March 2008
Volume 49, Issue 3
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Immunology and Microbiology  |   March 2008
Milk Components Inhibit Acanthamoeba-Induced Cytopathic Effect
Author Affiliations
  • Chandrassegar Saravanan
    From the Department of Anatomy and Cell Biology, Sackler School of Graduate Biomedical Sciences,
    Department of Ophthalmology, Center for Vision Research and The New England Eye Center, Tufts University School of Medicine, Boston, Massachusetts;
  • Zhiyi Cao
    Department of Ophthalmology, Center for Vision Research and The New England Eye Center, Tufts University School of Medicine, Boston, Massachusetts;
  • Janardan Kumar
    Department of Ophthalmology, Center for Vision Research and The New England Eye Center, Tufts University School of Medicine, Boston, Massachusetts;
  • Jiazhou Qiu
    Tufts-New England Medical Center, Boston, Massachusetts; and
  • Andrew G. Plaut
    Tufts-New England Medical Center, Boston, Massachusetts; and
  • David S. Newburg
    Program in Glycobiology, Pediatric Gastroenterology and Nutrition, Massachusetts General Hospital, Charlestown, Massachusetts.
  • Noorjahan Panjwani
    From the Department of Anatomy and Cell Biology, Sackler School of Graduate Biomedical Sciences,
    Department of Ophthalmology, Center for Vision Research and The New England Eye Center, Tufts University School of Medicine, Boston, Massachusetts;
Investigative Ophthalmology & Visual Science March 2008, Vol.49, 1010-1015. doi:10.1167/iovs.07-1130
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      Chandrassegar Saravanan, Zhiyi Cao, Janardan Kumar, Jiazhou Qiu, Andrew G. Plaut, David S. Newburg, Noorjahan Panjwani; Milk Components Inhibit Acanthamoeba-Induced Cytopathic Effect. Invest. Ophthalmol. Vis. Sci. 2008;49(3):1010-1015. doi: 10.1167/iovs.07-1130.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

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.

Acanthamoebae provoke a debilitating, vision-threatening corneal infection known as Acanthamoeba keratitis (AK). 1 2 3 4 In the developed world, roughly 85% of the cases are diagnosed in contact lens wearers; thus, use of contact lenses is thought to be the leading risk factor. 5 6 7 Since the initial description of AK in 1973, many cases have been diagnosed, and, more recently, an increase in its incidence has been reported. 4 8 9 10 Occurrence of the disease is relatively low, however, considering that more than 24 million persons in the United States alone wear contact lenses and that the amebae are commonly distributed throughout the environment. The first step in the pathogenesis of AK is the adhesion of the parasite to the host cell. 11 12 13 14 15 Subsequent to the adhesion, parasites produce a potent cytopathic effect (CPE) that involves killing of the host cells, degradation of the epithelial basement membrane and the underlying stromal matrix, and penetration into the deeper layers of the cornea (for a review, see Clarke and Niederkorn 3 ). 
Why cornea is selectively at risk for Acanthamoeba infection is unclear. In vitro, pathogenic strains of Acanthamoebae adhere to and produce CPEs on host cells derived not only from cornea but also from a variety of nonocular tissues (e.g., kidney, brain, ovary, esophagus, and bone) 16 17 18 19 20 21 that, in healthy, immunocompetent persons, are resistant to infection by Acanthamoeba. This suggests that protective factors must be present in vivo, most notably in mucosal secretions such as tear fluid, saliva, and breast milk. In fact, normal human tears, saliva, and milk contain Acanthamoeba-specific IgA antibodies capable of inhibiting the adhesion of the parasites to host cells 22 23 24 25 (Panjwani et al., unpublished observations, 2006), a key first step in the pathogenesis of infection. 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. Clearly, in-depth studies requiring large quantities of starting material for characterization of the CPE-inhibiting factors will have to rely on mucosal secretions such as milk, which is clean, uncontaminated, and readily available in large quantities. Ocular tears and breast milk share many components, and, indeed, important lessons about the protective role of tear fluid have been learned from in-depth investigations on milk. In the present study, using milk as a model, we demonstrated 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. 
Materials and Methods
Parasites and Epithelial Cells
An Acanthamoeba castellanii strain derived from an infected human cornea (MEEI 0184) was used throughout this study. The amebae were axenically cultured. 27 Immortalized rabbit corneal epithelial cells were used as host cells. 13  
Breast Milk Samples
Human milk samples from 40 healthy lactating mothers at all stages of lactation were pooled. Their use for research was approved by Partners Human Research Committee (Massachusetts General Hospital, Boston, MA). Samples were placed on ice immediately after collection and were stored at −80°C. To remove lipids (cream) from the aqueous portion (skimmed milk), aliquots of pooled milk samples were centrifuged (1400g, 30 minutes, 4°C), and the creamy layer consisting largely of fat was removed by filtration through a glass wool plug in a Pasteur pipette. 
Preparation of IgA-Depleted Milk
A 10-mL aliquot of milk containing 121 mg protein was chromatographed on an anti–human IgA-Sepharose (Sigma, St. Louis, MO) column (bed volume, 1.5 mL). The unbound fraction eluted from the column and the unfractionated milk were studied for the presence of IgA by Western blot analysis using horseradish peroxidase-conjugated rabbit anti–human heavy chain IgA (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA). 
Analysis of the Effect of IgA-Depleted Milk on Acanthamoeba-Induced Cytopathic Effect
CPE assays were performed as described previously. 13 Briefly, the parasites (more than 95% trophozoites) were rinsed three times in a serum-free medium supplemented with 0.4% bovine serum albumin (SFB medium), and aliquots of the parasite suspension (2 × 105 parasites/mL; 300 μL/well of 24-well cell culture plates) were added to wells of confluent cultures of rabbit corneal epithelium that had been rinsed and preincubated in the SFB medium for 2 hours. The plates were then incubated at 37°C and periodically examined under a phase-contrast microscope for up to 18 hours to asses the CPE, as detected by the presence of cell-free plaques in the monolayer. At the end of the incubation period, the cells were stained with Giemsa (Diff-Quick; Dade Diagnostic Inc., Aguada, PR) and scanned in a digital imaging system (FluorChem 8800; Alpha Innotech Corp, San Leandro, CA), and the approximate cell density of each well was estimated with the use of software (ImageQuant; Molecular Dynamics, Sunnyvale, CA). To study the effect of milk on ameba-induced CPE, 300-μL aliquots of SFB media containing IgA-depleted milk (50–200 μg protein) were added to each well (four wells/group), and the cultures were incubated in a CO2 incubator. To determine whether the CPE inhibitory activity was heat resistant, the CPE assays were performed using milk that had been heated for 5 minutes at 100°C. To determine whether the CPE inhibitory components were proteins, the milk proteins were incubated with proteinase K (Invitrogen, Carlsbad, CA; 2 μL proteinase K [20 mg/mL]/100 μL of milk [8 mg/mL]; 65°C, overnight). At the end of the incubation period, the enzyme was inactivated by boiling at 100°C for 5 minutes. Aliquots of digested and control samples (incubated with the enzyme buffer) were run on SDS polyacrylamide gels in reducing conditions and stained with Coomassie blue to confirm that the milk proteins were digested. The digests were subsequently tested for their ability to influence the ameba-induced CPE. In some experiments, CPE assays were performed in the presence of the commercially available purified milk components lactoferrin, α-lactalbumin, and casein (up to 100 μg/well; Sigma, St. Louis, MO). 
To investigate whether the CPE inhibitory activity in milk exists in fractions of molecular weight greater or less than 100 kDa, 1 mL milk was filtered through tubes (Centricon YM-100; Millipore, Bedford, MA) by centrifugation (1000g, 4°C). The filtrate (<100 kDa) and retentate (≥100 kDa) fractions obtained after centrifugation were collected and tested for CPE inhibitory activity using the procedure described. To determine whether the CPE inhibitory activity of IgA-depleted milk was localized in a specific fraction isolated based on molecular weight, the IgA-depleted milk (38 mg protein) was chromatographed on a gel filtration (Sephadex G200; GE Healthcare, Piscataway, NJ) column (1 cm × 130 cm), fractions of 1 mL were collected, and effluent was monitored by reading each fraction at 280 nm. Fractions comprising peaks were pooled and tested for the presence of the CPE inhibitory activity. Protein profiles of the pooled fraction exhibiting the CPE inhibitory activity were further analyzed by two-dimensional gel electrophoresis using pI 3–10 IPG strips (GE Healthcare) for the first dimension and SDS-polyacrylamide gradient gels (4%–20%) for the second dimension. 
Analysis of the Effect of Milk Proteins on Acanthamoeba Proteinases by Zymography
Conditioned media obtained by incubating the amebae with cultures of corneal epithelium for 16 to 18 hours was harvested. Either 10 μL of the elution buffer used for gel filtration or 10 μL each (adjusted to contain 27 μg protein) of various milk fractions obtained by gel filtration was added to 25-μL aliquots of the coculture media. The samples were incubated for 1 hour at 37°C and were then analyzed by zymography for the presence of proteases. Zymography was performed using separating gels containing 10% acrylamide and 2 mg/mL gelatin, essentially as described by Kleiner et al. 28 Samples to be analyzed were diluted in the electrophoresis sample buffer 29 without mercaptoethanol and were then applied to the gels. After electrophoresis, the proteinases separated on gels were renatured by incubating the gels in 2.5% Triton X-100 in 50 mM Tris-HCl, pH 7.5 to remove SDS. After this, the gels were incubated for 18 hours in a developing buffer (50 mM Tris-HCl, pH 7.5, containing 10 mM CaCl2) and were then stained with Coomassie blue. Areas of digestion were visualized as nonstaining regions of the gel. 
Results
IgA-Depleted Milk Inhibits Acanthamoeba-Induced CPE
To determine whether one or more protective factors distinct from sIgA are present in human milk, experiments were performed to investigate whether the IgA-depleted milk had the ability to inhibit the ameba-induced CPE. To remove IgA from human milk, aliquots of milk samples were chromatographed on an anti–human IgA-conjugated agarose column. The unbound fraction eluted from the column and the unfractionated milk were analyzed by Western blot for the presence of IgA. In unfractionated milk, a 56-kDa anti–IgA reactive component was detected (Fig. 1A , right panel, lane M). In contrast, the unbound fraction did not contain detectable levels of IgA (Fig. 1A , right panel, lane UB). The unbound fraction lacking the IgA was subsequently tested for its ability to influence the ameba-induced CPE. 
To test the effect of milk components on ameba-induced CPE, Acanthamoebae were incubated with monolayer cultures of corneal epithelial cells in the presence and the absence of the IgA-depleted milk. As expected, 13 Acanthamoebae produced extensive CPE on corneal epithelial cells. Within 4 to 6 hours of incubation with the parasites, small cell-free plaques were seen in the monolayer (Fig. 1B , 4 hours). With continued incubation with the amebae, the size of the cell-free areas increased (Fig. 1B , 8 hours), and eventually the monolayer surrounding the large plaques lifted entirely from the culture plates, resulting in almost complete loss of the cell layer (Fig. 1B , 16 hours). The IgA-depleted milk inhibited the Acanthamoeba-induced CPE in a concentration-dependent manner, with nearly complete inhibition at 150 μg protein/well concentration or greater (Fig. 1C) . Several major milk components, including lactoferrin, α-lactalbumin, and casein, did not exhibit CPE inhibitory activity, even when tested at high concentrations (50–100 μg/well; not shown). 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 (data not shown). 
To investigate whether the CPE inhibitory factor in milk exists in fractions of molecular weight greater or less than 100 kDa, filtrate and retentate obtained after filtering skimmed milk in tubes (Centricon Y-100; Amicon, Bedford, MA) were tested for their ability to inhibit the ameba-induced CPE. Almost all the CPE-inhibitory activity was recovered in the retentate obtained after filtration (Centricon Y-100; Amicon), whereas the filtrate lacked CPE inhibitory activity (not shown). This suggested that the molecular weight of the CPE inhibitory factor was 100 kDa or greater. On gel filtration, IgA-depleted milk proteins were separated into four major fractions (F1-F4; Fig. 2A ) and one minor fraction (F5; Fig. 2A ). Percentages of total protein eluted in each fraction were as follows: F1, 9.5%; F2, 23.4%; F3, 33%; F4, 33%; F5, 0.4%. Analysis of various milk fractions on nondenaturing polyacrylamide gels confirmed that gel filtration effectively separated milk proteins based on molecular weight (Fig. 2B) . Various milk fractions eluted from the gel filtration column were tested for their ability to influence the ameba-induced CPE. Of the four fractions (F1-F4) tested, CPE inhibitory activity was detected largely in fraction F3 (Fig. 3A) . In contrast, fractions F1, F2, and F4 exhibited weak or no CPE inhibitory activity. The CPE inhibitory activity of fraction F5 was not tested because of an insufficient yield of proteins. CPE assays performed in the presence and the absence of varying concentrations (3–66 μg protein/well) of IgA-depleted milk in fraction F3 revealed that compared with unfractionated IgA-depleted milk, CPE inhibitory activity was enriched 10-fold in this fraction (Fig. 3B) . Analysis by two-dimensional PAGE revealed that fraction F3 contained numerous proteins (not shown). 
Milk Components Inhibit Amebic Proteases
To determine the effect of milk components on the activity of amebic proteinases, conditioned media obtained after amebae were incubated with the host cells were incubated with various milk fractions and were then analyzed for expression levels of proteinases by zymography on gelatin gels. After overnight incubation, two components (∼98 kDa and ∼55 kDa) were detected in the samples obtained after amebae were incubated with cultures of corneal epithelium in media alone (Fig. 4 ; lane, E+A). Both these components are serine proteinases, as determined by susceptibility to phenylmethylsulfonyl fluoride (PMSF). 13 Of particular significance is our finding that the 98-kDa component was not detected in cocultures incubated with milk or fraction F3, which provided protection against ameba-induced CPE (Fig. 4 , lanes M and F3). In contrast, in cocultures incubated with factions F1, F2, and F4, which did not provide protection against CPE, a significant level of the 98-kDa component was detected (Fig. 4 , lanes F1, F2, F4). In addition, compared with the control coculture medium, the coculture medium incubated with milk and fraction F3 contained reduced levels of the 55-kDa component (Fig. 4) . When the cocultures were incubated for a shorter period (∼6 hours), two additional components (230 and 80 kDa) were also detected in addition to the 98-kDa and the 55-kDa components (not shown). However, these two components were unstable and degraded quickly. Therefore, it was not possible to determine in a reproducible manner whether they too were inhibited by milk or by fraction F3. The molecular weights of various proteinases reported here are rough estimates. It is known that molecular masses cannot be accurately determined in gelatin-containing SDS-PAGE gels because of a variety of reasons, including the sieving effect of gelatin and the affinity interactions between gelatin and the active site of enzyme. 30 Moreover, the apparent molecular mass on gelatin gels also depends on the amount of protease loaded on the gel. 31  
Discussion
It is thought that Acanthamoeba-specific IgA antibodies present in human mucosal secretions provide protection against Acanthamoeba infections by inhibiting the adhesion of parasites to host cells. 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. The putative inhibitory factor is likely to be 100 kDa or greater in molecular weight because the inhibitory activity was recovered in the retentate obtained after the filtration of milk through filtration tubes (Centricon YM-100; Millipore). Analysis of four fractions (F1–F4) separated by molecular sieve chromatography of IgA-depleted milk revealed that the CPE inhibitory activity was largely eluted in fraction F3, which contained components ranging in molecular weight up to approximately 500 kDa. 
Regarding the mechanism by which Acanthamoebae produce CPE, it is known that subsequent to the adhesion of parasites to host cells, Acanthamoebae secrete cytotoxic proteinases responsible for the killing of host cells and the degradation of the underlying extracellular matrix. 13 32 That the proteinases play an important role in the ameba-induced CPE has been further demonstrated by studies showing that PMSF, 13 33 34 a serine protease inhibitor, and Galardin (Cao and Panjwani, unpublished observations, 2002), a metalloproteinase inhibitor, almost completely block the ameba-induced CPE despite the adhesion of parasites to host cells. It was therefore of interest to determine whether IgA-depleted milk and fraction F3 have the capacity to inhibit the amebic proteinases. Zymography experiments revealed that the IgA-depleted milk and fraction F3 inhibited the activity of a 98-kDa amebic proteinase. In contrast, fractions F1, F2, and F4, which lacked CPE inhibitory activity, did not inhibit the 98-kDa amebic proteinase. These data, in conjunction with the published findings showing that amebic proteinases are responsible for the induction of Acanthamoeba CPE, 13 33 34 suggest that the CPE inhibitory activity of milk and fraction F3 observed in this study is, at least in part, caused by their ability to inhibit the activity of the cytotoxic amebic proteinases. Studies aimed at the characterization of protease inhibitors of milk have shown that the main protease inhibitors in human milk are α1-antitrypsin (MWt ∼50 kDa) and α1-antichymotrypsin (MWt 65 kDa). 35 36 37 In addition, two major proteins of milk, lactoferrin and β-casein, have been shown to inhibit cysteine protease inhibitors. 38 However, none of these molecules was likely to be responsible for the Acanthamoeba CPE inhibitory activity observed in this study because, as described, the CPE inhibitory factor was likely to have a molecular weight greater than 100 kDa, and neither lactoferrin nor casein was found to have CPE inhibitory activity. It is known that human milk also contains trace amounts of a number of other broad-specificity protease inhibitors, including α2-macroglobulin, α2-antiplasmin, and anti-thrombin III. 37 It remains to be determined whether the CPE inhibitory activity of milk and fraction F3 results from one of these inhibitors and whether human milk contains specific inhibitors against amebic proteinases. Given that fraction F3 contained numerous proteins as detected by two-dimensional gel electrophoresis, subtractive chromatographic fractionation methods guided by the presence of the CPE inhibitory activity are likely to be more rewarding in identifying and characterizing the putative CPE-inhibitory factors of milk. 
The presence of IgA-dependent and IgA-independent CPE inhibitory factors in tears helps us understand why the incidence of Acanthamoeba keratitis is low. In nonocular secretions, it helps explain, at least in part, why almost all nonocular tissues are resistant to Acanthamoeba infections in healthy persons. Characterization of the CPE inhibitory factors of human mucosal secretions such as milk, tears, and saliva should lead to a better understanding of the mechanism by which the tissues of the host resist the infection and also help decipher circumstances that predispose to Acanthamoeba infections. Whether the CPE inhibitory components present in milk and tears proves to be the same or different, their identification and characterization should help in the development of novel, rationally designed strategies to manage and protect against Acanthamoeba infections. 
 
Figure 1.
 
IgA-depleted human milk inhibits Acanthamoeba-induced CPE. (A) Preparation of IgA-depleted milk. An aliquot of milk containing 121 mg protein was chromatographed on an IgA-Sepharose column. Ten-microgram protein aliquots of the unbound fraction eluted from the affinity column and unfractionated milk were electrophoresed in SDS-polyacrylamide gels in reducing conditions. After electrophoresis, the proteins were transferred onto nitrocellulose membranes; the protein blots were stained with Ponceau S (left) and then processed for immunostaining with anti-IgA (right). Note that an intensely stained 56-kDa anti-IgA reactive component is present in the whole milk (M) but not in the unbound fraction (UB). (B) Acanthamoeba-induced CPE on host cells. Acanthamoebae (2 × 105 parasites/mL) were added to confluent cultures of corneal epithelium in 24-well plates, and the cultures were incubated in a CO2 incubator for varying periods. At the end of the incubation period, the plates were stained with Giemsa and photographed. Cont, epithelial cells incubated in media alone. Acanthamoebae, epithelial cells incubated with the parasites for varying periods of time. Clear, unstained regions indicate loss of epithelial cells (8 hours and 16 hours). Dark, stained areas indicate the presence of cells (Cont). (C) IgA-depleted human milk inhibits Acanthamoeba-induced CPE. Epithelial cells were incubated overnight with Acanthamoebae in the presence or the absence of varying concentrations (50–200 μg/well) of IgA-depleted milk. At the end of the incubation period, the monolayers were washed, stained, and scanned to estimate approximate cell density. A value of 1.0 was assigned to the cell density of the plates incubated in media alone without parasites (Cont). The values for cultures incubated with amebae in the presence and absence of milk are expressed as change in the density with respect to control plates. Photographs of the plates are shown in panel C(i); quantification of data is shown in panel C(ii). Note that CPE is inhibited by IgA-depleted milk in a concentration-dependent manner. Cont, corneal epithelial cells incubated with media alone. Group A, cultures incubated with amebae in serum-free media containing 0.4% BSA. A+IgA-depleted milk, cultures incubated with amebae in media containing varying concentration of IgA-depleted milk. Data are expressed as mean ± SE (N = 4 in each group). *P < 0.05 compared with group A and 50 μg/well groups.
Figure 1.
 
IgA-depleted human milk inhibits Acanthamoeba-induced CPE. (A) Preparation of IgA-depleted milk. An aliquot of milk containing 121 mg protein was chromatographed on an IgA-Sepharose column. Ten-microgram protein aliquots of the unbound fraction eluted from the affinity column and unfractionated milk were electrophoresed in SDS-polyacrylamide gels in reducing conditions. After electrophoresis, the proteins were transferred onto nitrocellulose membranes; the protein blots were stained with Ponceau S (left) and then processed for immunostaining with anti-IgA (right). Note that an intensely stained 56-kDa anti-IgA reactive component is present in the whole milk (M) but not in the unbound fraction (UB). (B) Acanthamoeba-induced CPE on host cells. Acanthamoebae (2 × 105 parasites/mL) were added to confluent cultures of corneal epithelium in 24-well plates, and the cultures were incubated in a CO2 incubator for varying periods. At the end of the incubation period, the plates were stained with Giemsa and photographed. Cont, epithelial cells incubated in media alone. Acanthamoebae, epithelial cells incubated with the parasites for varying periods of time. Clear, unstained regions indicate loss of epithelial cells (8 hours and 16 hours). Dark, stained areas indicate the presence of cells (Cont). (C) IgA-depleted human milk inhibits Acanthamoeba-induced CPE. Epithelial cells were incubated overnight with Acanthamoebae in the presence or the absence of varying concentrations (50–200 μg/well) of IgA-depleted milk. At the end of the incubation period, the monolayers were washed, stained, and scanned to estimate approximate cell density. A value of 1.0 was assigned to the cell density of the plates incubated in media alone without parasites (Cont). The values for cultures incubated with amebae in the presence and absence of milk are expressed as change in the density with respect to control plates. Photographs of the plates are shown in panel C(i); quantification of data is shown in panel C(ii). Note that CPE is inhibited by IgA-depleted milk in a concentration-dependent manner. Cont, corneal epithelial cells incubated with media alone. Group A, cultures incubated with amebae in serum-free media containing 0.4% BSA. A+IgA-depleted milk, cultures incubated with amebae in media containing varying concentration of IgA-depleted milk. Data are expressed as mean ± SE (N = 4 in each group). *P < 0.05 compared with group A and 50 μg/well groups.
Figure 2.
 
Fractionation of IgA-depleted milk by gel filtration. (A) An aliquot of IgA-depleted milk containing 38 mg protein was chromatographed on a Sephadex G200 column (1 cm × 130 cm), fractions of 1.0 mL were collected, effluent was monitored by reading each fraction at 280 nm, and fractions comprising peaks (F1-F5) were pooled. (B) Proteins eluted in various fractions were electrophoresed in nondenaturing/nonreducing 4% to 20% polyacrylamide gels and visualized by staining with Coomassie blue.
Figure 2.
 
Fractionation of IgA-depleted milk by gel filtration. (A) An aliquot of IgA-depleted milk containing 38 mg protein was chromatographed on a Sephadex G200 column (1 cm × 130 cm), fractions of 1.0 mL were collected, effluent was monitored by reading each fraction at 280 nm, and fractions comprising peaks (F1-F5) were pooled. (B) Proteins eluted in various fractions were electrophoresed in nondenaturing/nonreducing 4% to 20% polyacrylamide gels and visualized by staining with Coomassie blue.
Figure 3.
 
Acanthamoeba CPE inhibitory activity of IgA-depleted milk is enriched in fraction F3. (A) CPE assays were performed in the presence of 66 μg protein each of fractions F1 to F4. A value of 1.0 was assigned to the cell density of the plates incubated in media alone (Cont). Values for cultures incubated with amebae in the presence and the absence of various milk fractions are expressed as change in the density with respect to control plates. Top: photographs of the plates. Note that only fraction F3 inhibited the ameba-induced CPE. Cont, corneal cultures incubated with media alone. Group A cultures incubated with amebae in serum-free media containing 0.4% BSA. F1 to F4, cultures incubated with amebae in media containing 66 μg of each fraction obtained by gel filtration of IgA-depleted milk (see Fig. 2 ). Data are expressed as mean ± SE (N = 4 in each group). P < 0.05 compared with fractions F1, F2, and F4. (B) Acanthamoeba CPE inhibitory activity is enriched 10-fold in fraction F3. CPE assays were performed in the presence of varying concentrations (3–66 μg protein/well) of IgA-depleted milk and fraction F3. Results were calculated as described in (A). Note that fraction F3 inhibited CPE at a much lower concentration (6 μg/well) than unfractionated IgA-depleted milk (66 μg/well). Data are expressed as mean ± SE (N = 4 in each group). *P < 0.05 compared with all other groups in A + milk. **P < 0.05 compared with all other groups in A+F3 group.
Figure 3.
 
Acanthamoeba CPE inhibitory activity of IgA-depleted milk is enriched in fraction F3. (A) CPE assays were performed in the presence of 66 μg protein each of fractions F1 to F4. A value of 1.0 was assigned to the cell density of the plates incubated in media alone (Cont). Values for cultures incubated with amebae in the presence and the absence of various milk fractions are expressed as change in the density with respect to control plates. Top: photographs of the plates. Note that only fraction F3 inhibited the ameba-induced CPE. Cont, corneal cultures incubated with media alone. Group A cultures incubated with amebae in serum-free media containing 0.4% BSA. F1 to F4, cultures incubated with amebae in media containing 66 μg of each fraction obtained by gel filtration of IgA-depleted milk (see Fig. 2 ). Data are expressed as mean ± SE (N = 4 in each group). P < 0.05 compared with fractions F1, F2, and F4. (B) Acanthamoeba CPE inhibitory activity is enriched 10-fold in fraction F3. CPE assays were performed in the presence of varying concentrations (3–66 μg protein/well) of IgA-depleted milk and fraction F3. Results were calculated as described in (A). Note that fraction F3 inhibited CPE at a much lower concentration (6 μg/well) than unfractionated IgA-depleted milk (66 μg/well). Data are expressed as mean ± SE (N = 4 in each group). *P < 0.05 compared with all other groups in A + milk. **P < 0.05 compared with all other groups in A+F3 group.
Figure 4.
 
Fraction F3 but not fraction F1, F2, or F4 inhibited amebic proteinases. Conditioned media obtained after incubating amebae with the host cells were incubated with various milk fractions (27 μg protein each, 37°C, 1 hour) and were then analyzed for expression levels of proteinases by zymography on gelatin gels. Note that two major components (98 kDa, 55 kDa) were seen in conditioned media prepared by incubating Acanthamoebae with corneal epithelial cells (E+A) and that the 98-kDa component was not detected or was detected in trace amounts when unfractionated IgA-depleted milk (M) or fraction F3 was added to the coculture; in contrast, in cocultures incubated with factions F1, F2, and F4, significant levels of the 98-kDa component were detected.
Figure 4.
 
Fraction F3 but not fraction F1, F2, or F4 inhibited amebic proteinases. Conditioned media obtained after incubating amebae with the host cells were incubated with various milk fractions (27 μg protein each, 37°C, 1 hour) and were then analyzed for expression levels of proteinases by zymography on gelatin gels. Note that two major components (98 kDa, 55 kDa) were seen in conditioned media prepared by incubating Acanthamoebae with corneal epithelial cells (E+A) and that the 98-kDa component was not detected or was detected in trace amounts when unfractionated IgA-depleted milk (M) or fraction F3 was added to the coculture; in contrast, in cocultures incubated with factions F1, F2, and F4, significant levels of the 98-kDa component were detected.
The authors thank Casilda Mura for help with polyacrylamide gel electrophoresis. 
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Figure 1.
 
IgA-depleted human milk inhibits Acanthamoeba-induced CPE. (A) Preparation of IgA-depleted milk. An aliquot of milk containing 121 mg protein was chromatographed on an IgA-Sepharose column. Ten-microgram protein aliquots of the unbound fraction eluted from the affinity column and unfractionated milk were electrophoresed in SDS-polyacrylamide gels in reducing conditions. After electrophoresis, the proteins were transferred onto nitrocellulose membranes; the protein blots were stained with Ponceau S (left) and then processed for immunostaining with anti-IgA (right). Note that an intensely stained 56-kDa anti-IgA reactive component is present in the whole milk (M) but not in the unbound fraction (UB). (B) Acanthamoeba-induced CPE on host cells. Acanthamoebae (2 × 105 parasites/mL) were added to confluent cultures of corneal epithelium in 24-well plates, and the cultures were incubated in a CO2 incubator for varying periods. At the end of the incubation period, the plates were stained with Giemsa and photographed. Cont, epithelial cells incubated in media alone. Acanthamoebae, epithelial cells incubated with the parasites for varying periods of time. Clear, unstained regions indicate loss of epithelial cells (8 hours and 16 hours). Dark, stained areas indicate the presence of cells (Cont). (C) IgA-depleted human milk inhibits Acanthamoeba-induced CPE. Epithelial cells were incubated overnight with Acanthamoebae in the presence or the absence of varying concentrations (50–200 μg/well) of IgA-depleted milk. At the end of the incubation period, the monolayers were washed, stained, and scanned to estimate approximate cell density. A value of 1.0 was assigned to the cell density of the plates incubated in media alone without parasites (Cont). The values for cultures incubated with amebae in the presence and absence of milk are expressed as change in the density with respect to control plates. Photographs of the plates are shown in panel C(i); quantification of data is shown in panel C(ii). Note that CPE is inhibited by IgA-depleted milk in a concentration-dependent manner. Cont, corneal epithelial cells incubated with media alone. Group A, cultures incubated with amebae in serum-free media containing 0.4% BSA. A+IgA-depleted milk, cultures incubated with amebae in media containing varying concentration of IgA-depleted milk. Data are expressed as mean ± SE (N = 4 in each group). *P < 0.05 compared with group A and 50 μg/well groups.
Figure 1.
 
IgA-depleted human milk inhibits Acanthamoeba-induced CPE. (A) Preparation of IgA-depleted milk. An aliquot of milk containing 121 mg protein was chromatographed on an IgA-Sepharose column. Ten-microgram protein aliquots of the unbound fraction eluted from the affinity column and unfractionated milk were electrophoresed in SDS-polyacrylamide gels in reducing conditions. After electrophoresis, the proteins were transferred onto nitrocellulose membranes; the protein blots were stained with Ponceau S (left) and then processed for immunostaining with anti-IgA (right). Note that an intensely stained 56-kDa anti-IgA reactive component is present in the whole milk (M) but not in the unbound fraction (UB). (B) Acanthamoeba-induced CPE on host cells. Acanthamoebae (2 × 105 parasites/mL) were added to confluent cultures of corneal epithelium in 24-well plates, and the cultures were incubated in a CO2 incubator for varying periods. At the end of the incubation period, the plates were stained with Giemsa and photographed. Cont, epithelial cells incubated in media alone. Acanthamoebae, epithelial cells incubated with the parasites for varying periods of time. Clear, unstained regions indicate loss of epithelial cells (8 hours and 16 hours). Dark, stained areas indicate the presence of cells (Cont). (C) IgA-depleted human milk inhibits Acanthamoeba-induced CPE. Epithelial cells were incubated overnight with Acanthamoebae in the presence or the absence of varying concentrations (50–200 μg/well) of IgA-depleted milk. At the end of the incubation period, the monolayers were washed, stained, and scanned to estimate approximate cell density. A value of 1.0 was assigned to the cell density of the plates incubated in media alone without parasites (Cont). The values for cultures incubated with amebae in the presence and absence of milk are expressed as change in the density with respect to control plates. Photographs of the plates are shown in panel C(i); quantification of data is shown in panel C(ii). Note that CPE is inhibited by IgA-depleted milk in a concentration-dependent manner. Cont, corneal epithelial cells incubated with media alone. Group A, cultures incubated with amebae in serum-free media containing 0.4% BSA. A+IgA-depleted milk, cultures incubated with amebae in media containing varying concentration of IgA-depleted milk. Data are expressed as mean ± SE (N = 4 in each group). *P < 0.05 compared with group A and 50 μg/well groups.
Figure 2.
 
Fractionation of IgA-depleted milk by gel filtration. (A) An aliquot of IgA-depleted milk containing 38 mg protein was chromatographed on a Sephadex G200 column (1 cm × 130 cm), fractions of 1.0 mL were collected, effluent was monitored by reading each fraction at 280 nm, and fractions comprising peaks (F1-F5) were pooled. (B) Proteins eluted in various fractions were electrophoresed in nondenaturing/nonreducing 4% to 20% polyacrylamide gels and visualized by staining with Coomassie blue.
Figure 2.
 
Fractionation of IgA-depleted milk by gel filtration. (A) An aliquot of IgA-depleted milk containing 38 mg protein was chromatographed on a Sephadex G200 column (1 cm × 130 cm), fractions of 1.0 mL were collected, effluent was monitored by reading each fraction at 280 nm, and fractions comprising peaks (F1-F5) were pooled. (B) Proteins eluted in various fractions were electrophoresed in nondenaturing/nonreducing 4% to 20% polyacrylamide gels and visualized by staining with Coomassie blue.
Figure 3.
 
Acanthamoeba CPE inhibitory activity of IgA-depleted milk is enriched in fraction F3. (A) CPE assays were performed in the presence of 66 μg protein each of fractions F1 to F4. A value of 1.0 was assigned to the cell density of the plates incubated in media alone (Cont). Values for cultures incubated with amebae in the presence and the absence of various milk fractions are expressed as change in the density with respect to control plates. Top: photographs of the plates. Note that only fraction F3 inhibited the ameba-induced CPE. Cont, corneal cultures incubated with media alone. Group A cultures incubated with amebae in serum-free media containing 0.4% BSA. F1 to F4, cultures incubated with amebae in media containing 66 μg of each fraction obtained by gel filtration of IgA-depleted milk (see Fig. 2 ). Data are expressed as mean ± SE (N = 4 in each group). P < 0.05 compared with fractions F1, F2, and F4. (B) Acanthamoeba CPE inhibitory activity is enriched 10-fold in fraction F3. CPE assays were performed in the presence of varying concentrations (3–66 μg protein/well) of IgA-depleted milk and fraction F3. Results were calculated as described in (A). Note that fraction F3 inhibited CPE at a much lower concentration (6 μg/well) than unfractionated IgA-depleted milk (66 μg/well). Data are expressed as mean ± SE (N = 4 in each group). *P < 0.05 compared with all other groups in A + milk. **P < 0.05 compared with all other groups in A+F3 group.
Figure 3.
 
Acanthamoeba CPE inhibitory activity of IgA-depleted milk is enriched in fraction F3. (A) CPE assays were performed in the presence of 66 μg protein each of fractions F1 to F4. A value of 1.0 was assigned to the cell density of the plates incubated in media alone (Cont). Values for cultures incubated with amebae in the presence and the absence of various milk fractions are expressed as change in the density with respect to control plates. Top: photographs of the plates. Note that only fraction F3 inhibited the ameba-induced CPE. Cont, corneal cultures incubated with media alone. Group A cultures incubated with amebae in serum-free media containing 0.4% BSA. F1 to F4, cultures incubated with amebae in media containing 66 μg of each fraction obtained by gel filtration of IgA-depleted milk (see Fig. 2 ). Data are expressed as mean ± SE (N = 4 in each group). P < 0.05 compared with fractions F1, F2, and F4. (B) Acanthamoeba CPE inhibitory activity is enriched 10-fold in fraction F3. CPE assays were performed in the presence of varying concentrations (3–66 μg protein/well) of IgA-depleted milk and fraction F3. Results were calculated as described in (A). Note that fraction F3 inhibited CPE at a much lower concentration (6 μg/well) than unfractionated IgA-depleted milk (66 μg/well). Data are expressed as mean ± SE (N = 4 in each group). *P < 0.05 compared with all other groups in A + milk. **P < 0.05 compared with all other groups in A+F3 group.
Figure 4.
 
Fraction F3 but not fraction F1, F2, or F4 inhibited amebic proteinases. Conditioned media obtained after incubating amebae with the host cells were incubated with various milk fractions (27 μg protein each, 37°C, 1 hour) and were then analyzed for expression levels of proteinases by zymography on gelatin gels. Note that two major components (98 kDa, 55 kDa) were seen in conditioned media prepared by incubating Acanthamoebae with corneal epithelial cells (E+A) and that the 98-kDa component was not detected or was detected in trace amounts when unfractionated IgA-depleted milk (M) or fraction F3 was added to the coculture; in contrast, in cocultures incubated with factions F1, F2, and F4, significant levels of the 98-kDa component were detected.
Figure 4.
 
Fraction F3 but not fraction F1, F2, or F4 inhibited amebic proteinases. Conditioned media obtained after incubating amebae with the host cells were incubated with various milk fractions (27 μg protein each, 37°C, 1 hour) and were then analyzed for expression levels of proteinases by zymography on gelatin gels. Note that two major components (98 kDa, 55 kDa) were seen in conditioned media prepared by incubating Acanthamoebae with corneal epithelial cells (E+A) and that the 98-kDa component was not detected or was detected in trace amounts when unfractionated IgA-depleted milk (M) or fraction F3 was added to the coculture; in contrast, in cocultures incubated with factions F1, F2, and F4, significant levels of the 98-kDa component were detected.
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