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.

Characterization of dendritic cell phenotype in allergic conjunctiva: increased expression of Fc(epsilon)RI, the high-affinity receptor for immunoglobulin E.

PURPOSE: Dendritic cells (DCs) express the high-affinity receptor for IgE (Fc(epsilon)RI) on their surface, which may enhance their ability to capture and internalize antigens for presentation to T-lymphocytes. The aim of this study was to determine if expression of Fc(epsilon)RI(+) DCs is increased in the conjunctivae of vernal keratoconjunctivitis (VKC) patients compared with those of normal controls. METHODS: Conjunctival biopsies were obtained from non-atopic and VKC patients. Double immunohistochemical staining was carried out using antibodies against Fc(epsilon)RI and the CD1a antigen, a DC marker. The double-positive cells were counted in five representative fields of view for each conjunctival sample. RESULTS: Fc(epsilon)RI(+) CD1a(+) cells were present in significantly higher numbers in VKC conjunctivae compared with normal controls (mean cell count of 21.3 in VKC vs5.0 in controls, P<0.005). In normal patients the Fc(epsilon)RI-expressing DCs tended to be confined to the epithelial layer or the superficial substantia propria, but in the VKC samples these Fc(epsilon)RI(+) cells were mainly concentrated in the deeper substantia propria. CONCLUSIONS: Fc(epsilon)RI(+) DC numbers are elevated in the conjunctivae of VKC patients, a finding consistent with the results of other studies focusing on atopic conditions. Elevated expression of Fc(epsilon)RI on DCs would facilitate antigen presentation and enhance T-cell priming, thereby contributing to ocular symptoms.

Increased mast cell numbers in the conjunctiva of glaucoma patients: a possible indicator of preoperative glaucoma surgery inflammation.

PURPOSE: Inflammation is a risk factor for scarring after trabeculectomy surgery. Mast cells are important mediators of inflammation and scarring in allergic eye disease. This exploratory project investigated the presence of mast cells in the conjunctiva of glaucoma patients having trabeculectomy surgery. METHODS: Conjunctival biopsies from glaucoma patients belonging to specific groups: medically treated glaucoma (M, n=6), repeat glaucoma surgery (S, n=8), and uveitic glaucoma (U, n=7). The control group (C, n=8) was retinal detachment patients undergoing repair surgery for the first time. Immunohistochemistry techniques stained for the presence of the intracellular mast cell enzyme tryptase. RESULTS: The median mast cell tryptase-positive counts for all glaucoma groups (M, S, and U) ranged from 0.102-0.113 cells/mm2 compared to 0.064 cells/mm2 for group C. This was statistically significant comparing group S to group C (P=0.0063), but not when comparing groups U or M to group C. The mast cell tryptase-positive counts did not significantly differ among the groups. CONCLUSIONS: Mast cell numbers were significantly increased in glaucoma patients who have previously undergone surgery (group S). Mast cell activity may contribute to the scarring process and the increased risk of excessive conjunctival scarring after trabeculectomy surgery. Further investigation needs to be performed to evaluate this potential role.

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.

Does the polymorphism of MHC class II promoters matter?

The promoters of genes of the major histocompatibility complex vary not only because of linkage disequilibrium with their coding sequences but also, we argue, because of natural selection that acts particularly strongly on MHC II gene promoters. Thus, the promoter of H2Eb varies more than that of H2K, to an extent that cannot be accounted for by coding variation, and the same applies to HLA.DRB1 in comparison with H2D. We discuss how transduction by lentivirus vectors followed by adoptive transfer of monoclonal T cells could be used to test the functional activity of variant mouse promoters in vivo, and how homologous recombination in suitable cell lines might provide a short cut to obtaining promoter knock-ins.

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.

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.

Genetics of allergic disease: evidence for organ-specific susceptibility genes.

BACKGROUND: While previous studies have probed the genetics of asthma and serum IgE levels, there have been no studies on the genetics of allergic conjunctivitis. This paper describes the initial phase of a genetic study of allergic conjunctivitis. METHODS: Approximately 117 families with probands with allergic conjunctivitis were recruited generating 245 affected sib pairs. Each family member completed a detailed questionnaire on atopic symptoms, and results from skin testing were obtained. Genomic DNA was extracted and genotyped using thirty-five polymorphic markers spanning human chromosomes 5, 6, 11, 12, 16 and 17. Heritability was assessed using the POINTER program, and nonparametric linkage analysis using the BETA program. RESULTS: Evidence for genetic linkage of allergic conjunctivitis was obtained for chromosomes 5, 16 and 17. Weak linkage was detected for chromosome 6 when studies were restricted to specific allergens. No evidence for linkage was detected for chromosomes 11 and 12. CONCLUSIONS: Consistent with heritability analysis discussed in this paper, genetic linkage for allergic conjunctivitis is shown to differ from that reported for atopic asthma. This indicates that there are likely to be organ-specific disease susceptibility genes, which together with general atopy genes target the allergic response to specific mucosal tissues.

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.

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.

Identification and characterization of a critical CP2-binding element in the human interleukin-4 promoter.

Expression of cytokine genes in T cells is thought to result from a complex network of antigen- and mitogen-activated transcriptional regulators. CP2, a factor homologous to Drosophila Elf-1 and previously found to be a critical regulator of several viral and cellular genes in response to developmental signals, is rapidly activated in T helper (Th) cells in response to mitogenic stimulation. Here we show that overexpression of CP2 enhances interleukin (IL)-4 promoter-driven chloramphenicol acetyltransferase expression, while repressing IL-2 promoter activity, in transiently transfected Jurkat cells. A CP2-protected element, partially overlapping the nuclear factor of activated T cell-binding P2 sequence, was required for IL-4 promoter activation in CP2-overexpressing Jurkat cells. This CP2-response element is the site of a cooperative interaction between CP2 and an inducible heteromeric co-factor(s). Mutation of conserved nucleotide contacts within the CP2-response element prevented CP2 binding and significantly reduced constitutive and induced IL-4 promoter activity. Expression of a CP2 mutant lacking the Elf-1-homology region of the DNA-binding domain inhibited IL-4 promoter activity in a dominant negative fashion in transiently transfected Jurkat cells. Moreover, overexpressed CP2 markedly enhanced, while its dominant negative mutant consistently suppressed, expression of the endogenous IL-4 gene in the murine Th2 cell line D10. Taken together, these findings point to CP2 as a critical IL-4 transactivator in Th cells.

Functional domains of the cone-rod homeobox (CRX) transcription factor.

The paired-like homeodomain transcription factor CRX (cone-rod homeobox) is involved in regulating photoreceptor gene expression and rod outer segment development. Mutations in CRX have been associated with several retinal degenerative diseases. These conditions range from Leber congenital amaurosis (a severe cone and rod degeneration of childhood onset) to adult onset cone-rod dystrophy and retinitis pigmentosa (an adult onset condition that primarily affects rods). The goal of this study is to better understand the molecular basis of CRX function and to provide insight into how mutations in CRX cause such a variety of clinical phenotypes. We performed deletion analysis in conjunction with DNA binding and transient transfection-based transactivation studies to identify the functional domains within CRX. DNA binding requires a complete homeodomain. Furthermore, truncated proteins that did not contain an intact homeodomain failed to demonstrate detectable expression in tissue culture upon transfection. Transactivation analysis indicated that both the OTX tail and the WSP domain are important for controlling positive regulatory activity of CRX. Interestingly, the mapped CRX transactivation domains were also critical when coexpressed with NRL. Specifically, the synergy between CRX and NRL was constant regardless of which CRX variant was used.

Molecular genetics of allergic diseases.

Allergic diseases affect approximately one third of the general population. This class of disease, characterized by elevated serum IgE levels and hypersensitivity to normally innocuous antigen, can manifest in practically any mucosal tissue or as a systemic response. A few examples of serious allergic diseases include asthma, dermatitis, bee sting allergy, food allergy, conjunctivitis, and severe systemic anaphylaxis. Taken together, allergic diseases constitute one of the major problems of modern day medicine. A considerable portion of the healthcare budget is expended in the treatment of allergic disease, and morbidity rates of inner city asthmatics are rising steadily. Due to the enormity of the problem, there has been a worldwide effort to identify factors that contribute to the etiology of allergic diseases. Epidemiologic studies of multigeneration families and large numbers of twins clearly indicate a strong genetic component to atopic diseases. At least two independently segregating diseasesusceptibility genes are thought to come together with environmental factors to result in allergic inflammation in a particular tissue. On the basis of the strong genetic studies, multiple groups have attempted to identify disease-susceptibility genes via either a candidate gene approach or by genome-wide scans. Both of these approaches have implicated multiple regions in the human and mouse genomes, which are currently being evaluated as harboring putative atopy genes.

CD4+ T cell responses to self- and mutated p53 determinants during tumorigenesis in mice.

We analyzed CD4+ T helper responses to wild-type (wt) and mutated (mut) p53 protein in normal and tumor-bearing mice. In normal mice, we observed that although some self-p53 determinants induced negative selection of p53-reactive CD4+ T cells, other p53 determinants (cryptic) were immunogenic. Next, BALB/c mice were inoculated with J774 syngeneic tumor cell line expressing mut p53. BALB/c tumor-bearing mice mounted potent CD4+ T cell responses to two formerly cryptic peptides on self-p53. This response was characterized by massive production of IL-5, a Th2-type lymphokine. Interestingly, we found that T cell response was induced by different p53 peptides depending upon the stage of cancer. Mut p53 gene was shown to contain a single mutation resulting in the substitution of a tyrosine by a histidine at position 231 of the protein. Two peptides corresponding to wt and mutated sequences of this region were synthesized. Both peptides bound to the MHC class II-presenting molecule (Ed) with similar affinities. However, only mut p53.225-239 induced T cell responses in normal BALB/c mice, a result strongly suggesting that high-affinity wt p53.225-239 autoreactive T cells had been eliminated in these mice. Surprisingly, CD4+ T cell responses to both mut and wt p53.225-239 peptides were recorded in J774 tumor-bearing mice, a phenomenon attributed to the recruitment of low-avidity p53.225-239 self-reactive T cells.

Transgenic mice expressing a truncated form of the high mobility group I-C protein develop adiposity and an abnormally high prevalence of lipomas.

Chromosomal translocations in human lipomas frequently create fusion transcripts encoding high mobility group (HMG) I-C DNA-binding domains and C-terminal sequences from different presumed transcription factors, suggesting a potential role for HMG I-C in the development of lipomas. To evaluate the role of the HMG I-C component, the three DNA-binding domains of HMG I-C have now been expressed in transgenic mice. Despite the ubiquitous expression of the truncated HMG I-C protein, the transgenic mice develop a selective abundance of fat tissue early in life, show marked adipose tissue inflammation, and have an abnormally high incidence of lipomas. These findings demonstrate that the DNA-binding domains of HMG I-C, in the absence of a C-terminal fusion partner, are sufficient to perturb adipogenesis and predispose to lipomas. We provide data supporting the central utility of this animal model as a tool to understand the molecular mechanisms underlying the development of one of the most common kind of human benign tumors.

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.

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.