AECs secrete soluble factors that inhibit cells in both the innate and adaptive immune systems.

The pathogenesis of Acanthamoeba keratitis begins when Acanthamoeba trophozoites bind specifically to mannosylated glycoproteins upregulated on the surfaces of traumatized corneal epithelial cells. When Acanthamoeba castellanii trophozoites are grown in methyl-␣-D-mannopyranoside, they are induced to secrete a novel 133-kDa protein that is cytolytic to corneal epithelial cells. Clinical isolates of Acanthamoeba spp., and not the soil isolates, were proficient at producing a mannose-induced protein (MIP-133) and generating disease in Chinese hamsters. The purified protein was efficient at killing corneal epithelial cells, the first mechanistic barrier, by inducing apoptosis in a caspase 3-dependent pathway. Subsequent steps in pathogenesis require the amoebae to penetrate and degrade collagen. Only the clinical isolates tested were efficient at migrating through a collagenous matrix in vitro, presumably by MIP-133 degradation of both human type I and human type IV collagen. A chicken anti-MIP-133 antiserum effectively bound to the protein and blocked collagenolytic activity, migration, and cytopathic effects (CPE) against corneal cells in vitro. Chinese hamsters orally immunized with MIP-133 displayed a >30% reduction in disease. Immunoglobulin A isolated from immunized animals bound MIP-133 and blocked CPE on corneal cells in vitro. Animals induced to generate severe chronic infections displayed significant reductions in disease symptoms upon oral immunization postinfection. These data suggest that MIP-133 production might be necessary to initiate corneal disease and that it may play an important role in the subsequent steps of the pathogenic cascade of Acanthamoeba keratitis. Furthermore, as antibodies produced both prior to and after infection reduced clinical symptoms of disease, the protein may represent an important immunotherapeutic target for Acanthamoeba keratitis.

Our findings suggest that the RNA nuclear foci are pathognomonic for CTG18.1 expansion-mediated endothelial disease. The RNA nuclear foci have been previously found only in rare neurodegenerative disorders caused by repeat expansions. Our detection of abundant ribonuclear foci in FECD implicates a role for toxic RNA in this common disease.

The purpose of the present study was to investigate whether functional 20S and/or 26S proteasomes are present within mature human red blood cells (RBCs; depleted of reticulocytes and leukocytes). Double-immunofluorescence confocal microscopy showed the presence of immunoreactive 20S and 19S proteasomal subunit proteins and their partial co-localization within mature RBCs. Proteasomes isolated from mature RBCs displayed 20S activity in vitro; atomic-force and transmission electron microscopy of isolated proteasomes revealed abundant 20S core particles and very few 26S particles. A two-dimensional differential in-gel electrophoresis (2D-DIGE) approach was used to determine if proteasome-dependent protein degradation occurs within mature RBCs. Twenty-eight proteins were identified with altered protein content in response to lactacystin. Seven cytosolic proteins showed an increase and 16 showed a decrease; five membrane proteins showed a decrease. We conclude that the proteins showing increased abundance are either primary or secondary targets of the 20S proteasome and that putatively degraded proteins are secondary targets. Therefore, functional 20S proteasomes exist within mature RBCs. Our study did not detect 26S proteasome activity using the 2D-DIGE approach.

The ocular surface is continuously exposed to potential pathogens, including free-living amoebae. Acanthamoeba species are among the most ubiquitous amoebae, yet Acanthamoeba keratitis is remarkably rare. The pathogenesis of Acanthamoeba keratitis is a complex, sequential process. Here we show that Acanthamoeba keratitis is profoundly affected by mannosylated proteins on the ocular surface, which stimulate the amoebae to elaborate a 133-kDa pathogenic protease. The mannose-induced protease (MIP133) mediates apoptosis of the corneal epithelium, facilitates corneal invasion, and degrades the corneal stroma. We show that contact lens wear upregulates mannosylated proteins on the corneal epithelium, stimulates MIP133 secretion, and exacerbates corneal disease. Corynebacterium xerosis, a constituent of the ocular flora, contains large amounts of mannose and is associated with Acanthamoeba keratitis. The present results show that amoebae exposed to C. xerosis produce increased amounts of MIP133 and more severe corneal disease. Oral immunization with MIP133 mitigates Acanthamoeba keratitis and demonstrates the feasibility of antidisease vaccines for pathogens that resist immune elimination.Acanthamoeba keratitis is a rare but potentially blinding infection of the cornea that is caused by free-living amoebae belonging to the genus Acanthamoeba. Acanthamoeba spp. are ubiquitous organisms that can be isolated from a wide variety of environments, including public water supplies, swimming pools, freshwater reservoirs, salt water, hot tubs, ventilation ducts, soil, bottled water, and even eyewash stations (2, 18). The disease is closely associated with contact lens wear, which appears to be an important risk factor in infection. Previous studies showed that more than 80% of the cases of Acanthamoeba keratitis occurred in contact lens wearers (11,22). Acanthamoeba spp. have been isolated from the contact lens cases of Acanthamoeba keratitis patients, and it is widely believed that contact lenses serve as vectors for transmitting infectious Acanthamoeba trophozoites to the eye. However, contact lenses can stimulate the expression of glycoproteins on the corneal epithelium (12). This in turn might exacerbate the infectious process, as mannosylated proteins promote the binding of Acanthamoeba trophozoites to the corneal epithelium via a mannose-binding protein (mannose receptor) that is expressed on the Acanthamoeba cell membrane. However, adhesion can be inhibited by the addition of free methyl-␣-D-mannopyranoside (4, 25). Although engagement of the mannose receptor blocks adhesion, it does not prevent Acanthamoebamediated cytolysis of corneal cells (8,9,14). We have recently shown that methyl-␣-D-mannopyranoside stimulates Acanthamoeba trophozoites to elaborate a 133-kDa protease, designated MIP133, which is produced by pathogenic strains of Acanthamoeba spp. (5) and mediates cell contact-independent apoptosis of corneal epithelial cells and degradation of the collagenous matrix that forms the corneal stroma (8, 9).In addition...

PurposeSevering corneal nerves during corneal transplantation does not affect first corneal transplants, but abolishes immune privilege of subsequent corneal allografts. This abrogation of immune privilege is attributable to the disabling of T regulatory cells (T regs) induced by corneal transplantation. The goal of this study was to determine if severing corneal nerves induces the development of contrasuppressor (CS) cells, which disable T regs that impair other forms of immune tolerance.MethodsEffect of corneal nerve ablation on immune tolerance was assessed in four forms of immune tolerance: anterior chamber–associated immune deviation (ACAID); oral tolerance; corneal transplantation, and intravenously (IV) induced immune tolerance. T regulatory cell activity was assessed by adoptive transfer and by local adoptive transfer (LAT) of suppression assays.ResultsCorneal nerve ablation prevented ACAID and oral tolerance, but did not affect IV-induced immune tolerance. Contrasuppressor cells blocked the action of T regs that were generated by anterior chamber injection, oral tolerance, or orthotopic corneal transplantation. The neuropeptide substance P (SP) was crucial for contrasuppressor activity as CS cells could not be induced in SP−/− mice and the SP receptor inhibitor, Spantide II, prevented the expression of CS cell activity in vivo. Contrasuppressor cells expressed CD11c surface marker that identifies dendritic cells (DC).ConclusionsThe loss of immune privilege produced by corneal nerve ablation following corneal transplantation extends beyond the eye and also affects immune tolerance induced through mucosal surfaces and appears to be mediated by a novel cell population of CD11c+ CS cells that disables T regs.

Promoting the expansion of adult stem cell populations offers the potential to ameliorate radiation or chemotherapy-induced bone marrow failure and allows for expedited recovery for patients undergoing these therapies. Previous genetic studies suggested a pivotal role for SH2 domain-containing inositol-5-phosphatase 1 (SHIP1) in limiting the size of the hematopoietic stem cell (HSC) compartment. The aim of this study was to determine whether our recent development of small molecule SHIP1 inhibitors offers the potential for pharmacological expansion of the HSC compartment in vivo. We show here that treatment of mice with aminosteroid inhibitors of SHIP1 (SHIPi) more than doubles the size of the adult mesenchymal stem cell (MSC) compartment while simultaneously expanding the HSC pool sixfold. Consistent with its ability to target SHIP1 function in vivo, SHIPi also significantly increases plasma granulocyte colony-stimulating factor (G-CSF) levels, a growth factor that supports proliferation of HSC. Here, we show that SHIPi-induced G-CSF production mediates HSC and MSC expansion, as in vivo neutralization of G-CSF abrogates the SHIPi-induced expansion of both the HSC and MSC compartments. Due to its expansionary effect on adult stem cell compartments, SHIPi represents a potential novel strategy to improve declining stem cell function in both therapy induced and genetically derived bone marrow failure syndromes. STEM CELLS 2015;33:848-858

These data suggest that once trophozoites invade the cornea, MIP-133 production is necessary to initiate corneal disease and plays an important role in the subsequent steps of the pathogenic cascade of Acanthamoeba keratitis.