Effect of Human Tears on Acanthamoeba-Induced Cytopathic Effect

Abstract

To determine whether tears of healthy individuals provide protection against Acanthamoebainduced cytopathic effect (CPE) in vitro.Methods: Acanthamoebae were added to confluent cultures of corneal epithelium in 24-well plates, and co-cultures were incubated overnight in a serum-free medium containing varying amounts of tears or immunoglobulin A (IgA)-depleted tears. At the end of the incubation period, the cells were stained with Giemsa, and the extent of target cell damage (ie, CPE) was quantified.Results: Acanthamoebae produced extensive CPE. The presence of even a low concentration of tears (10 µL of undiluted tears per milliliter of media) almost completely inhibited Acanthamoeba-induced CPE. The CPE was inhibited by pretreatment of the parasites with tears.In contrast, the pretreatment of host cells with tears was not protective. This finding suggests that the target of the inhibitory factor is the parasite. IgA-depleted tears also inhibited Acanthamoeba-induced CPE, albeit with a lower potency than total tears. Conclusion:In addition to known IgA-dependent protective factors, human tears contain factors that inhibit Acanthamoeba-induced CPE independently of IgA.Clinical Relevance: Identification and characterization of factors that protect against Acanthamoebainduced CPE should help in the development of novel, rationally designed strategies to manage and protect against keratitis.

Pathogenesis of Acanthamoeba Keratitis

“…We, therefore, tested IgA-depleted human tear fluid for its ability to inhibit Acanthamoeba -induced CPE. We demonstrated that IgA-depleted tears inhibit the amoeba-induced CPE, but unfractionated complete tears are three times more potent in inhibiting the amoeba-induced CPE than the IgA-depleted tears 60. Additional studies using breast milk as a model of human mucosal secretions revealed that the IgA-depleted milk also inhibited the Acanthamoeba -induced CPE 61.…”

Pathogenesis of Acanthamoeba Keratitis

“…We, therefore, tested IgA-depleted human tear fluid for its ability to inhibit Acanthamoeba -induced CPE. We demonstrated that IgA-depleted tears inhibit the amoeba-induced CPE, but unfractionated complete tears are three times more potent in inhibiting the amoeba-induced CPE than the IgA-depleted tears 60. Additional studies using breast milk as a model of human mucosal secretions revealed that the IgA-depleted milk also inhibited the Acanthamoeba -induced CPE 61.…”

Milk Components InhibitAcanthamoeba-Induced Cytopathic Effect

“…Thus far, secretory IgA (sIgA) antibody is the only recognized component that is widely thought to account for the protection afforded by mucosal secretions. A recent study 26 in our laboratory revealed that IgA-depleted tears of healthy persons also inhibit Acanthamoeba-induced CPE, albeit with a lower potency than total tears. In that study, because of the limited availability of tears, it was not possible to characterize the mechanism by which the IgA-depleted tears inhibit the ameba-induced CPE.…”

“…[23][24][25] In the present study, using milk as a model, we performed studies to determine whether human mucosal secretions have the potential to provide protection against Acanthamoeba-induced CPE by an additional mechanism that is independent of IgA. We demonstrated here that, as do IgA-depleted tears, 26 IgA-depleted milk has the capacity to inhibit Acanthamoeba-induced CPE. Our findings that proteinase K treatment abolished the CPE inhibitory activity of milk whereas heating the milk (100°C, 5 minutes) did not destroy the CPE inhibitory activity suggested that the CPE inhibitory factor of milk is composed of one or more heat-resistant protein moieties.…”

Taurine, a Component of the Tear Film, Exacerbates the Pathogenic Mechanisms of Acanthamoeba castellanii in the Ex Vivo Amoebic Keratitis Model

“…Moreover, faced with corneal invasion by Acanthamoeba or any other pathogen that attempts to colonize the ocular surface, there are host defense mechanisms in which the first line of defense between the external environment and the ocular surface is the tear film [ 10 ]. The lgA is the major immunoglobulin isotype in normal tears constituting a frontline defense against microorganisms [ 11 , 12 ]. Several studies have suggested that the mucosal immune system provides protection against Acanthamoeba keratitis [ 13 , 14 ].…”

Pathogenesis of Acanthamoeba Keratitis

“…We, therefore, tested IgA-depleted human tear fluid for its ability to inhibit Acanthamoeba -induced CPE. We demonstrated that IgA-depleted tears inhibit the amoeba-induced CPE, but unfractionated complete tears are three times more potent in inhibiting the amoeba-induced CPE than the IgA-depleted tears 60. Additional studies using breast milk as a model of human mucosal secretions revealed that the IgA-depleted milk also inhibited the Acanthamoeba -induced CPE 61.…”

Milk Components InhibitAcanthamoeba-Induced Cytopathic Effect

“…Thus far, secretory IgA (sIgA) antibody is the only recognized component that is widely thought to account for the protection afforded by mucosal secretions. A recent study 26 in our laboratory revealed that IgA-depleted tears of healthy persons also inhibit Acanthamoeba-induced CPE, albeit with a lower potency than total tears. In that study, because of the limited availability of tears, it was not possible to characterize the mechanism by which the IgA-depleted tears inhibit the ameba-induced CPE.…”

“…[23][24][25] In the present study, using milk as a model, we performed studies to determine whether human mucosal secretions have the potential to provide protection against Acanthamoeba-induced CPE by an additional mechanism that is independent of IgA. We demonstrated here that, as do IgA-depleted tears, 26 IgA-depleted milk has the capacity to inhibit Acanthamoeba-induced CPE. Our findings that proteinase K treatment abolished the CPE inhibitory activity of milk whereas heating the milk (100°C, 5 minutes) did not destroy the CPE inhibitory activity suggested that the CPE inhibitory factor of milk is composed of one or more heat-resistant protein moieties.…”

Taurine, a Component of the Tear Film, Exacerbates the Pathogenic Mechanisms of Acanthamoeba castellanii in the Ex Vivo Amoebic Keratitis Model

“…Moreover, faced with corneal invasion by Acanthamoeba or any other pathogen that attempts to colonize the ocular surface, there are host defense mechanisms in which the first line of defense between the external environment and the ocular surface is the tear film [ 10 ]. The lgA is the major immunoglobulin isotype in normal tears constituting a frontline defense against microorganisms [ 11 , 12 ]. Several studies have suggested that the mucosal immune system provides protection against Acanthamoeba keratitis [ 13 , 14 ].…”

Pathogenesis of Acanthamoeba Keratitis

“…We, therefore, tested IgA-depleted human tear fluid for its ability to inhibit Acanthamoeba -induced CPE. We demonstrated that IgA-depleted tears inhibit the amoeba-induced CPE, but unfractionated complete tears are three times more potent in inhibiting the amoeba-induced CPE than the IgA-depleted tears 60. Additional studies using breast milk as a model of human mucosal secretions revealed that the IgA-depleted milk also inhibited the Acanthamoeba -induced CPE 61.…”

Milk Components InhibitAcanthamoeba-Induced Cytopathic Effect

“…Thus far, secretory IgA (sIgA) antibody is the only recognized component that is widely thought to account for the protection afforded by mucosal secretions. A recent study 26 in our laboratory revealed that IgA-depleted tears of healthy persons also inhibit Acanthamoeba-induced CPE, albeit with a lower potency than total tears. In that study, because of the limited availability of tears, it was not possible to characterize the mechanism by which the IgA-depleted tears inhibit the ameba-induced CPE.…”

“…[23][24][25] In the present study, using milk as a model, we performed studies to determine whether human mucosal secretions have the potential to provide protection against Acanthamoeba-induced CPE by an additional mechanism that is independent of IgA. We demonstrated here that, as do IgA-depleted tears, 26 IgA-depleted milk has the capacity to inhibit Acanthamoeba-induced CPE. Our findings that proteinase K treatment abolished the CPE inhibitory activity of milk whereas heating the milk (100°C, 5 minutes) did not destroy the CPE inhibitory activity suggested that the CPE inhibitory factor of milk is composed of one or more heat-resistant protein moieties.…”

Taurine, a Component of the Tear Film, Exacerbates the Pathogenic Mechanisms of Acanthamoeba castellanii in the Ex Vivo Amoebic Keratitis Model

“…Moreover, faced with corneal invasion by Acanthamoeba or any other pathogen that attempts to colonize the ocular surface, there are host defense mechanisms in which the first line of defense between the external environment and the ocular surface is the tear film [ 10 ]. The lgA is the major immunoglobulin isotype in normal tears constituting a frontline defense against microorganisms [ 11 , 12 ]. Several studies have suggested that the mucosal immune system provides protection against Acanthamoeba keratitis [ 13 , 14 ].…”