Anti-Angiogenic and Anti-Scarring Dual Effect of Galectin-3 Inhibition in Mouse Models of Corneal Wound Healing.

Corneal scarring is the third leading cause of global blindness. Neovascularization of ocular tissues is a major predisposing factor in scar development. Although corneal transplantation is effective in restoring vision, some patients are at high risk for graft rejection due to the presence of blood vessels in the injured cornea. Current treatment options for controlling corneal scarring are limited, and outcomes are typically poor. In this study, topical application of a small-molecule inhibitor of galectin-3, GB1265, in mouse models of corneal wound healing, led to the reduction of the following in injured corneas: i) corneal angiogenesis; ii) corneal fibrosis; iii) infiltration of immune cells; and iv) expression of the proinflammatory cytokine IL-1ß. Four independent techniques (RNA sequencing, NanoString, real-time quantitative RT-PCR, and Western blot analysis) determined that decreased corneal opacity in the galectin-3 inhibitor-treated corneas was associated with decreases in the numbers of genes and signaling pathways known to promote fibrosis. These findings allowed for a high level of confidence in the conclusion that galectin-3 inhibition by the small-molecule inhibitor GB1265 has dual anti-angiogenic and anti-scarring effects. Targeting galectin-3 by GB1265 is, thus, attractive for the development of innovative therapies for a myriad of ocular and nonocular diseases characterized by pathologic angiogenesis and fibrosis.

Galectin-8 Downmodulates TLR4 Activation and Impairs Bacterial Clearance in a Mouse Model of Pseudomonas aeruginosa Keratitis.

Pseudomonas aeruginosa provokes a painful, sight-threatening corneal infection. It progresses rapidly and is difficult to treat. In this study, using a mouse model of P. aeruginosa keratitis, we demonstrate the importance of a carbohydrate-binding protein, galectin-8 (Gal-8), in regulation of the innate immune response. First, using two distinct strains of P. aeruginosa, we established that Gal-8-/- mice are resistant to P. aeruginosa keratitis. In contrast, mice deficient in Gal-1, Gal-3, and Gal-9 were fully susceptible. Second, the addition of exogenous rGal-8 to LPS (TLR4 ligand)-stimulated bone marrow-derived macrophages suppressed 1) the activation of the NF-κB pathway, and 2) formation of the MD-2/TLR4 complex. Additionally, the expression level of reactive oxygen species was substantially higher in infected Gal-8-/- bone marrow neutrophils (BMNs) compared with the Gal-8+/+ BMNs, and the P. aeruginosa killing capacity of Gal-8-/- BMNs was considerably higher compared with that of Gal-8+/+ BMNs. In the bacterial killing assays, the addition of exogenous rGal-8 almost completely rescued the phenotype of Gal-8-/- BMNs. Finally, we demonstrate that a subconjunctival injection of a Gal-8 inhibitor markedly reduces the severity of infection in the mouse model of P. aeruginosa keratitis. These data lead us to conclude that Gal-8 downmodulates the innate immune response by suppressing activation of the TLR4 pathway and clearance of P. aeruginosa by neutrophils. These findings have broad implications for developing novel therapeutic strategies for treatment of conditions resulting from the hyperactive immune response both in ocular as well as nonocular tissues.

Collagen VII deficient mice show morphologic and histologic corneal changes that phenotypically mimic human dystrophic epidermolysis bullosa of the eye.

BACKGROUND: Absence of collagen VII causes blistering of the skin, eyes and many other tissues. This disease is termed dystrophic epidermolysis bullosa (DEB). Corneal fibrosis occurs in up to 41% and vision loss in up to 64% of patients. Standard treatments are supportive and there is no cure. The hypomorphic mouse model for DEB shows production of collagen VII at 10% of wild type levels in skin and spleen, but the eyes have not been described. Our purpose is to characterize the corneas to determine if this is an appropriate model for study of ocular therapeutics. METHODS: Western blot analysis (WB) and immunohistochemistry (IHC) were performed to assess presence and location of collagen VII protein within the hypomorphic mouse cornea. Additional IHC for inflammatory and fibrotic biomarkers transforming growth factor-beta-1 (TGF-ß1), alpha-smooth muscle actin (α-SMA), connective tissue growth factor (CTGF), proteinase 3, tenascin C and collagen III were performed. Clinical photographs documenting corneal opacification were assessed and scored independently by 2 examiners. Histology was then used to investigate morphologic changes. RESULTS: IHC and WB confirmed that hypomorphic mice produce less collagen VII production at the level of the basement membrane when compared with wild-types. IHC showed anomalous deposition of collagen III throughout the stroma. Of the 5 biomarkers tested, TGF-ß1 showed the strongest and most consistently staining. Photographs documented corneal opacities only in mice older than 10 weeks, opacities were not seen in younger animals. Histology showed multiple abnormalities, including epithelial hyperplasia, ulceration, fibrosis, edema, dysplasia, neovascularization and bullae formation. CONCLUSIONS: The collagen VII hypomorphic mouse shows reduced collagen VII production at the level of the corneal basement membrane. Corneal changes are similar to pathology seen in humans with this disease. The presence of anomalous stromal collagen III and TGF-ß1 appear to be the most consistent and strongest staining biomarkers in diseased mice. This mouse appears to mimic human corneal disease. It is an appropriate model for testing of therapeutics to treat EB ocular disease.

IL-17A-mediated protection against Acanthamoeba keratitis.

Acanthamoeba keratitis (AK) is a very painful and vision-impairing infection of the cornea that is difficult to treat. Although past studies have indicated a critical role of neutrophils and macrophages in AK, the relative contribution of the proinflammatory cytokine, IL-17A, that is essential for migration, activation, and function of these cells into the cornea is poorly defined. Moreover, the role of the adaptive immune response, particularly the contribution of CD4(+) T cell subsets, Th17 and regulatory T cells , in AK is yet to be understood. In this report, using a mouse corneal intrastromal injection-induced AK model, we show that Acanthamoeba infection induces a strong CD4(+) T effector and regulatory T cell response in the cornea and local draining lymph nodes. We also demonstrate that corneal Acanthamoeba infection induces IL-17A expression and that IL-17A is critical for host protection against severe AK pathology. Accordingly, IL-17A neutralization in Acanthamoeba-infected wild-type mice or Acanthamoeba infection of mice lacking IL-17A resulted in a significantly increased corneal AK pathology, increased migration of inflammatory cells at the site of inflammation, and a significant increase in the effector CD4(+) T cell response in draining lymph nodes. Thus, in sharp contrast with other corneal infections such as herpes and Pseudomonas aeruginosa keratitis where IL-17A exacerbates corneal pathology and inflammation, the findings presented in this article suggest that IL-17A production after Acanthamoeba infection plays an important role in host protection against invading parasites.

Molecular basis for MMP9 induction and disruption of epithelial cell-cell contacts by galectin-3.

Dynamic modulation of the physical contacts between neighboring cells is integral to epithelial processes such as tissue repair and cancer dissemination. Induction of matrix metalloproteinase (MMP) activity contributes to the disassembly of intercellular junctions and the degradation of the extracellular matrix, thus mitigating the physical constraint to cell movement. Using the cornea as a model, we show here that a carbohydrate-binding protein, galectin-3, promotes cell-cell detachment and redistribution of the tight junction protein occludin through its N-terminal polymerizing domain. Notably, we demonstrate that galectin-3 initiates cell-cell disassembly by inducing matrix metalloproteinase expression in a manner that is dependent on the interaction with and clustering of the matrix metalloproteinase inducer CD147 (also known as EMMPRIN and basigin) on the cell surface. Using galectin-3-knockout mice in an in vivo model of wound healing, we further show that increased synthesis of MMP9 at the leading edge of migrating epithelium is regulated by galectin-3. These findings establish a new galectin-3-mediated regulatory mechanism for induction of metalloproteinase expression and disruption of cell-cell contacts required for cell motility in migrating epithelia.

Galectin-8 promotes regulatory T-cell differentiation by modulating IL-2 and TGFß signaling.

Galectins (Gals) have emerged as potent immunoregulatory molecules that control chronic inflammation through distinct mechanisms. Gal-8, a tandem-repeat type Gal with unique preference for α2,3-sialylated glycans, is ubiquitously expressed, but little is known about its role in T-cell differentiation. Here we report that Gal-8 promotes the polyclonal differentiation of primary mouse regulatory T (Treg) cells in vitro. We further show that Gal-8 also facilitates antigen-specific differentiation of Treg cells, and that Treg cells polarized in the presence of Gal-8 express cytotoxic T-lymphocyte antigen-4 and interleukin (IL)-10 at a higher frequency than control Treg cells, and efficiently inhibit proliferation of activated T-cells in vitro. Investigation of the mechanism by which Gal-8 promotes Treg conversion revealed that Gal-8 activates transforming growth factor-ß signaling and promotes sustained IL-2R signaling. Taken together, these data suggest that Gal-8 promotes the differentiation of highly suppressive Treg cells, which has implications for the treatment of inflammatory and autoimmune diseases.

Verteporfin without light stimulation inhibits YAP activation in trabecular meshwork cells: Implications for glaucoma treatment.

Verteporfin, a photosensitizer, is used in photodynamic therapy to treat age-related macular degeneration. In a glaucoma mouse model, Verteporfin without light stimulation has been shown to reduce intraocular pressure (IOP) but the mechanism is unknown. Recent studies have shown that Verteporfin inhibits YAP without light stimulation in cancer cells. Additionally, YAP has emerged as an important molecule in the pathogenesis of glaucoma. We hypothesize that YAP inactivation by Verteporfin in trabecular meshwork (TM) may be related to the reduced IOP observed in vivo. As contractility of TM tissues is associated with IOP, collagen gel contraction assay was used to assess the effect of Verteporfin on contractility of TM cells. Human TM cells were embedded in collagen gel and treated with Verteporfin for 48 h. Areas of collagen gel sizes were quantified by ImageJ. To assess the effect of Verteporfin on the expression of YAP, human TM cells were treated with Verteporfin for 24 h and the expression of YAP was determined by Western blotting. To determine the cytotoxic effect of Verteporfin, human TM cells were treated with Verteporfin for 24 h, and then the cell viability was assessed by WST-1. We demonstrated here that Verteporfin (i) abolishes TM cell-mediated collagen gel contraction in a dose-dependent manner, (ii) attenuates expression of YAP and CTGF (connective tissue growth factor, a direct YAP target gene) in a dose-dependent manner, and (iii) has no significant cytotoxicity below 2 µM. Taken together, Verteporfin may facilitate aqueous humor outflow through the conventional outflow system and reduce IOP by inactivating YAP.

Galectin-1-mediated suppression of Pseudomonas aeruginosa-induced corneal immunopathology.

Corneal infection with Pseudomonas aeruginosa leads to a severe immunoinflammatory lesion, often causing vision impairment and blindness. Although past studies have indicated a critical role for CD4(+) T cells, particularly Th1 cells, in corneal immunopathology, the relative contribution of recently discovered Th17 and regulatory T cells is undefined. In this study, we demonstrate that after corneal P. aeruginosa infection, both Th1 and Th17 cells infiltrate the cornea with increased representation of Th17 cells. In addition to Th1 and Th17 cells, regulatory T cells also migrate into the cornea during early as well as late stages of corneal pathology. Moreover, using galectin-1 (Gal-1), an immunomodulatory carbohydrate-binding molecule, we investigated whether shifting the balance among various CD4(+) T cell subsets can modulate P. aeruginosa-induced corneal immunopathology. We demonstrate in this study that local recombinant Gal-1 (rGal-1) treatment by subconjunctival injections significantly diminishes P. aeruginosa-mediated corneal inflammation through multiple mechanisms. Specifically, in our study, rGal-1 treatment significantly diminished corneal infiltration of total CD45(+) T cells, neutrophils, and CD4(+) T cells. Furthermore, rGal-1 treatment significantly reduced proinflammatory Th17 cell response in the cornea as well as local draining lymph nodes. Also, rGal-1 therapy promoted anti-inflammatory Th2 and IL-10 response in secondary lymphoid organs. Collectively, our results indicate that corneal P. aeruginosa infection induces a strong Th17-mediated corneal pathology, and treatment with endogenously derived protein such as Gal-1 may be of therapeutic value for the management of bacterial keratitis, a prevalent cause of vision loss and blindness in humans worldwide.

Glycobiology of ocular angiogenesis.

Ocular neovascularization can affect almost all the tissues of the eye: the cornea, the iris, the retina, and the choroid. Pathological neovascularization is the underlying cause of vision loss in common ocular conditions such as diabetic retinopathy, retinopathy of prematurity and age-related macular neovascularization. Glycosylation is the most common covalent posttranslational modification of proteins in mammalian cells. A growing body of evidence demonstrates that glycosylation influences the process of angiogenesis and impacts activation, proliferation, and migration of endothelial cells as well as the interaction of angiogenic endothelial cells with other cell types necessary to form blood vessels. Recent studies have provided evidence that members of the galectin class of ß-galactoside-binding proteins modulate angiogenesis by novel carbohydrate-based recognition systems involving interactions between glycans of angiogenic cell surface receptors and galectins. This review discusses the significance of glycosylation and the role of galectins in the pathogenesis of ocular neovascularization.

The NLRP3 Inflammasome Is Required for Protection Against Pseudomonas Keratitis.

Purpose: The current study was designed to examine the role of the NLRP3 inflammasome pathway in the clearance of Pseudomonas aeruginosa (PA) infection in mouse corneas. Methods: Corneas of wild type and NLRP3-/- mice were infected with PA. The severity of bacterial keratitis was graded on days 1 and 3 post-infection by slit lamp, and then corneas were harvested for: (i) bacterial enumeration, (ii) immune cell analysis by flow cytometry, (iii) immunoblotting analysis of cleaved caspase-1 and IL-1ß, and (iv) IL-1ß quantification by ELISA. In parallel experiments, severity of keratitis was examined in the wild-type mice receiving a subconjunctival injection of a highly selective NLRP3 inhibitor immediately prior to infection. Results: Compared to wild type mice, NLRP3-/- mice exhibited more severe infection, as indicated by an increase in opacity score and an increase in bacterial load. The hallmark of inflammasome assembly is the activation of proinflammatory caspase-1 and IL-1ß by cleavage of their precursors, pro-caspase-1 and pro-IL-1ß, respectively. Accordingly, increased severity of infection in the NLRP3-/- mice was associated with reduced levels of cleaved forms of caspase-1 and IL-1ß and reduced IL-1ß+ neutrophil infiltration in infected corneas. Likewise, corneas of mice receiving subconjunctival injections of NLRP3 inhibitor exhibited increased bacterial load, and reduced IL-1ß expression. Conclusions: Activation of NLRP3 pathway is required for the clearance of PA infection in mouse corneas.

Galectin-3 protein modulates cell surface expression and activation of vascular endothelial growth factor receptor 2 in human endothelial cells.

Angiogenesis is heavily influenced by VEGF-A and its family of receptors, particularly VEGF receptor 2 (VEGF-R2). Like most cell surface proteins, VEGF-R2 is glycosylated, although the function of VEGF-R2 with respect to its glycosylation pattern is poorly characterized. Galectin-3, a glycan binding protein, interacts with the EGF and TGFß receptors, retaining them on the plasma membrane and altering their signal transduction. Because VEGF-R2 is glycosylated and both galectin-3 and VEGF-R2 are involved with angiogenesis, we hypothesized that galectin-3 binds VEGF-R2 and modulates its signal transduction as well. Employing a Western blot analysis approach, we found that galectin-3 induces phosphorylation of VEGF-R2 in endothelial cells. Knockdown of galectin-3 and Mgat5, an enzyme that synthesizes high-affinity glycan ligands of galectin-3, reduced VEGF-A mediated angiogenesis in vitro. A direct interaction on the plasma membrane was detected between galectin-3 and VEGF-R2, and this interaction was dependent on the expression of Mgat5. Using immunofluorescence and cell surface labeling, we found an increase in the level of internalized VEGF-R2 in both Mgat5 and galectin-3 knockdown cells, suggesting that galectin-3 retains the receptor on the plasma membrane. Finally, we observed reduced suture-induced neovascularization in the corneas of Gal3(-/-) and Mgat5(-/-) mice. These findings are consistent with the hypothesis that, like its role with the EGF and TGFß receptors, galectin-3 contributes to the plasma membrane retention and proangiogenic function of VEGF-R2.

Galectin-3 promotes lamellipodia formation in epithelial cells by interacting with complex N-glycans on alpha3beta1 integrin.

Recent studies have shown that galectin-3 (Gal-3; also known as LGALS3), a beta-galactoside-binding lectin, promotes cell migration during re-epithelialization of corneal wounds. The goal of this study was to characterize the molecular mechanism by which Gal-3 stimulates cell migration. We demonstrate here that exogenous Gal-3, but not Gal-1 or Gal-8, promotes cell scattering and formation of lamellipodia in human corneal epithelial cells in a beta-lactose-inhibitable manner. alpha3beta1 integrin was identified as the major Gal-3-binding protein in corneal epithelial cells by affinity chromatography of cell lysates on a Gal-3-Sepharose column. Preincubation of cells with anti-alpha3 integrin function-blocking antibody significantly inhibited the induction of lamellipodia by Gal-3. Furthermore, exogenous Gal-3 activated both focal adhesion kinase, a key regulator of integrin-dependent intracellular signaling, and Rac1 GTPase, a member of the family of Rho GTPases, well known for its role in the reorganization of the actin cytoskeleton and formation of lamellipodial extensions. Experiments involving knockdown of beta-1,6-N-acetylglucosaminytransferase V, an enzyme that synthesizes high-affinity glycan ligands for Gal-3, revealed that carbohydrate-mediated interaction between Gal-3 and complex N-glycans on alpha3beta1 integrin plays a key role in Gal-3-induced lamellipodia formation. We propose that Gal-3 promotes epithelial cell migration by cross-linking MGAT5-modified complex N-glycans on alpha3beta1 integrin and subsequently activating alpha3beta1-integrin-Rac1 signaling to promote lamellipodia formation.

Induction of CXCL10-Mediated Cell Migration by Different Types of Galectins.

Chemokines are an extended group of chemoattractant cytokines responsible for the recruitment of leukocytes into tissues. Among them, interferon-γ-inducible protein 10 (CXCL10) is abundantly expressed following inflammatory stimuli and participates in the trafficking of monocytes and activated T cells into sites of injury. Here, we report that different members of the galectin family of carbohydrate-binding proteins promote the expression and synthesis of CXCL10 independently of interferon-γ. Interestingly, CXCL10 induction was observed when galectins came in contact with stromal fibroblasts isolated from human cornea but not other cell types such as epithelial, monocytic or endothelial cells. Induction of CXCL10 by the tandem repeat galectin-8 was primarily associated with the chemotactic migration of THP-1 monocytic cells, whereas the prototype galectin-1 promoted the CXCL10-dependent migration of Jurkat T cells. These results highlight the potential importance of the galectin signature in dictating the recruitment of specific leukocyte populations into precise tissue locations.

Association of cell surface mucins with galectin-3 contributes to the ocular surface epithelial barrier.

Maintenance of an intact mucosal barrier is critical to preventing damage to and infection of wet-surfaced epithelia. The mechanism of defense has been the subject of much investigation, and there is evidence now implicating O-glycosylated mucins on the epithelial cell surface. Here we investigate a new role for the carbohydrate-binding protein galectin-3 in stabilizing mucosal barriers through its interaction with mucins on the apical glycocalyx. Using the surface of the eye as a model system, we found that galectin-3 colocalized with two distinct membrane-associated mucins, MUC1 and MUC16, on the apical surface of epithelial cells and that both mucins bound to galectin-3 affinity columns in a galactose-dependent manner. Abrogation of the mucin-galectin interaction in four different mucosal epithelial cell types using competitive carbohydrate inhibitors of galectin binding, beta-lactose and modified citrus pectin, resulted in decreased levels of galectin-3 on the cell surface with concomitant loss of barrier function, as indicated by increased permeability to rose bengal diagnostic dye. Similarly, down-regulation of mucin O-glycosylation using a stable tetracycline-inducible RNA interfering system to knockdown c1galt1 (T-synthase), a critical galactosyltransferase required for the synthesis of core 1 O-glycans, resulted in decreased cell surface O-glycosylation, reduced cell surface galectin-3, and increased epithelial permeability. Taken together, these results suggest that galectin-3 plays a key role in maintaining mucosal barrier function through carbohydrate-dependent interactions with cell surface mucins.

Analysis of differential expression of glycosyltransferases in healing corneas by glycogene microarrays.

It is generally accepted that the glycans on the cell surface and extracellular matrix proteins play a pivotal role in the events that mediate re-epithelialization of wounds. Yet, the global alteration in the structure and composition of glycans, specifically occurring during corneal wound closure remains unknown. In this study, GLYCOv2 glycogene microarray technology was used for the first time to identify the differentially expressed glycosylation-related genes in healing mouse corneas. Of approximately 2000 glycogenes on the array, the expression of 11 glycosytransferase and glycosidase enzymes was upregulated and that of 19 was downregulated more than 1.5-fold in healing corneas compared with the normal, uninjured corneas. Among them, notably, glycosyltransferases, beta3GalT5, T-synthase, and GnTIVb, were all found to be induced in the corneas in response to injury, whereas, GnTIII and many sialyltransferases were downregulated. Interestingly, it appears that the glycan structures on glycoproteins and glycolipids, expressed in healing corneas as a result of differential regulation of these glycosyltransferases, may serve as specific counter-receptors for galectin-3, a carbohydrate-binding protein, known to play a key role in re-epithelialization of corneal wounds. Additionally, many glycogenes including a proteoglycan, glypican-3, cell adhesion proteins dectin-1 and -2, and mincle, and mucin 1 were identified for the first time to be differentially regulated during corneal wound healing. Results of glycogene microarray data were confirmed by qRT-PCR and lectin blot analyses. The differentially expressed glycogenes identified in the present study have not previously been investigated in the context of wound healing and represent novel factors for investigating the role of carbohydrate-mediated recognition in corneal wound healing.

Pathogenesis of acanthamoeba keratitis.

Acanthamoeba keratitis (AK) is a serious infection of the cornea. At present, diagnosis of the disease is not straightforward and treatment is very demanding. While contact lens wear is the leading risk factor for A K, Acanthamoeba parasites are increasingly recognized as an important cause of keratitis in non-contact lens wearers. The first critical step in the pathogenesis of infection is the adhesion of the microbe to the surface of the host tissues. Acanthamoebae express a major virulence protein, the mannose-binding protein (MBP), which mediates the adhesion of amoebae to the surface of the cornea. The MBP is a transmembrane protein with characteristics of a typical cell surface receptor. Subsequent to the MBP-mediated adhesion to host cells, the amoebae produce a contact-dependent metalloproteinase and several contact-independent serine proteinases. These proteinases work in concert to produce a potent cytopathic effect (CPE ) involving killing of the host cells, degradation of epithelial basement membrane and underlying stromal matrix, and penetration into the deeper layers of the cornea. In the hamster animal model, oral immunization with the recombinant MBP protects against AK, and this protection is associated with an increased level of anti-MBP IgA in tears of protected animals. Normal human tear fluid contains IgA antibodies against Acanthamoeba MBP that is likely to provide protection by inhibiting the adhesion of parasites to host cells. Indeed, in in vitro CPE assays, even a low concentration of tears (10 microL of undiluted tears per milliliter of media) almost completely inhibits Acanthamoeba-induced CPE . In addition to adherence-inhibiting, IgA-mediated protection, human tears also contain IgA-independent factors that provide protection against Acanthamoeba-induced CPE by inhibiting the activity of cytotoxic proteinases. Characterization of the CPE-inhibitory factors of human tears should lead to a better understanding of the mechanism by which the tissues of the host resist the infection and also help decode circumstances that predispose to Acanthamoeba infections.

The role of integrin glycosylation in galectin-8-mediated trabecular meshwork cell adhesion and spreading.

Primary open angle glaucoma (POAG) is a major blindness-causing disease, characterized by elevated intraocular pressure due to an insufficient outflow of aqueous humor. The trabecular meshwork (TM) lining the aqueous outflow pathway modulates the aqueous outflow facility. TM cell adhesion, cell-matrix interactions, and factors that influence Rho signaling in TM cells are thought to play a pivotal role in the regulation of aqueous outflow. In a recent study, we demonstrated that galectin-8 (Gal8) modulates the adhesion and cytoskeletal arrangement of TM cells and that it does so through binding to beta(1) integrins and inducing Rho signaling. The current study is aimed at the characterization of the mechanism by which Gal8 mediates TM cell adhesion and spreading. We demonstrate here that TM cells adhere to and spread on Gal8-coated wells but not on galectin-1 (Gal1)- or galectin-3 (Gal3)-coated wells. The adhesion of TM cells to Gal8-coated wells was abolished by a competing sugar, beta-lactose, but not by a noncompeting sugar, sucrose. Also, a trisaccharide, NeuAcalpha2-3Galbeta1-4GlcNAc, which binds specifically to the N-CRD of Gal8, inhibited the spreading of TM cells to Gal8-coated wells. In contrast, NeuAcalpha2-6Galbeta1-4GlcNAc which lacks affinity for Gal8 had no effect. Affinity chromatography of cell extracts on a Gal8-affinity column and binding experiments with plant lectins, Maakia Amurensis and Sambucus Nigra, revealed that alpha(3)beta(1), alpha(5)beta(1), and alpha(v)beta(1) integrins are major counterreceptors of Gal8 in TM cells and that TM cell beta(1) integrins carry predominantly alpha2-3-sialylated glycans, which are high-affinity ligands for Gal8 but not for Gal1 or Gal3. These data lead us to propose that Gal8 modulates TM cell adhesion and spreading, at least in part, by interacting with alpha2-3-sialylated glycans on beta(1) integrins.

Milk components inhibit Acanthamoeba-induced cytopathic effect.

PURPOSE: Acanthamoebae provoke a vision-threatening corneal infection known as Acanthamoeba keratitis (AK). It is thought that Acanthamoeba-specific IgA antibodies present in mucosal secretions such as human tears, milk, and saliva provide protection against infection by inhibiting the adhesion of parasites to host cells. The goal of the present study was to determine whether human mucosal secretions have the potential to provide protection against the Acanthamoeba-induced cytopathic effect (CPE) by an additional mechanism that is independent of IgA. METHODS: Breast milk was used as a model of human mucosal secretions. In vitro CPE assays were used to examine the CPE inhibitory effect of IgA-depleted milk and various milk fractions obtained by gel filtration. The activity of amebic proteinases was examined by zymography. RESULTS: IgA-depleted milk inhibited the Acanthamoeba-induced CPE in a concentration-dependent manner. Milk proteins were separated into four major fractions (F1-F4) by gel filtration. Of these four fractions, CPE inhibitory activity was detected largely in fraction F3. In contrast, fractions F1, F2, and F4 lacked CPE inhibitory activity. Moreover, fraction F3, but not F1, F2, or F4, inhibited amebic proteinases. CONCLUSIONS: These data, in conjunction with published findings showing that amebic proteinases are responsible for the induction of Acanthamoeba CPE, led us to propose that human mucosal secretions have the potential to provide protection against Acanthamoeba-induced CPE by an additional mechanism that is independent of IgA and that involves the inhibition of cytotoxic proteinases of amebae.