Sudha Neelam

Pathogenic Acanthamoeba spp. Secrete a Mannose-Induced Cytolytic Protein That Correlates with the Ability To Cause Disease

ABSTRACT 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-α-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.

Role of Contact Lens Wear, Bacterial Flora, and Mannose-Induced Pathogenic Protease in the Pathogenesis of Amoebic Keratitis

ABSTRACT 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.

Oral immunization with Acanthamoeba castellanii mannose-binding protein ameliorates amoebic keratitis

ABSTRACT Acanthamoeba castellanii mannose-binding protein (MBP) mediates adhesion of the amoebae to corneal epithelial cells, a key first step in the pathogenesis of Acanthamoeba keratitis (AK), a devastating corneal infection. In the present study, we demonstrate that oral immunization with recombinant MBP ameliorates AK in a hamster animal model and that this protection is associated with the presence of elevated levels of anti-MBP immunoglobulin A in the tear fluid of the immunized animals.

Modulation of corneal and stromal matrix metalloproteinase by the mannose-induced Acanthamoeba cytolytic protein

The involvement of the mannose-induced Acanthamoeba cytopathic protein (MIP-133) in tissue injury and activation of metalloproteinase of corneal and stromal cells was examined in vitro. Activation of MMP-1, MMP-2, MMP-3, and MMP-9 induced by MIP-133 on human corneal epithelial and stromal cell cultures was examined by reverse transcriptase polymerase chain reaction (RT-PCR) and ELISA. MMP-1, MMP-2, MMP-3, and MMP-9 mRNA were expressed in both cultured human corneal epithelial and stromal cells. When the epithelial cells were exposed to MIP-133 protein, the mRNA expression for MMP-1 and MMP-9 was unchanged. However, the transcript for MMP-2 and MMP-3 was decreased by 2-fold. By contrast, the expression of MMP-2 and MMP-3 was significantly upregulated (2- to 4-fold) in the corneal stromal cells 1, 4, and 8h after MIP-133 stimulation. At the protein level, there was no significant difference in the level of MMPs between the corneal epithelial cells before and after stimulation with MIP-133. By contrast, the levels of MMP-2 and MMP-3 were significantly higher in the corneal stromal cells stimulated with MIP-133. The supernatants from corneal stromal cells stimulated with MIP-133 were incubated with PMSF and MIP-133 antibody and the level of MMP-2 was measured by ELISA. Activation of MMP-2 by MIP-133 was inhibited in the supernatants pretreated with the serine protease inhibitor, PMSF, and anti-MIP-133. Supernatants pretreated with the cysteine protease inhibitor E6 or control antibody produced the same amount of MMP-2 as the untreated supernatants. To verify possible homology between MMPs and Acanthamoeba castellanii proteases, the mRNA from A. castellanii was prepared and analyzed for the expression of MMP genes by PT-PCR. The results showed that A. castellanii did not express mRNA for MMP-1, MMP-2, MMP-3, or MMP-9. Thus, A. castellanii mRNA does not cross-react with human MMPs. Furthermore, ELISA was used to determine the cross-reactivity of MMP antibodies with the MIP-133 protein. Monoclonal antibodies against MMPs did not cross-react with either the MIP-133 protein or BSA (negative control antigen). The results indicate that the MIP-133 protein modulates MMP-2 and -3 expression differently in human corneal epithelial and stromal cells.

Amoebicidal Activities of Alexidine Against 3 Pathogenic Strains of Acanthamoeba

Objectives: Effective pharmacotherapy for Acanthamoeba keratitis has been hampered because of the marked resistance of various stains to a variety of antimicrobial agents. In view of the fact that topical Brolene (propamidine isethionate) and neosporin are currently considered to be the first-line medical treatment of choice in Europe, we sought to determine whether Alexidine is equally effective, because the latter drug is more readily available in the United States. Methods: Trophozoites and cysts from 3 pathogenic corneal isolates (A. castellanii, A. polyphaga, and A. rhysodes) were incubated in peptone-yeast extract-glucose medium containing different concentrations of Alexidine for 24 hr. The number of trophozoites was counted by hemocytometer. The cysts were plated in to nonnutrient agar plates precoated with Escherichia coli and observed for viability or excystment over a period of 2 weeks. The capacity of different concentrations of Alexidine to induce cytolysis of corneal epithelial cells was tested in vitro. Chinese hamster corneas were treated with 5 &mgr;L of Alexidine topically, every hour; 6 times a day and the corneas were stained with fluorescein to asses the epithelial defects in vivo. Results: Alexidine was effective in killing the trophozoites at a concentration of 10 &mgr;g/mL. However, a higher concentration of Alexidine (100 &mgr;g/mL) is required to kill Acanthamoeba cysts and the cytotoxic activities of Alexidine are comparable with chlorhexidine. We have also demonstrated that both Alexidine and chlorhexidine at 100 &mgr;g/mL induced significant cytopathic effect on the corneal epithelial cells in vitro. In vivo results indicate that Alexidine at a concentration of 100 &mgr;g/mL is less toxic than chlorhexidine when applied topically to the Chinese hamster cornea. Conclusions: Our study has identified Alexidine as a novel anti-Acanthamoeba drug and suggests that Alexidine may be an effective therapeutic option because of its potency and low toxicity to the corneal tissues when applied topically in vivo.

ROLE OF ACTIVATED MACROPHAGES IN ACANTHAMOEBA KERATITIS

The purpose of this study was to determine whether activating the conjunctival macrophages would affect the course of Acanthamoeba spp. keratitis in a Chinese hamster model of this disease. Chinese hamster spleen cells were stimulated with concanavalin A (Con A), and interferon gamma (IFN-γ) –containing supernatants were collected 24 hr later. The IFN-γ–containing supernatants were loaded into liposomes, which were fed to peritoneal macrophages in vitro. Macrophage activation was assessed by testing for production of nitric oxide (NO) with the use of Griess reagent. Conjunctival macrophages were activated in situ by subconjunctival injection of liposomes containing Con A–activated spleen cell culture supernatants. Control liposomes were loaded with phosphate-buffered saline (PBS). Macrophages exposed to supernatants from Con A–stimulated spleen cells produced 4-fold-higher amounts of NO than unstimulated macrophages. Activation of macrophages via subconjunctival injection of liposomes containing supernatants from Con A–stimulated spleen cell cultures resulted in rapid resolution of the corneal infection. Approximately 80% of animals treated with PBS-containing liposomes demonstrated evidence of corneal disease at day 14 compared to 10% incidence of infection in the Con A–treated group. Moreover, at all time points examined, the clinical appearance of the keratitis in animals treated with liposomes containing Con A supernatant was significantly reduced compared to the group treated with liposomes containing PBS (P < 0.05). Macrophages stimulated with IFN-γ–containing supernatants killed significant numbers of the trophozoites in vitro (P < 0.05). Killing was inhibited by cytochalasin d, but not by l-N6-1-iminoethyl-l-lysine dihydrochloride (l-NIL), which is a selective inhibitor of inducible NO synthase (INOS).

Lenticular cytoprotection. Part 1: the role of hypoxia inducible factors-1α and -2α and vascular endothelial growth factor in lens epithelial cell survival in hypoxia.

Two new phenolic compounds, epicaesalpin J and 7,10,11-trihydroxydracaenone, were isolated from the heartwood of Caesalpinia sappan L. Their structures were identified by spectroscopic analysis methods, such as 1D and 2D NMR, along with the high resolution mass spectral data. The NO inhibition activities of two new compounds and six known compounds were tested.

Effect of Corneal Nerve Ablation on Immune Tolerance Induced by Corneal Allografts, Oral Immunization, or Anterior Chamber Injection of Antigens

Purpose Severing 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. Methods Effect 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. Results Corneal 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). Conclusions The 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.

Induction of Contrasuppressor Cells and Loss of Immune Privilege Produced by Corneal Nerve Ablation

Purpose Severing of corneal nerves in preparation of corneal transplantation abolishes immune privilege of subsequent corneal transplants placed into either eye: a phenomenon termed sympathetic loss of immune privilege (SLIP). SLIP is due to the disabling of T regulatory cells (Tregs) by CD11c+ contrasuppressor (CS) cells. This study characterized the induction, function, and manipulation of CS cell activity and the effect of these cells on Tregs induced by anterior chamber-associated immune deviation (ACAID). Methods CS cells were induced using a 2.0-mm trephine to score the corneal epithelium. CD11c+ CS cells were evaluated by adoptive transfer and by their capacity to disable CD8+ ACAID Tregs in local adoptive transfer (LAT) of suppression assays. CD11c+ cells were deleted from the ocular surface by subconjunctival injection of clodronate-containing liposomes. Results CD11c+ CS cell were radiosenstive and long lived. As few as 1000 CS cells blocked the suppressive activity of previously generated CD8+ ACAID Tregs, indicating that CS cells act at the efferent arm of the immune response. Depletion of resident CD11c+ cells at the ocular surface prevented the generation of CS cells. Conclusions Corneal nerve injury that occurs during keratoplasty converts ocular surface CD11c+ cells into CS cells that block CD8+ Tregs, which are induced by introducing antigens into the anterior chamber (i.e., ACAID Tregs). Depletion of CD11c+ cells at the ocular surface prevents the generation of CS cells and may be a useful strategy for preventing SLIP and enhancing the survival of second corneal transplants.

Genetics of Corneal Endothelial Dystrophies: An Asian Perspective

Corneal endothelial dystrophies are a group of disorders marked by dysfunction and loss of the corneal endothelial cells. The three primary endothelial dystrophies are congenital hereditary endothelial dystrophy, posterior polymorphous corneal dystrophy, and Fuchs’ endothelial corneal dystrophy. Recent advances in human genetics have led to the discovery of several genes associated with these endothelial dystrophies. In this chapter, we review these corneal endothelial dystrophies with specific emphasis on the molecular genetic studies performed on subjects from Asia.