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Immunology and Microbiology  |   September 2013
In Vitro and In Vivo Uptake Study of Escherichia coli Nissle 1917 Bacterial Ghosts: Cell-Based Delivery System to Target Ocular Surface Diseases
Author Affiliations & Notes
  • Elisabeth Stein
    Medical University Vienna, OCUVAC-Laura Bassi Centres of Expertise, Institute of Specific Prophylaxis and Tropical Medicine, Centre of Pathophysiology, Infectiology and Immunology, Vienna, Austria
  • Aleksandra Inic-Kanada
    Medical University Vienna, OCUVAC-Laura Bassi Centres of Expertise, Institute of Specific Prophylaxis and Tropical Medicine, Centre of Pathophysiology, Infectiology and Immunology, Vienna, Austria
  • Sandra Belij
    Medical University Vienna, OCUVAC-Laura Bassi Centres of Expertise, Institute of Specific Prophylaxis and Tropical Medicine, Centre of Pathophysiology, Infectiology and Immunology, Vienna, Austria
  • Jacqueline Montanaro
    Medical University Vienna, OCUVAC-Laura Bassi Centres of Expertise, Institute of Specific Prophylaxis and Tropical Medicine, Centre of Pathophysiology, Infectiology and Immunology, Vienna, Austria
  • Nora Bintner
    Medical University Vienna, OCUVAC-Laura Bassi Centres of Expertise, Institute of Specific Prophylaxis and Tropical Medicine, Centre of Pathophysiology, Infectiology and Immunology, Vienna, Austria
  • Simone Schlacher
    Medical University Vienna, OCUVAC-Laura Bassi Centres of Expertise, Institute of Specific Prophylaxis and Tropical Medicine, Centre of Pathophysiology, Infectiology and Immunology, Vienna, Austria
  • Ulrike Beate Mayr
    BIRD-C GmbH & CoKG, Kritzendorf, Austria
  • Werner Lubitz
    BIRD-C GmbH & CoKG, Kritzendorf, Austria
  • Marijana Stojanovic
    Institute of Virology, Vaccines and Sera – TORLAK, Belgrade, Serbia
  • Hristo Najdenski
    Stephan Angeloff Institute of Microbiology, BAS, Sofia, Bulgaria
  • Talin Barisani-Asenbauer
    Medical University Vienna, OCUVAC-Laura Bassi Centres of Expertise, Institute of Specific Prophylaxis and Tropical Medicine, Centre of Pathophysiology, Infectiology and Immunology, Vienna, Austria
  • Correspondence: Talin Barisani-Asenbauer, OCUVAC - Centre of Ocular Inflammation and Infection, Laura Bassi Centres of Expertise, Institute of Specific Prophylaxis and Tropical Medicine, Centre of Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Kinderspitalgasse 15, A - 1090 Vienna, Austria; talin.barisani@meduniwien.ac.at
Investigative Ophthalmology & Visual Science September 2013, Vol.54, 6326-6333. doi:10.1167/iovs.13-12044
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      Elisabeth Stein, Aleksandra Inic-Kanada, Sandra Belij, Jacqueline Montanaro, Nora Bintner, Simone Schlacher, Ulrike Beate Mayr, Werner Lubitz, Marijana Stojanovic, Hristo Najdenski, Talin Barisani-Asenbauer; In Vitro and In Vivo Uptake Study of Escherichia coli Nissle 1917 Bacterial Ghosts: Cell-Based Delivery System to Target Ocular Surface Diseases. Invest. Ophthalmol. Vis. Sci. 2013;54(9):6326-6333. doi: 10.1167/iovs.13-12044.

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

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Abstract

Purpose.: For the successful topical administration of drugs or vaccines to treat ocular surface diseases, efficient and well-tolerated delivery systems/carriers for conjunctival delivery are crucial in the development of new treatment strategies. The present study investigated the efficiency of internalization of bacterial ghosts (BGs) produced from probiotic Escherichia coli Nissle 1917 (EcN) by human conjunctival epithelial (HCjE) cell line, the EcN BGs cytotoxicity for HCjE cells, and in vivo uptake of EcN BGs by conjunctival guinea pig epithelial cells.

Methods.: The uptake of EcN BGs by HCjE cells was analyzed by laser scanning microscopy and flow cytometry. Immunohistochemistry was used to localize the EcN BGs in the guinea pig conjunctival tissue. Cytotoxicity of EcN BGs on HCjE cells was evaluated by measurement of LDH.

Results.: Laser scanning microscopy and flow cytometry revealed that EcN BGs internalization by HCjE cells was time- and dose dependent. No cytotoxic effect on HCjE cells was observed after EcN BGs inoculation for 30 and 120 minutes, as well as 24 hours. In addition, the uptake of EcN BGs was detected in the conjunctival cells after in vivo administration of EcN BGs into the eye of the guinea pig.

Conclusions.: The findings that EcN BGs are nontoxic and effectively internalized in vitro by human and in vivo by guinea pig conjunctival cells comprise an important contribution to the future use of BGs as a system for conjunctival delivery of drugs and vaccines, either to treat or prevent ocular surface diseases.

Introduction
Ocular surface diseases encompass a plethora of pathologies with overlapping conditions that lead to common outcomes: dysfunction of the ocular tear film and/or the integrity of the ocular surface. 1 Furthermore, concomitant inflammation can cause damage to the various structures of the ocular surface. Moreover, in severe cases, chronic surface damage may result in mild-to-profound decreases in vision, as in severe dry eye syndromes, 2 vernal keratoconjunctivitis (VKC), 3 or infectious diseases, such as trachoma. 4  
Although affecting the majority of individuals with ocular symptoms, ocular surface diseases are globally under-recognized and neglected. This situation also is clearly apparent in drug and vaccine development, as the current topical anti-inflammatory drugs used in the management of ocular surface diseases are optimized mostly for intraocular delivery. 5 This approach simply means that the novel drugs are not developed to reach the ocular surface, but instead to overcome its function as a barrier. The use of these drugs eventually contributes to increased ocular surface dysfunction as observed in the chronic use of topical therapeutics prescribed for ocular conditions, such as glaucoma. Therefore, the development of delivery platforms targeting the ocular surface cells would be helpful to overcome these sequelae. 
Bacterial ghosts (BGs) are empty, nonliving, Gram-negative bacterial envelopes produced by controlled expression of the cloned gene E, which causes the fusion of the inner and outer membranes to form a transmembrane lysis tunnel structure through which the cytoplasmic content is expelled. 6 One of the main characteristics of BGs is that they possess all cell membrane structures, like native bacteria (e.g., adhesins, lipopolysaccharide [LPS], and peptidoglycan), which give a potential for an adjuvant delivery platform, while not posing any infectious threat. 7  
It has been shown that BGs can be internalized efficiently by various types of cells. 8,9  
Moreover, the inner space of BGs can be loaded with single components, or combinations of peptides, drugs, or DNA. 10,11 This method provides an opportunity to design new types of (polyvalent) drug delivery vehicles. In Caco-2 cells, uptake of BGs loaded with doxorubicin (DOX) led to the DOX release from endo-lysosomal compartments that was two magnitudes of orders more effective than DOX provided in the medium at the same concentration. In these experiments Paukner et al. could show that BGs, after successfully delivering DOX or calcein into the targeted cells, were degraded intercellularly. 12,13  
Further studies have demonstrated that DNA-loaded BGs were phagocytosed efficiently and internalized by professional antigen-presenting cells (APCs) and tumor cells, with up to 82% of cells expressing the plasmid-encoded reporter gene. 14,15  
In an attempt to target ocular surface diseases, preliminary experiments have shown a promising uptake of BGs in primary human conjunctiva-derived epithelial cell line (HCDECs) and CCL-20.2 cells. 16  
The purpose of our study first was to investigate the in vitro uptake and safety of Escherichia coli Nissle 1917 (EcN) BGs by immortalized human conjunctival epithelial (HCjE) cell line, since these cells retained several of the unique characteristics of their native tissue. 17 Our second goal was to establish the guinea pig animal model of short-term in vivo uptake, as this could be a good model that reflects the situation in humans. 
Materials and Methods
Cell Culture
HCjE cells, kindly provided by Ilene Gipson (Schepens Eye Research Institute, Harvard Medical School, Boston, MA), were maintained in keratinocyte serum-free medium (Life Technologies, Paisley, UK) at 37°C/5% CO2 and 95% humidity. The medium was changed every second day, and the cells were passaged at 70% confluence. Cells were harvested by trypsinization (0.05% Trypsin/0.02% EDTA in PBS; PAA Laboratories GmbH, Pasching, Austria), and seeded at a density of 30,000 cells/well in 6-well plates (Greiner Bio-One, Kremsmünster, Austria) for the flow cytometric analysis and in 24-well imaging plates (PAA Laboratories GmbH) for laser scanning microscopy. 
BGs Production and Labeling With Atto488 Dye
EcN BGs were produced by the controlled expression of the phage-derived lysis gene E, as described previously. 18 Lyophilized EcN BGs were reconstituted in 0.1 M sodium bicarbonate buffer (pH 8.5; PAA Laboratories GmbH) and incubated with Atto488 dye (2 mg/mL in dimethylsulfoxide [DMSO]; ATTO-TEC GmbH, Siegen, Germany) for 1 hour while stirring at 900g. Atto488-labeled EcN BGs were washed 5 times with PBS to remove unbound dye and diluted in keratinocyte serum-free medium (Life Technologies). 
Live Cell Staining for Laser Scanning Microscopy
Atto488-labeled EcN BGs (100, 1000, and 10,000 BGs per HCjE cell) were applied to confluent cultures of HCjE cells and plates were centrifuged at 315g for 15 minutes to accelerate the attachment. After this initial step, cells were incubated at 37°C/5% CO2 for another 15 minutes and 105 minutes, respectively (incubation time in total was 30 and 120 minutes). HCjE cells were washed six times with PBS, and cells on imaging plates, stained with CellMask plasma membrane stain (5 μg/ml; Molecular Probes, Inc., Eugene, OR) for 5 minutes at 37°C, were subjected directly to live cell staining. 4′,6-diamidino-2-phenylindole (1 μg/ml; Sigma-Aldrich, St. Louis, MO) was used as a counterstain. To assess only the signal of internalized EcN BGs, the fluorescent signal of BGs attached to cell surfaces was quenched by incubation with 0.4% trypan blue solution (0.4%; Sigma-Aldrich) in PBS for 5 minutes. Plates were mounted and examined by laser scanning microscopy (Zeiss Axiovert 100; Carl Zeiss GmbH, Vienna, Austria). 
Flow Cytometry Analysis of the Atto488-Labeled EcN BGs Internalization by HCjE Cells
The uptake of EcN BGs by HCjE cells also was analyzed by flow cytometry. The cells were prepared the same way as described in the section “Live Cell Staining for Laser Scanning Microscopy.” Cells then were incubated at 37°C/5% CO2 for another 15 and 105 minutes, respectively (control cells were incubated either with nonlabeled EcN BGs at 37°C/5% CO2 or with Atto488-labeled EcN BGs on ice). HCjE cells were washed six times with PBS to remove the excess of EcN BGs. HCjE cells were detached by trypsinization (0.05% Trypsin/0.02% EDTA; PAA Laboratories GmbH), washed three times with 2% BSA/PBS (Sigma-Aldrich), and resuspended in 0.5 mL of this solution. To eliminate signals from the attached EcN BGs and to ensure the fluorescence was only internal, trypan blue solution (0.4%; Sigma-Aldrich) was added to a final concentration of 0.2% directly before analysis. Cells were analyzed on a BD FACScan (BD Biosciences, San Jose, CA). Dead cells were excluded according to their forward and side scatter properties. Data were analyzed using Flow Jo Software version 9.3.2 (Tree Star, Inc., Ashland, OR). 
Cytotoxicity Assay
For the assessment of possible cytotoxic effects of EcN BGs on HCjE cells, cells were seeded at a density of 4 × 104 cells in 96-well plates. BGs were applied at 100, 1000, and 10,000 EcN BGs/cell, and plates were centrifuged at 315g for 15 minutes. Cells were incubated under standard conditions for 15 and 105 minutes to reach total co-incubation times of 30 and 120 minutes. Supernatants were collected and stored at −80°C until the measurements were taken. An additional number of cells, after washing with PBS, was incubated for another 24 hours for detection of possible delayed cell death. Cytotoxicity was evaluated by measurement of lactate dehydrogenase (LDH) that was released to the culture medium upon damage of the plasma membrane with a commercially available WST-based assay (LDH Cytotoxicity Assay Kit II; BioVision, Milpitas, CA) according to the manufacturer's instructions. 
Inoculation of EcN BGs Into Guinea Pig Eyes
To detect the uptake of EcN BGs in vivo, the EcN BGs were applied into the conjunctival sac of guinea pigs eyes. All experiments were conducted in agreement with the International and National Guidelines for the Care and Use of Laboratory Animals, approved by the Animal Care Committee at the Institute of Microbiology, Sofia, and adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Female Hartley strain guinea pigs (300–350 g) were obtained from Charles River Laboratories, Inc. (Frederick, MD) and were housed separately in cages covered with fiberglass filter tops. Six guinea pigs were anesthetized with a mixture of ketamine (30 mg/kg) and xylazine (2 mg/kg) administered intramuscularly, and 25 μL PBS containing 1 × 107 EcN BGs particles were instilled directly into the conjunctival sacs of both eyes. Six guinea pigs were used as sham controls and received only PBS. After 120 minutes, the animals were euthanized by intramuscular administration of an overdose mixture of xylazine and ketamine. Conjunctival tissue was removed and fixed in 1% formaldehyde in PHEM buffer, and paraffin-embedded sections were processed for immunohistochemistry. Additionally, the sections were stained routinely with hematoxylin and eosin (H&E) for a pathology evaluation. 
Eye Pathology Examination
In accordance with Organisation for Economic Cooperation and Development 405 (OECD 405) guidelines for the testing of chemicals (in vivo test for acute eye irritation/corrosion), 19 120 minutes postinoculation the animal discomfort, and clinical signs in the conjunctiva, corneas, and lids were evaluated macroscopically by a trained clinician for possible acute toxicity of the EcN BGs. Additionally, conjunctival sections (4 μm) were evaluated in a masked fashion to determine eventual modification of the corneal, limbal, or conjunctival epithelia; edema in lid tissues; presence of inflammatory neutrophils, eosinophils, mast cells, or lymphocytes; and any other abnormalities. 
Immunohistochemistry Assay
To localize the EcN BGs in the guinea pig conjunctival tissue and confirm BG uptake in vivo, immunohistochemistry studies were performed. Paraffin sections of guinea pig conjunctival tissue taken 120 minutes after inoculation with EcN BGs were deparaffinized and brought to water. Antigen retrieval was performed with 1 mg/mL Trypsin (Sigma-Aldrich) in PBS for 10 minutes at 37°C. After washing in PBS with 0.025% Triton X-100, sections were blocked with 10% normal goat serum in PBS for 2 hours at room temperature and incubated with cell-adsorbed polyclonal rabbit anti-EcN BGs antisera (Institute of Virology, Vaccines and Sera Torlak, Belgrade, Serbia), 1:500 dilution in 1% BSA in PBS for 30 minutes at room temperature. Control sections were incubated with antibody diluent alone. After washing three times with PBS with 0.025% Triton X-100 (Sigma-Aldrich) sections were incubated with fluorophore-labeled secondary antibody (CF488A goat anti-rabbit; Sigma-Aldrich), 1:1000 dilution in PBS containing 1% BSA. A phalloidin conjugate (Phalloidin-Atto 565; Sigma-Aldrich) was added to the secondary antibody cocktail (dilution 1:20) to visualize the actin filaments in the tissue. Sections were incubated for 30 minutes at room temperature and washed extensively with PBS with 0.025% Triton X-100. Then, 4′,6-diamidino-2-phenylindole (1 μg/ml; Sigma-Aldrich) was used for counterstaining (5 minutes at room temperature). After rinsing in PBS and brief wash in distilled water, sections were mounted in DAKO fluorescent mounting medium (DAKO Corp., Carpinteria, CA). Sections were examined by fluorescence microscopy using the TissueFAXSsi plus system and TissueFAXS software (TissueGnostics GmbH, Vienna, Austria). 
Results
HCjE Cells Efficiently Internalize EcN BGs
Laser scanning microscopy of HCjE cells co-incubated with EcN BGs confirmed the internalization of BGs (Fig. 1). The presence of internalized EcN BGs within the cytoplasm was proved by quenching of the fluorescent signal coming from BGs eventually attached to HCjE cells surface by using trypan blue solution. Samples that did not undergo the quenching procedure showed distinctively more BGs attached and/or internalized than samples that underwent quenching. In addition, the analysis of HCjE cells co-incubated with Atto488-labeled EcN BGs on ice either for 30 or 120 minutes showed no internalization of BGs (data not shown). Representative pictures of EcN internalization (1000 EcN BGs per HCjE cell) are displayed in Figure 1 (Fig. 1A shows internalization after 30 minutes, Fig. 1B shows internalization after 120 minutes). Quantification of the uptake efficiencies showed that EcN BGs are internalized into HCjE cells in a time- and dose-dependent manner. Uptake of EcN BGs by HCjE cells was detected 30 minutes after administration of Atto488-labeled EcN BGs into human conjunctival cell culture. Prolonged (120 minutes) incubation of HCjE with EcN BGs led to significantly increased number of cells efficiently internalizing EcN BGs (Fig. 1C). Irrespective to the EcN BGs amount, their internalization by HCjE cells after 120 minutes of co-incubation was significantly higher than the internalization observed after 30 minutes. The fact that percentages of HCjE cells that internalized BGs in cultures incubated for 30 minutes with 10,000 EcN BGs/HCjE cell and 120 minutes with 1000 EcN BGs/HCjE cell were similar (P = 0.301) implies that, in the respect of percentage of HCjE cells that internalized EcN BGs, the lower amount of EcN BGs could be compensated by prolongation of incubation time and vice versa. FACS analysis revealed results similar to those from the microscopic evaluation. Representative histograms are shown in Figure 2
Figure 1
 
Analysis of Atto488-labeled EcN BGs uptake by HCjE cells by laser scanning microscopy. HCjE cells were incubated for 30 minutes (A) and 120 minutes (B) with Atto488-EcN BGs (1000 per cell), washed with PBS, and cells on imaging plates were subjected directly to live cell staining. Scale bar: 10 μm. EcN BGs found in HCjE cells are marked by arrows (A, B). Values indicated on histogram y-axes (C) were calculated as the percentage of cells incubated on ice with Atto488-labeled EcN BGs subtracted from the percentage of positive cells incubated with Atto488-labeled EcN BGs at 37°C. Data represent the mean of four independent samples ± SD. The significance of the differences between specific groups defined by the incubation time and the ratio of EcN BGs to HCjE was determined by t-test (*P < 0.05, **P < 0.005, ****P < 0.00005). Compared groups are indicated by double-head arrow.
Figure 1
 
Analysis of Atto488-labeled EcN BGs uptake by HCjE cells by laser scanning microscopy. HCjE cells were incubated for 30 minutes (A) and 120 minutes (B) with Atto488-EcN BGs (1000 per cell), washed with PBS, and cells on imaging plates were subjected directly to live cell staining. Scale bar: 10 μm. EcN BGs found in HCjE cells are marked by arrows (A, B). Values indicated on histogram y-axes (C) were calculated as the percentage of cells incubated on ice with Atto488-labeled EcN BGs subtracted from the percentage of positive cells incubated with Atto488-labeled EcN BGs at 37°C. Data represent the mean of four independent samples ± SD. The significance of the differences between specific groups defined by the incubation time and the ratio of EcN BGs to HCjE was determined by t-test (*P < 0.05, **P < 0.005, ****P < 0.00005). Compared groups are indicated by double-head arrow.
Figure 2
 
Flow cytometry results displaying the time- and dose-dependent internalization of EcN BGs after incubation with HCjE cells. Cells were incubated for 30 (left) and 120 (right) minutes at 37°C with different concentrations of EcN BGs: 100 EcN BGs/cell (dotted), 1000 EcN BGs/cell (dashed), and 10,000 EcN BGs/cell (long dashed). HCjE cells incubated without EcN BGs served as controls (shaded area). Trypan blue solution was added to a final concentration of 0.2% directly before analysis to eliminate signals from the attached BGs. Each sample is analyzed in duplicate and two independent experiments were performed. Representative plots are provided.
Figure 2
 
Flow cytometry results displaying the time- and dose-dependent internalization of EcN BGs after incubation with HCjE cells. Cells were incubated for 30 (left) and 120 (right) minutes at 37°C with different concentrations of EcN BGs: 100 EcN BGs/cell (dotted), 1000 EcN BGs/cell (dashed), and 10,000 EcN BGs/cell (long dashed). HCjE cells incubated without EcN BGs served as controls (shaded area). Trypan blue solution was added to a final concentration of 0.2% directly before analysis to eliminate signals from the attached BGs. Each sample is analyzed in duplicate and two independent experiments were performed. Representative plots are provided.
EcN BGs Have No Cytotoxic Effect on HCjE Cells
The measurement of LDH release as a consequence to cytotoxic effects decreasing cell viability was performed to elucidate the effect of EcN BGs in contact with HCjE cells. Co-incubation of HCjE cells with EcN BGs did not result in any adverse effects on cellular metabolic activities. The LDH release levels were less than in the low control, indicating that there were no cytotoxic effects, neither after treatment with EcN BGs for 30 and 120 minutes, respectively, nor after 24 hours of incubation at 37°C/5% CO2 after washing off EcN BGs from the cells (Fig. 3). 
Figure 3
 
Influence of EcN BGs on the viability of HCjE cells measured through the LDH release. Cells were seeded at a density of 4 × 104 cells in 96-well plates, and incubated with EcN BGs at three different methods of inoculation (MOIs): 100, 1000, and 10,000 EcN BGs/cell. Total co-incubation times were 30 and 120 minutes under standard conditions (5% CO2/37°C). LDH release was measured after 30 minutes, 120 minutes, and 24 hours. Each bar represents the mean of three independent experiments performed in triplicate (±SD).
Figure 3
 
Influence of EcN BGs on the viability of HCjE cells measured through the LDH release. Cells were seeded at a density of 4 × 104 cells in 96-well plates, and incubated with EcN BGs at three different methods of inoculation (MOIs): 100, 1000, and 10,000 EcN BGs/cell. Total co-incubation times were 30 and 120 minutes under standard conditions (5% CO2/37°C). LDH release was measured after 30 minutes, 120 minutes, and 24 hours. Each bar represents the mean of three independent experiments performed in triplicate (±SD).
EcN BGs Do Not Cause Ocular Pathology in Guinea Pig
Seven days before the experiments began, the animals presented no clinical signs of diseases, and ocular surface structures were normal. Pathologic analysis of the guinea pig eyelids, confirmed the presence of normal ocular surface structures in those with EcN BGs–treated eyes (Fig. 4A) and control (Fig. 4B). Some of the guinea pigs (n = 4) were monitored additionally for 7 days and no signs of any ocular pathology were noticed at any time point (data not shown). Conjunctival, limbal, and corneal epithelia displayed normal numbers of cell layers composed of cells with appropriate morphologies in treated eyes (Fig. 4C) and EcN BGs control (Fig. 4D) without the presence of inflammatory cells of any type. 
Figure 4
 
Pictures of guinea pig eyes inoculated with 1 × 107 EcN BGs (A) or PBS as control (B) 120 minutes after inoculation. No signs of irritation, redness, or exaggerated lacrimation could be detected. Histologic examination (H&E staining) of conjunctival tissue performed at sections of the tissue collected 120 minutes after inoculation showed no differences between the BG-treated group (C) and the controls (D). Scale bars: 200 μm.
Figure 4
 
Pictures of guinea pig eyes inoculated with 1 × 107 EcN BGs (A) or PBS as control (B) 120 minutes after inoculation. No signs of irritation, redness, or exaggerated lacrimation could be detected. Histologic examination (H&E staining) of conjunctival tissue performed at sections of the tissue collected 120 minutes after inoculation showed no differences between the BG-treated group (C) and the controls (D). Scale bars: 200 μm.
EcN BGs Are Localized Into Conjunctival Epithelial Cells in the Guinea Pig
Positive staining for EcN BGs was detected in all samples to which EcN BGs were added (Fig. 5A), and in none of the samples originated from nontreated guinea pigs (Fig. 5B). We detected EcN BGs within guinea pig conjunctival epithelial cells 120 minutes after inoculation of EcN BGs into outbred guinea pigs. Analysis of lymph nodes, spleen, eye, and brain sections from these guinea pigs showed no evidence of EcN BGs (data not shown). 
Figure 5
 
Immunofluorescent analysis of guinea pig conjunctival tissue performed 120 minutes after EcN BGs (A) and PBS (B) inoculation (25 μL of PBS containing 1 × 107 EcN BGs particles or 25 μL of PBS alone, respectively, was instilled directly into the conjunctival sacs). EcN BGs are found in the conjunctival epithelium (marked by arrows). Paraffin sections of guinea pig conjunctiva were stained with rabbit anti-EcN BGs polyclonal antibodies, followed by goat CF488-labeled anti-rabbit IgG. The guinea pigs receiving only PBS were used as sham controls. Data are representative of observations from three guinea pigs in each experimental group. Scale bars: 20 μm.
Figure 5
 
Immunofluorescent analysis of guinea pig conjunctival tissue performed 120 minutes after EcN BGs (A) and PBS (B) inoculation (25 μL of PBS containing 1 × 107 EcN BGs particles or 25 μL of PBS alone, respectively, was instilled directly into the conjunctival sacs). EcN BGs are found in the conjunctival epithelium (marked by arrows). Paraffin sections of guinea pig conjunctiva were stained with rabbit anti-EcN BGs polyclonal antibodies, followed by goat CF488-labeled anti-rabbit IgG. The guinea pigs receiving only PBS were used as sham controls. Data are representative of observations from three guinea pigs in each experimental group. Scale bars: 20 μm.
Discussion
For the successful topical administration of either drugs or vaccines to treat/prevent ocular surface diseases, efficient and well-tolerated carriers for conjunctival delivery are crucial in the development of new treatment concepts. An optimal delivery platform would penetrate the ocular surface cells with the minimum of properties capable of making trans- and paracellular transport into the eye impossible. 
In our study, we investigated the efficiency of internalization of BGs produced from probiotic EcN by HCjE cell line, the EcN BGs cytotoxicity for HCjE, and in vivo uptake of EcN BGs by conjunctival guinea pig epithelial cells. 
In vitro models to mimic the conjunctival surface as closely as possible are important tools in the development of innovative approaches to treat ocular surface diseases. Primary cultures of human conjunctival cells exhibit numerous disadvantages, including limited availability of donor material, the quick turnover, and senescence primary conjunctival epithelial cells, and the change of differentiation characteristics over time. The previously reported Chang conjunctival cell line American Type Culture Collection 20.2, the model used in many previous studies regarding ocular immunity, 16,20,21 is reported to show altered responses to inflammatory cytokines, such as TNF-α and IFN-γ, 22 but the most striking reservation against using it might be the reported contamination with HeLa cells. 23  
Immortalized epithelial cell lines are able to overcome these problems because they retain their differentiation characteristics and exhibit gene expression profiles resembling native epithelial cells. 17 Hence, we used the hTERT-immortalized human conjunctival epithelial cell line described previously by Rheinwald et al. 24 and Gipson et al. 17 This HCjE cell line has been shown to express the epithelial marker K19, and the membrane-associated mucins MUC1, MUC4, and MUC16, which are characteristic for conjunctival epithelium. 17  
We have demonstrated that HCjE cells were able to internalize EcN BGs efficiently and in a quite short time period, in a time- and dose-dependent manner, which strengthens the potential of EcN-derived BGs as delivery platforms for ocular drug/vaccine delivery, where the efficient and quick uptake of a carrier is of critical importance. As reported earlier, administration of vaccines via the conjunctival route counted as a promising alternative route for inducing local and systemic immune responses. In addition, a protection against challenge with influenza, virulent Salmonella, 25 Brucellaovis, 26 and tetanus 27 was achieved. It is known that the type of vaccine formulation is critical for achieving the appropriate immune response, 28,29 and could be tailored to activate specific immune-responsive cells in the conjunctival associated lymphoid tissue (CALT) to increase the efficacy of conjunctival immunization against mucosal pathogens. CALT detects antigens from the ocular surface, presents the antigens, and generates protective effector cells; together, these properties signify the presence of a mucosal immune system at the conjunctiva. On the other hand, recent studies suggest that different epithelial cells are involved in direct antigen sampling. 30 Hence, the evidence of EcN BGs internalization by human conjunctival epithelial cells is a step further toward a development of eyedrop immunization using the BGs as a carrier/adjuvant. 
In addition, we noted that there were no detectable negative EcN BGs effects on HCjE cell viability, which is in accordance with data obtained from in vitro studies performed over the past years that BGs produced from different bacterial origins represent a safe vaccine and drug delivery platform. 15,16,31  
We also demonstrated that EcN BGs were internalized efficiently in vivo, as EcN BGs were found not only in superficial, but also in deeper layers of conjunctival epithelium. The guinea pig was chosen as an animal model of in vivo uptake due to several reasons. First, the guinea pig has a particularly stable tear film. Periods of up to 20 minutes without blinking have been observed without any signs of ocular discomfort or corneal dryness. 32 Second, guinea pigs have greater numbers of conjunctival lymphoid follicles than any other small animal models, 33 and third, it has been shown that guinea pigs have fully functioning antigen-sampling M cells in the follicle-associated epithelium (FAE) above organized lymphoid tissue. 34 Therefore, stability of tear film and complexity of conjunctival lymphoid tissue, organized in the form of CALT, could be a good model that reflects the situation in humans. 
The bacterial species that was used for the generation of the BGs in our study consisted of the probiotic EcN. In general, probiotics are defined as nonpathogenic living microorganisms that, when administered in adequate amounts, confer a health benefit on the host. 35  
Recently published data encourage further research on probiotics as a carrier/delivery system to either prevent or treat ocular diseases. Namely, live Lactobacillus acidophilus, may offer a safe and well-tolerated support to standard anti-allergic therapy for the treatment of mild-to-moderate cases of VKC. 36 It also was shown that patients with VKC had an improvement in the signs and symptoms of ocular allergy associated with the down-regulation of ICAM-1 and TLR4 after 4 weeks of treatment with Lactobacillus acidophilus eyedrops. However, the use of live probiotics carries a very small risk of either changes in phenotype or translocation to other sites. A risk-reducing solution is to use the same probiotic strain as a BG, the empty shell of the bacteria instead of the bacteria itself, since BGs do retain the surface properties of the live bacteria they were derived from. As the BGs are nonliving, this would confer all the benefits of the BGs without safety concerns for immunocompromised hosts. 
Several reports have elucidated the beneficial effects of EcN as a treatment for inflammatory bowel disease, 37 in the prevention of allergies, 38,39 and as a treatment for severe diarrhea in infants and toddlers. 40,41 Recently, it was shown that EcN is able to activate dendritic cells by a TLR4-dependent pathway, 39 and to ameliorate colitis in mice via TLR2- and TLR4-dependent signaling. 42  
Our results on EcN BGs are in accordance with prior findings that BGs derived from different bacterial species induced specific humoral and cellular immune responses by various methods of administration in in vitro studies 13,16 and in different animal models using different routes of immunisation. 4347 Previous studies on the safety and tolerability of BGs indicated that without regard to the bacterial species, no cytotoxic or genotoxic effects could be detected in a broad spectrum of human cells. 48 Our results clearly showed no adverse effects of EcN BGs on HCjE cells in vitro and optimal tolerability in conjunctival administration in vivo. 
Paukner et al. showed that BGs are degraded within cellular compartments and the reported substance was released, thus, demonstrating the BGs as drug delivery and targeting vesicles. 14 Although BGs show very promising drug delivery characteristics, all eukaryotic cells do not take them up with the same capacity. As reported previously, the percentage of BGs adherent to human colorectal adenocarcinoma cells and internalized BGs was 78% vs. 50% at a BG-to-cell ratio of 100% and 95%, versus 47% at a BG-to-cell ratio of 500 after 2 hours of co-incubation, respectively. 13 Therefore, in our interest to introduce BGs as drug delivery platform for ocular surface conditions, it was very important first to prove successful uptake of BGs by conjunctival cells in vitro and in vivo, and gain tolerability and safety data. Contrary to nanoparticles, 49 we have data that BGs are not transported into the eye, brain, and other major organs. This probably is due to their size, as a typical BG is considered a cylinder with a length of 3.5 μL and a diameter of 0.5 μm, and the size of nanoparticles is ≤100 nm, typically. 50  
The findings that EcN BGs are nontoxic, and effectively internalized in vitro by human and in vivo by guinea pig conjunctival cells comprise an important contribution to the future use of BGs as a system for conjunctival delivery of drugs and vaccines to treat/prevent ocular surface diseases. 
Acknowledgments
The authors thank Pavol Kudela for his help in reviewing the manuscript. 
Supported by the Laura Bassi Centers of Expertise (FFG Project Number 822768, available in the public domain at http://www.ffg.at/en) and the Republic of Austria. 
Disclosure: E. Stein, None; A . Inic-Kanada, None; S. Belij, None; J. Montanaro, None; N. Bintner, None; S. Schlacher, None; U.B. Mayr, Bird-C (E); W. Lubitz, Bird-C (I), P; M. Stojanovic, None; H. Najdenski, None; T. Barisani-Asenbauer, None 
References
Leung EW Medeiros FA Weinreb RN. Prevalence of ocular surface disease in glaucoma patients. J Glaucoma . 2008; 17: 350–355. [CrossRef] [PubMed]
Stevenson W Chauhan SK Dana R. Dry eye disease: an immune-mediated ocular surface disorder. Arch Ophthalmol . 2012; 130: 90–100. [CrossRef] [PubMed]
De Smedt S Wildner G Kestelyn P. Vernal keratoconjunctivitis: an update. Br J Ophthalmol . 2013; 97: 9–14. [CrossRef] [PubMed]
Burton MJ. Trachoma: an overview. Br Med Bull . 2007; 84: 99–116. [CrossRef] [PubMed]
Edelhauser HF Rowe-Rendleman CL Robinson MR Ophthalmic drug delivery systems for the treatment of retinal diseases: basic research to clinical applications. Invest Ophthalmol Vis Sci . 2010; 51: 5403–5420. [CrossRef] [PubMed]
Witte A Wanner G Blasi U Halfmann G Szostak M Lubitz W. Endogenous transmembrane tunnel formation mediated by phi X174 lysis protein E. J Bacteriol . 1990; 172: 4109–4114. [PubMed]
Mayr UB Walcher P Azimpour C Riedmann E Haller C Lubitz W. Bacterial ghosts as antigen delivery vehicles. Adv Drug Deliv Rev . 2005; 57: 1381–1391. [CrossRef] [PubMed]
Haslberger AG Kohl G Felnerova D Mayr UB Fürst-Ladani S Lubitz W. Activation, stimulation and uptake of bacterial ghosts in antigen presenting cells. J Biotechnol . 2000; 83: 57–66. [CrossRef] [PubMed]
Abtin A Kudela P Mayr UB Escherichia coli ghosts promote innate immune responses in human keratinocytes. Biochem Biophys Res Commun . 2010; 400: 78–82. [CrossRef] [PubMed]
Kudela P Koller VJ Lubitz W. Bacterial ghosts (BGs)--advanced antigen and drug delivery system. Vaccine . 2010; 28: 5760–5767. [CrossRef] [PubMed]
Muhammad A Champeimont J Mayr UB Lubitz W Kudela P. Bacterial ghosts as carriers of protein subunit and DNA-encoded antigens for vaccine applications. Expert Rev Vaccines . 2012; 11: 97–116. [CrossRef] [PubMed]
Kudela P Paukner S Mayr UB Effective gene transfer to melanoma cells using bacterial ghosts. Cancer Lett . 2008; 262: 54–63. [CrossRef] [PubMed]
Paukner S Kohl G Lubitz W. Bacterial ghosts as novel advanced drug delivery systems: antiproliferative activity of loaded doxorubicin in human Caco-2 cells. J Control Release . 2004; 94: 63–74. [CrossRef] [PubMed]
Paukner S Kohl G Jalava K Lubitz W. Sealed bacterial ghosts–novel targeting vehicles for advanced drug delivery of water-soluble substances. J Drug Target . 2003; 11: 151–161. [PubMed]
Kudela P Paukner S Mayr UB Bacterial ghosts as novel efficient targeting vehicles for DNA delivery to the human monocyte-derived dendritic cells. J Immunother . 2005; 28: 136–143. [CrossRef] [PubMed]
Kudela P Koller VJ Mayr UB Nepp J Lubitz W Barisani-Asenbauer T. Bacterial ghosts as antigen and drug delivery system for ocular surface diseases: effective internalization of bacterial ghosts by human conjunctival epithelial cells. J Biotechnol . 2011; 153: 167–175. [CrossRef] [PubMed]
Gipson IK Spurr-Michaud S Argueso P Tisdale A Ng TF Russo CL. Mucin gene expression in immortalized human corneal-limbal and conjunctival epithelial cell lines. Invest Ophthalmol Vis Sci . 2003; 44: 2496–2506. [CrossRef] [PubMed]
Langemann T Koller VJ Muhammad A Kudela P Mayr UB Lubitz W. The bacterial ghost platform system: production and applications. Bioeng Bugs . 2010; 1: 326–336. [CrossRef] [PubMed]
Organisation for Economic Cooperation and Development: Test No. 405: acute eye irritation/corrosion. In: OECD Guidelines for the Testing of Chemicals, Section 4: Health Effects . Paris, France: OECD Publishing; 2002: 1–19.
Guenoun JM Baudouin C Rat P Pauly A Warnet JM Brignole-Baudouin F. In vitro comparison of cytoprotective and antioxidative effects of latanoprost, travoprost, and bimatoprost on conjunctiva-derived epithelial cells. Invest Ophthalmol Vis Sci . 2005; 46: 4594–4599. [CrossRef] [PubMed]
Talreja J Dileepan K Puri S Human conjunctival epithelial cells lack lipopolysaccharide responsiveness due to deficient expression of MD2 but respond after interferon-gamma priming or soluble MD2 supplementation. Inflammation . 2005; 29: 170–181. [CrossRef] [PubMed]
De Saint Jean M Baudouin C Di Nolfo M Comparison of morphological and functional characteristics of primary-cultured human conjunctival epithelium and of Wong-Kilbourne derivative of Chang conjunctival cell line. Exp Eye Res . 2004; 78: 257–274. [CrossRef] [PubMed]
Lacroix M. Persistent use of “false” cell lines. Int J Cancer . 2008; 122: 1–4. [CrossRef] [PubMed]
Rheinwald JG Hahn WC Ramsey MR A two-stage, p16INK4A- and p53-dependent keratinocyte senescence mechanism that limits replicative potential independent of telomere status. Mol Cell Biol . 2002; 22: 5157–5172. [CrossRef] [PubMed]
Seo KY Han SJ Cha HR Eye mucosa: an efficient vaccine delivery route for inducing protective immunity. J Immunol . 2010; 185: 3610–3619. [CrossRef] [PubMed]
Da Costa Martins R, Gamazo C, Sanchez-Martinez M, Barberan M, Penuelas I, Irache JM. Conjunctival vaccination against Brucella ovis in mice with mannosylated nanoparticles. J Control Release . 2012; 162: 553–560. [CrossRef] [PubMed]
Barisani-Asenbauer T Inic-Kanada A Belij S The ocular conjunctiva as a mucosal immunization route: a profile of the immune response to the model antigen tetanus toxoid. PLoS One . 2013; 8: e60682. [CrossRef] [PubMed]
Lycke N Bemark M. Mucosal adjuvants and long-term memory development with special focus on CTA1-DD and other ADP-ribosylating toxins. Mucosal Immunol . 2010; 3: 556–566. [CrossRef] [PubMed]
Perrie Y Mohammed AR Kirby DJ McNeil SE Bramwell VW. Vaccine adjuvant systems: enhancing the efficacy of sub-unit protein antigens. Int J Pharm . 2008; 364: 272–280. [CrossRef] [PubMed]
Holmgren J Svennerholm AM. Vaccines against mucosal infections. Curr Opin Immunol . 2012; 24: 343–353. [CrossRef] [PubMed]
Koller VJ Dirsch VM Beres H Modulation of bacterial ghosts—induced nitric oxide production in macrophages by bacterial ghost-delivered resveratrol. FEBS J . 2013; 280: 1214–1225. [CrossRef] [PubMed]
Trost K Skalicky M Nell B. Schirmer tear test, phenol red thread tear test, eye blink frequency and corneal sensitivity in the guinea pig. Vet Ophthalmol . 2007; 10: 143–146. [CrossRef] [PubMed]
Chodosh J Nordquist RE Kennedy RC. Comparative anatomy of mammalian conjunctival lymphoid tissue: a putative mucosal immune site. Dev Comp Immunol . 1998; 22: 621–630. [CrossRef] [PubMed]
Meagher CK Liu H Moore CP Phillips TE. Conjunctival M cells selectively bind and translocate Maackia amurensis leukoagglutinin. Exp Eye Res . 2005; 80: 545–553. [CrossRef] [PubMed]
Troge A Scheppach W Schroeder BO More than a marine propeller--the flagellum of the probiotic Escherichia coli strain Nissle 1917 is the major adhesin mediating binding to human mucus. Int J Med Microbiol . 2012; 302: 304–314. [CrossRef] [PubMed]
Iovieno A Lambiase A Sacchetti M Stampachiacchiere B Micera A Bonini S. Preliminary evidence of the efficacy of probiotic eye-drop treatment in patients with vernal keratoconjunctivitis. Graefes Arch Clin Exp Ophthalmol . 2008; 246: 435–441. [CrossRef] [PubMed]
Jonkers D Penders J Masclee A Pierik M. Probiotics in the management of inflammatory bowel disease: a systematic review of intervention studies in adult patients. Drugs . 2012; 72: 805–823. [CrossRef]
Weise C Zhu Y Ernst D Kuhl AA Worm M. Oral administration of Escherichia coli Nissle 1917 prevents allergen-induced dermatitis in mice. Exp Dermatol . 2011; 20: 805–809. [CrossRef] [PubMed]
Adam E Delbrassine L Bouillot C Probiotic Escherichia coli Nissle 1917 activates DC and prevents house dust mite allergy through a TLR4-dependent pathway. Eur J Immunol . 2010; 40: 1995–2005. [CrossRef] [PubMed]
Henker J Laass M Blokhin BM The probiotic Escherichia coli strain Nissle 1917 (EcN) stops acute diarrhoea in infants and toddlers. Eur J Ped . 2007; 166: 311–318. [CrossRef]
Henker J Laass MW Blokhin BM Probiotic Escherichia coli Nissle 1917 versus placebo for treating diarrhea of greater than 4 days duration in infants and toddlers. Pediatr Infect Dis J . 2008; 27: 494–499. [CrossRef] [PubMed]
Grabig A Paclik D Guzy C Escherichia coli strain Nissle 1917 ameliorates experimental colitis via toll-like receptor 2- and toll-like receptor 4-dependent pathways. Infect Immun . 2006; 74: 4075–4082. [CrossRef] [PubMed]
Eko FO Lubitz W McMillan L Recombinant Vibrio cholerae ghosts as a delivery vehicle for vaccinating against Chlamydia trachomatis . Vaccine . 2003; 21: 1694–1703. [CrossRef] [PubMed]
Eko FO Schukovskaya T Lotzmanova EY Evaluation of the protective efficacy of Vibrio cholerae ghost (VCG) candidate vaccines in rabbits. Vaccine . 2003; 21: 3663–3674. [CrossRef] [PubMed]
Walcher P Cui X Arrow JA Bacterial ghosts as a delivery system for zona pellucida-2 fertility control vaccines for brushtail possums (Trichosurus vulpecula). Vaccine . 2008; 26: 6832–6838. [CrossRef] [PubMed]
Hensel A Huter V Katinger A Intramuscular immunization with genetically inactivated (ghosts) Actinobacillus pleuropneumoniae serotype 9 protects pigs against homologous aerosol challenge and prevents carrier state. Vaccine . 2000; 18: 2945–2955. [CrossRef] [PubMed]
Huter V Hensel A Brand E Lubitz W. Improved protection against lung colonization by Actinobacillus pleuropneumoniae ghosts: characterization of a genetically inactivated vaccine. J Biotechnol . 2000; 83: 161–172. [CrossRef] [PubMed]
Mader HJ Szostak MP Hensel A Lubitz W Haslberger AG. Endotoxicity does not limit the use of bacterial ghosts as candidate vaccines. Vaccine . 1997; 15: 195–202. [CrossRef] [PubMed]
Henrich-Noack P Prilloff S Voigt N In vivo visualisation of nanoparticle entry into central nervous system tissue. Arch Toxicol . 2012; 86: 1099–1105. [CrossRef] [PubMed]
Xu Q Kambhampati SP Kannan RM. Nanotechnology approaches for ocular drug delivery. Middle East Afr J Ophthalmol . 2013; 20: 26–37. [CrossRef] [PubMed]
Footnotes
 ES and AI-K contributed equally to the work presented here and should therefore be regarded as equivalent authors.
Figure 1
 
Analysis of Atto488-labeled EcN BGs uptake by HCjE cells by laser scanning microscopy. HCjE cells were incubated for 30 minutes (A) and 120 minutes (B) with Atto488-EcN BGs (1000 per cell), washed with PBS, and cells on imaging plates were subjected directly to live cell staining. Scale bar: 10 μm. EcN BGs found in HCjE cells are marked by arrows (A, B). Values indicated on histogram y-axes (C) were calculated as the percentage of cells incubated on ice with Atto488-labeled EcN BGs subtracted from the percentage of positive cells incubated with Atto488-labeled EcN BGs at 37°C. Data represent the mean of four independent samples ± SD. The significance of the differences between specific groups defined by the incubation time and the ratio of EcN BGs to HCjE was determined by t-test (*P < 0.05, **P < 0.005, ****P < 0.00005). Compared groups are indicated by double-head arrow.
Figure 1
 
Analysis of Atto488-labeled EcN BGs uptake by HCjE cells by laser scanning microscopy. HCjE cells were incubated for 30 minutes (A) and 120 minutes (B) with Atto488-EcN BGs (1000 per cell), washed with PBS, and cells on imaging plates were subjected directly to live cell staining. Scale bar: 10 μm. EcN BGs found in HCjE cells are marked by arrows (A, B). Values indicated on histogram y-axes (C) were calculated as the percentage of cells incubated on ice with Atto488-labeled EcN BGs subtracted from the percentage of positive cells incubated with Atto488-labeled EcN BGs at 37°C. Data represent the mean of four independent samples ± SD. The significance of the differences between specific groups defined by the incubation time and the ratio of EcN BGs to HCjE was determined by t-test (*P < 0.05, **P < 0.005, ****P < 0.00005). Compared groups are indicated by double-head arrow.
Figure 2
 
Flow cytometry results displaying the time- and dose-dependent internalization of EcN BGs after incubation with HCjE cells. Cells were incubated for 30 (left) and 120 (right) minutes at 37°C with different concentrations of EcN BGs: 100 EcN BGs/cell (dotted), 1000 EcN BGs/cell (dashed), and 10,000 EcN BGs/cell (long dashed). HCjE cells incubated without EcN BGs served as controls (shaded area). Trypan blue solution was added to a final concentration of 0.2% directly before analysis to eliminate signals from the attached BGs. Each sample is analyzed in duplicate and two independent experiments were performed. Representative plots are provided.
Figure 2
 
Flow cytometry results displaying the time- and dose-dependent internalization of EcN BGs after incubation with HCjE cells. Cells were incubated for 30 (left) and 120 (right) minutes at 37°C with different concentrations of EcN BGs: 100 EcN BGs/cell (dotted), 1000 EcN BGs/cell (dashed), and 10,000 EcN BGs/cell (long dashed). HCjE cells incubated without EcN BGs served as controls (shaded area). Trypan blue solution was added to a final concentration of 0.2% directly before analysis to eliminate signals from the attached BGs. Each sample is analyzed in duplicate and two independent experiments were performed. Representative plots are provided.
Figure 3
 
Influence of EcN BGs on the viability of HCjE cells measured through the LDH release. Cells were seeded at a density of 4 × 104 cells in 96-well plates, and incubated with EcN BGs at three different methods of inoculation (MOIs): 100, 1000, and 10,000 EcN BGs/cell. Total co-incubation times were 30 and 120 minutes under standard conditions (5% CO2/37°C). LDH release was measured after 30 minutes, 120 minutes, and 24 hours. Each bar represents the mean of three independent experiments performed in triplicate (±SD).
Figure 3
 
Influence of EcN BGs on the viability of HCjE cells measured through the LDH release. Cells were seeded at a density of 4 × 104 cells in 96-well plates, and incubated with EcN BGs at three different methods of inoculation (MOIs): 100, 1000, and 10,000 EcN BGs/cell. Total co-incubation times were 30 and 120 minutes under standard conditions (5% CO2/37°C). LDH release was measured after 30 minutes, 120 minutes, and 24 hours. Each bar represents the mean of three independent experiments performed in triplicate (±SD).
Figure 4
 
Pictures of guinea pig eyes inoculated with 1 × 107 EcN BGs (A) or PBS as control (B) 120 minutes after inoculation. No signs of irritation, redness, or exaggerated lacrimation could be detected. Histologic examination (H&E staining) of conjunctival tissue performed at sections of the tissue collected 120 minutes after inoculation showed no differences between the BG-treated group (C) and the controls (D). Scale bars: 200 μm.
Figure 4
 
Pictures of guinea pig eyes inoculated with 1 × 107 EcN BGs (A) or PBS as control (B) 120 minutes after inoculation. No signs of irritation, redness, or exaggerated lacrimation could be detected. Histologic examination (H&E staining) of conjunctival tissue performed at sections of the tissue collected 120 minutes after inoculation showed no differences between the BG-treated group (C) and the controls (D). Scale bars: 200 μm.
Figure 5
 
Immunofluorescent analysis of guinea pig conjunctival tissue performed 120 minutes after EcN BGs (A) and PBS (B) inoculation (25 μL of PBS containing 1 × 107 EcN BGs particles or 25 μL of PBS alone, respectively, was instilled directly into the conjunctival sacs). EcN BGs are found in the conjunctival epithelium (marked by arrows). Paraffin sections of guinea pig conjunctiva were stained with rabbit anti-EcN BGs polyclonal antibodies, followed by goat CF488-labeled anti-rabbit IgG. The guinea pigs receiving only PBS were used as sham controls. Data are representative of observations from three guinea pigs in each experimental group. Scale bars: 20 μm.
Figure 5
 
Immunofluorescent analysis of guinea pig conjunctival tissue performed 120 minutes after EcN BGs (A) and PBS (B) inoculation (25 μL of PBS containing 1 × 107 EcN BGs particles or 25 μL of PBS alone, respectively, was instilled directly into the conjunctival sacs). EcN BGs are found in the conjunctival epithelium (marked by arrows). Paraffin sections of guinea pig conjunctiva were stained with rabbit anti-EcN BGs polyclonal antibodies, followed by goat CF488-labeled anti-rabbit IgG. The guinea pigs receiving only PBS were used as sham controls. Data are representative of observations from three guinea pigs in each experimental group. Scale bars: 20 μm.
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