth and numbers of cells infiltrating the host cornea were compared between groups. RESULTS: There were significantly higher numbers of CD11b(+) cells and LYVE-1(+) vessels in the host cornea at day 2 in allergic compared with naive recipients, but no differences between naive and allergic recipients at day 6. In allergic eyes, dexamethasone treatment significantly inhibited LYVE-1 expression at days 2 and 6, and significantly improved allograft survival in recipients with allergic conjunctivitis if maintained for a week. CONCLUSIONS: The innate immune response to allogeneic corneal tissue is more vigorous in the presence of allergic conjunctivitis than in naive eyes and is associated with accelerated lymphatic ingrowth to host cornea. Topical dexamethasone inhibits lymphatic ingrowth and this may be one mechanism by which topical steroid enhances graft survival.

Use of ultrasonic pachymetry for measurement of changes in corneal thickness in mouse corneal transplant rejection.

BACKGROUND/AIMS: Diagnosis of rejection in the mouse model of corneal transplantation is based on subjective judgement of loss of graft transparency. The aims of this study were to (1) evaluate a pachymetry technique to measure changes in mouse corneal thickness and (2) correlate increases in transplant thickness with clinical and histological features of rejection. METHODS: Orthotopic corneal allografts (C57BL/6 strain donor) and syngeneic grafts were performed in A/J mice. Graft transparency was graded and corneal thickness measured by pachymetry on alternate days. Transverse sections of donor cornea excised from eyes representative of clinical opacity grades 1-4 were prepared, photographed, graft section thickness measured and stromal graft-infiltrating cells counted. Intraobserver and interobserver variations in pachymetry were statistically tested. RESULTS: Graft thickness, as measured by pachymetry, increased with each clinical opacity grade. Thickness for opacity grades 0, 1 and 2 was less than 300 microm in all recipients. Graft thickness for grades 3 and 4 was greater than 300 microm in all cases. For measurements up to 400 mum, there was a good correlation between thickness as measured by in vivo pachymetry and in histopathological sections. The mean interobserver bias was -11.35 microm, while the mean intraobserver bias was +3.96 microm. Stromal cellularity increased with increasing corneal thickness up to approximately 300 microm. CONCLUSION: In vivo graft pachymetry provides a new and reliable way to objectively diagnose rejection in the mouse model of corneal transplantation.