Tanja Stachon

Photodynamic inactivation of multidrug-resistant Staphylococcus aureus by chlorin e6 and red light (λ = 670 nm)

Multidrug-resistant Staphylococcus aureus (MDR-SA) are a frequent cause of antibiotic treatment refractory bacterial corneal infections. Photodynamic therapy (PDT) is being discussed as a putative treatment option to cure this type of bacterial infection. Here we tested the in vitro susceptibility of a set of 12 clinically derived MDR-SA isolates with differing genetic backgrounds and antibiotic resistance profiles against photodynamic inactivation (PDI) by the porphyrin chlorin e6 (Ce6) and red light (λ=670nm). All tested clinical isolates displayed a 5-log10 reduction in viable cells by Ce6 and red light, when cells were preincubated with the photosensitizer at concentrations ≥128μM for 30min in the dark, and a subsequent irradiation with light at λ=670nm (power density: 31mW/cm(2), absorbed dose: 18,6J/cm(2)) was applied. Similarly, cells of the laboratory strain Newman required the same Ce6 pre-incubation and light dose for a 5-log10 reduction in cell viability. Inactivation of crtM in strain Newman, which interferes with pigment production in S. aureus, rendered the mutant more susceptible to this PDT procedure, indicating that the level of resistance of S. aureus to this therapy form is affected by ability of the pathogen to produce the carotenoid pigment staphyloxanthin. Incubation of freshly explanted porcine corneas with a 0.5% Ce6 gel demonstrated that the photosensitizer can diffuse into and accumulate within the stroma of the cornea in concentrations found to be sufficient to yield a 5-log10 reduction of the S. aureus cell pool in vitro. These data suggest that PDI with Ce6 and red light might be a promising new option for the treatment of MDR-SA induced corneal infections.

Impact of crosslinking/riboflavin-UVA-photodynamic inactivation on viability, apoptosis and activation of human keratocytes in vitro

Abstract Riboflavin-UVA photodynamic inactivation is a potential treatment alternative in therapy resistant infectious keratitis. The purpose of our study was to determine the impact of riboflavin-UVA photodynamic inactivation on viability, apoptosis and activation of human keratocytes in vitro. Primary human keratocytes were isolated from human corneal buttons and cultured in DMEM/Hams F12 medium supplemented with 10% fetal calf serum. Keratocytes underwent UVA light illumination (375 nm) for 4.10 minutes (2 J/cm2) during exposure to different concentrations of riboflavin. Twenty-four hours after treatment, cell viability was evaluated photometrically, whereas apoptosis, CD34 and alpha-smooth muscle actin (α-SMA) expression were assessed using flow cytometry. We did not detect significant changes in cell viability, apoptosis, CD34 and α-SMA expression in groups only treated with riboflavin or UVA light. In the group treated with riboflavin-UVA-photodynamic inactivation, viability of keratocytes decreased significantly at 0.1% riboflavin (P<0.01) while the percentage of CD34 (P<0.01 for both 0.05% and 0.1% riboflavin) and alpha-SMA positive keratocytes (P<0.01 and P<0.05 for 0.05% and 0.1% riboflavin, respectively) increased significantly compared to the controls. There was no significant change in the percentage of apoptotic keratocytes compared to controls at any of the used riboflavin concentrations (P = 0.09 and P = 0.13). We concluded that riboflavin-UVA-photodynamic-inactivation decreases viability of myofibroblastic transformation and multipotent haematopoietic stem cell transformation; however, it does not have an impact on apoptosis of human keratocytes in vitro.

Viability, Apoptosis, Proliferation, Activation, and Cytokine Secretion of Human Keratoconus Keratocytes after Cross-Linking

Purpose. The purpose of this study was to determine the impact of cross-linking (CXL) on viability, apoptosis, proliferation, activation, and cytokine secretion of human keratoconus (KC) keratocytes, in vitro. Methods. Primary KC keratocytes were cultured in DMEM/Hams F12 medium supplemented with 10% FCS and underwent UVA illumination (370 nm, 2 J/cm2) during exposure to 0.1% riboflavin and 20% Dextran in PBS. Twenty-four hours after CXL, viability was assessed using Alamar blue assay; apoptosis using APO-DIRECT Kit; proliferation using ELISA-BrdU kit; and CD34 and alpha-smooth muscle actin (α-SMA) expression using flow cytometry. Five and 24 hours after CXL, FGFb, HGF, TGFβ1, VEGF, KGF, IL-1β, IL-6, and IL-8 secretion was measured using enzyme-linked-immunoabsorbent assay (ELISA). Results. Following CXL, cell viability and proliferation decreased (P < 0.05; P = 0.009), the percentage of apoptotic keratocytes increased (P < 0.05) significantly, and CD34 and α-SMA expression remained unchanged (P > 0.06). Five hours after CXL, FGFb secretion increased significantly (P = 0.037); however no other cytokine secretion differed significantly from controls after 5 or 24 hours (P > 0.12). Conclusions. Cross-linking decreases viability, triggers apoptosis, and inhibits proliferation, without an impact on multipotent hematopoietic stem cell transformation and myofibroblastic transformation of KC keratocytes. CXL triggers FGFb secretion of KC keratocytes transiently (5 hours), normalizing after 24 hours.

Wachstumsfaktoren und Interleukine in Amniongewebehomogenat

PURPOSE Application of amniotic membrane homogenate eye drops may be a potential treatment alternative for therapy resistant corneal epithelial defects. The purpose of this study was to determine the concentrations of epidermal growth factor (EGF), fibroblast growth factor basic (bFGF), hepatocyte growth factor (HGF), keratinocyte growth factor (KGF), interleukin-6 (IL-6) and interleukin-8 (IL-8) in amniotic membrane homogenates. METHODS Amniotic membranes of 8 placentas were prepared and thereafter stored at - 80 °C using the standard methods of the LIONS Cornea Bank Saar-Lor-Lux, Trier/Westpfalz. Following defreezing, amniotic membranes were cut in two pieces and homogenized in liquid nitrogen. One part of the homogenate was prepared in cell-lysis buffer, the other part was prepared in PBS. The tissue homogenates were stored at - 20 °C until enzyme-linked immunosorbent assay (ELISA) analysis for EGF, bFGF, HGF, KGF, IL-6 and IL-8 concentrations. RESULTS Concentrations of KGF, IL-6 and IL-8 were below the detection limit using both preparation techniques. The EGF concentration in tissue homogenates treated with cell-lysis buffer (2412 pg/g tissue) was not significantly different compared to that of tissue homogenates treated with PBS (1586 pg/g tissue, p = 0.72). bFGF release was also not significantly different using cell-lysis buffer (3606 pg/g tissue) or PBS treated tissue homogenates (4649 pg/g tissue, p = 0.35). HGF release was significantly lower using cell-lysis buffer (23,555 pg/g tissue), compared to PBS treated tissue (47,766 pg/g tissue, p = 0.007). CONCLUSION Containing EGF, bFGF and HGF, and lacking IL-6 and IL-8, the application of amniotic membrane homogenate eye drops may be a potential treatment alternative for therapy-resistant corneal epithelial defects.

Human aniridia limbal epithelial cells lack expression of keratins K3 and K12

Abstract Aniridia is a rare disease of the eye that affects the iris, lens and the cornea. In about 90% of the cases, patients showed a loss of PAX6 function. Patients with aniridia often develop aniridia‐related keratopathy (ARK), due to limbal stem cell insufficiency. The aim of this study was to determine the differentiation status of limbal epithelial cells (LECs) in patients with ARK. Epithelial cells were isolated from the limbus region of two patients with aniridia and cultured in KSFM medium supplemented with EGF and BPE. Normal cells were obtained from limbus region of cadaveric control patients. Cells were analyzed with RT‐PCR, qPCR and Western blot to evaluate expression of the developmental transcription factor, PAX6, potential stem cell markers, &Dgr;Np63&agr; and ABCG2, and corneal differentiation markers, keratin 12 (K12) and K3. Conjunctival differentiation markers, keratin 13 (K13) and K19 were also investigated. Cells were immunostained to evaluate K3, PAX6, and p63&agr; protein expression. Protein coding sequence of PAX6 from patient LEC‐cDNA was cloned and sequenced. RT‐PCR showed that K3 and K12 transcripts were absent from patient cells, but present in healthy control preparations. Transcription levels of PAX6, ABCG2, and p63&agr; of aniridia patients show no differences compared to normal control cells. Western blot showed reduced PAX6, protein levels in aniridia‐LECs compared to control‐LECs. Immunostaining also showed reduced PAX6 and K3 expression in aniridia‐LECs compared to control‐LECs. One aniridia patient showed a loss of stop codon in half of the cloned transcripts. In the second aniridia patient mRNA degradation through nonsense mediated decay seems to be very likely since we could not identify the mutation c.174C > T (Refseq. NM_000280), or misspliced transcripts in cDNA. We identified decreased PAX6 protein levels in aniridia patients in addition to decreased K12 mRNA levels compared to control cells. This result indicates an altered differentiation of limbal epithelial cells of aniridia patients. Further studies are necessary to evaluate the mechanism of differentiation of limbal epithelial cells in aniridia. HighlightsEpithelial cells isolated from limbus region of aniridia patients can be expanded.Limbal epithelial cells of aniridia patients lack expression of K12 and K3.PAX6 mRNA expression may not correlate with protein expression in NMD mutants.Elongated PAX6 (loss of stop mutation) may be degraded in epithelial cells.

Urea, Uric Acid, Prolactin and fT4 Concentrations in Aqueous Humor of Keratoconus Patients

ABSTRACT Purpose: Keratoconus is a noninflammatory disease of the cornea associated with progressive thinning and conical shape. Metabolic alterations in the urea cycle, with changes in collagen fibril stability, oxidative stress, thyroid hormones and prolactin with regulatory effect on biosynthesis and biomechanical stability of corneal stroma, may all play a role in keratoconus etiology. Our purpose was to determine urea, uric acid, prolactin and free thyroxin (fT4) concentrations in human aqueous humor (hAH) of keratoconus and cataract patients. Methods: hAH was collected from 100 keratoconus (penetrating keratoplasty) (41.9 ± 14.9 years, 69 males) and 100 cataract patients (cataract surgery) (71.2 ± 12.4 years, 58 males). Urea, uric acid, prolactin and fT4 concentrations were measured by Siemens clinical chemistry or immunoassay system. For statistical analysis, a generalized linear model (GLM) was used. Results: Urea concentration was 11.88 ± 3.03 mg/dl in keratoconus and 16.44 ± 6.40 mg/dl in cataract patients, uric acid 2.04 ± 0.59 mg/dl in keratoconus and 2.18 ± 0.73 mg/dl in cataract groups. Prolactin concentration was 3.18 ± 0.34 ng/ml in keratoconus and 3.33 ± 0.32 ng/ml in cataract patients, fT4 20.57 ± 4.76 pmol/l in KC and 19.06 ± 3.86 pmol/l in cataract group. Urea concentration was effected through gender (p = 0.039), age (p = 0.001) and diagnosis (p = 0.025). Uric acid concentration was not effected through any of the analyzed parameters (p > 0.056). Prolactin and fT4 concentration were effected only through diagnosis (p = 0.009 and p = 0.006). Conclusions: Urea and prolactin concentrations are decreased, fT4 concentration is increased in aqueous humor of keratoconus patients, and uric acid concentration remains unchanged. Urea concentration in aqueous humor is also increased in older and male patients. Therefore, metabolic disorder and hormonal balance may both have an impact on keratoconus development. Further studies are necessary to assess the specific impact.

Growth Factor and Interleukin Concentrations in Amniotic Membrane-Conditioned Medium

ABSTRACT Purpose: Application of amniotic membrane-conditioned medium (AMM) eye drops is a potential treatment alternative for therapy-resistant corneal epithelial defects. Our purpose was to determine the concentration of growth factors epidermal growth factor (EGF), nerve growth factor (NGF), vascular endothelial growth factor (VEGF), transforming growth factor β1 (TGFβ1), fibroblast growth factor basic (FGFb), and interleukins, IL-1β, IL-6, IL-8, in AMM following different preparation methods. Methods: Amniotic membranes of 10 placentas were prepared and thereafter stored at −80°C using the standard method of our LIONS Cornea Bank. Following defreezing, amniotic membrane pieces with a standard size were inserted in a 12-well plate either complete or cut in small pieces, and 2000 µl DMEM culture medium was added. EGF, NGF, VEGF, TGFβ1, FGFb, IL-1β, IL-6, and IL-8 concentrations in the culture medium were determined following 8, 48, and 96 hours, and 1, 2, 3, and 4 weeks of incubation using enzyme-linked immunosorbent assay (ELISA). Results: Concentrations of NGF, VEGF, TGFβ1, and IL-1ß were beyond the detection limit at all time points. EGF concentrations were between 0.14 and 0.80 ng/g tissue, FGFb between 0.48 and 2.89 ng/g tissue, IL-6 between 0.11 and 1.41 ng/g tissue, and IL-8 between 0.32 and 6.18 ng/g tissue. A significant difference between both preparation methods was shown for the IL-6 concentration after 8 and 48 hours (p < 0.001; p = 0.01) and in IL-8 concentration after 8 and 96 hours and after 3 weeks (p = 0.02; p = 0.002; p = 0.027). Conclusion: AMM containing EGF and FGFb, and IL-6 and IL-8 AMM is a potential nonsurgical treatment alternative of therapy-resistant corneal epithelial defects. However, the most effective preparation method and the optimal harvesting time point are yet to be determined.

Effect of Keratocyte Supernatant on Epithelial Cell Migration and Proliferation After Corneal Crosslinking (CXL).

Abstract Purpose: To evaluate the effect of keratocyte supernatant (harvesting time, riboflavin concentration and UV-A-light illumination) on migration and proliferation of human corneal epithelial cells (HCECs) by CXL, in vitro. Methods: Primary human keratocytes isolated from 8 normal and 6 keratoconus corneas were cultured. Thereafter, keratocytes in 0%, 0.05% or 1% riboflavin solution were split into samples without and with 370 nm UVA-light-illumination. After removal of the riboflavin solution, keratocytes were incubated in the mentioned keratocyte culture medium at 37 °C and keratocyte supernatant was harvested after 5 and 24 hours. Keratocyte supernatant without riboflavin and UVA treatment, was used as control. HCECs were cultured until reaching confluence, the HCEC culture medium was replaced by the keratocyte supernatant and HCEC migration was analyzed using the wound-healing assay. HCEC proliferation was determined by the cell proliferation ELISA BrdU (colorimetric) kit. Statistical analysis was performed using a linear mixed model in the framework of a Generalized Estimating Equations (GEE) approach to analyze the effect of harvesting time, riboflavin concentration and UV-A-light illumination using IBM-SPSS version 22. Results: Riboflavin concentration, UVA-light illumination and harvesting time of normal or keratoconus keratocyte supernatant had no significant impact on HCEC proliferation (p > 0.10). Riboflavin concentration did not show significant impact on HCEC migration using normal or keratoconus keratocyte supernatant (p > 0.10), however, longer harvesting time of normal or keratoconus keratocyte supernatant significantly increased (p = 0.01 for both) and UVA-light illumination of keratoconus keratocyte supernatant (p < 0.001) significantly decreased HCEC migration. Conclusion: Harvesting time, riboflavin concentration and UV-A-light illumination of normal and keratoconus keratocyte cultures has no impact on proliferation of HCECs, in the short term. However, 24 hours harvesting time (both for normal and keratoconus keratocytes) increases and UVA-light-illumination of keratoconus keratocyte cultures decreases HCEC migration.

The Effect of Antiamoebic Agents on Viability, Proliferation and Migration of Human Epithelial Cells, Keratocytes and Endothelial Cells, In Vitro

ABSTRACT Purpose: To analyze the effect of diamidines (hexamidine-diisethionat (HD), propamidin-isethionate (PD), dibromopropamidine-diisethionat (DD)), and biguanides (polyhexamethylen biguanid (PHMB), chlorhexidine (CH)) on human corneal epithelial cell, keratocyte and endothelial cell viability, proliferation, and migration, in vitro. Methods: For epithelial and endothelial cells a human cell line and for keratocytes primary cultures were used (n = 6 each). We used 3.9x10−4–0.1% HD, PD or DD, 3.9x10−4–0.0125% PD, 7.8x10−5–0.02% PHMB or CH concentration for 24 h to determine viability (Cell Proliferation Kit XTT), proliferation (Cell Proliferation ELISA BrdU kit), and migration using wound healing assay. Viability/proliferation/migration values of each drug were summarized as “area under curve” (AUC) together with a Mann–Whitney test. Results: HCEC, keratocyte, and HCEC-12 viability AUC, comparing PD and PHMB (p ≤ 0.014 for all; PD better) or PD and HD (p ≤ 0.011 for all; PD better) differed significantly. Keratocyte and HCEC-12 viability AUC comparing CH and HD (p ≤ 0.027; CH better), HCEC-12 viability AUC comparing PD and HD (p = 0.005; PD better) and HCEC viability AUC comparing CH and PHMB (p = 0.014; CH better) differed significantly. HCEC proliferation AUC, comparing PD with PHMB, CH, DD, HD (p ≤ 0.016; PD worse for all) and keratocyte proliferation AUC, comparing PHMB with HD, PD (p = 0.004; p = 0.002; PHMB better for both), CH with HD, PD (p ≤ 0.001; CH better for both) and DD with PD (p = 0.043; DD better) differed significantly. Keratocyte migration AUC comparing PD with control, PHMB, CH, DD and HD differed significantly (p ≤ 0.012; PD worse for all). Conclusions: Propamidin-isethionate as diamidine and chlorhexidin as biguanide may be used clinically to reduce cytotoxicity of antiamoebic treatment on human corneal cells. Diamidines reduce proliferation of human epithelial cells and keratocytes more than biguanides and propamidin-isethionate reduces migration of keratocytes. Therefore, in spite of lower cytotoxicity, the inhibitory effect on proliferation and migration indicates that extended use of propamidin-isethionate should be avoided in patients.

Effect of Amniotic Membrane Suspension (AMS) and Amniotic Membrane Homogenate (AMH) on Human Corneal Epithelial Cell Viability, Migration and Proliferation In Vitro.

ABSTRACT Purpose: To analyze the effects of different concentrations of amniotic membrane suspension (AMS) or amniotic membrane homogenate (AMH) on human corneal epithelial cell (HCEC) viability, migration and proliferation. Methods: Amniotic membranes (AMs) of 13 placentas were prepared and stored at −80°C. For AMS preparation, following de-freezing, AM pieces were inserted in six-well plates and 5 ml Dulbecco’s Modified Eagle’s Medium (DMEM)/F12 (with 5% fetal bovine serum, FBS) per gram tissue was added for 96 h. After removal of the AM, the remaining supernatant was collected for experiments. For AMH preparation, following de-freezing, AMs were homogenized in liquid nitrogen and 5 ml DMEM/F12 (with 5% FBS) per gram tissue was added. Following centrifugation, the supernatant was collected for experiments. HCECs were expanded and incubated in DMEM/F12, 5% FBS supplemented by 15%, 30% or 100% AMS or 15% or 30% AMH. Viability was analyzed using Cell Proliferation Kit XTT, migration using wound healing assay and proliferation by the cell proliferation ELISA BrdU kit. Results: HCEC viability remained unchanged using 15% or 30% AMS (p = 1.0 for both); however, it decreased significantly using 100% AMS (p < 0.001) or 15% (p = 0.041) or 30% AMH (p < 0.001), compared to controls. Using 15% or 30% AMS, HCEC migration increased significantly (p < 0.001 for both). Using 15% or 30% AMH (p = 0.153; p = 0.083), HCEC migration remained unchanged and 100% AMS inhibited HCEC migration (p < 0.001). Next, 15% and 30% AMS had no effect on HCEC proliferation (p = 0.454 and p = 0.119), but 100% AMS (p < 0.001) and 15% (p = 0.002) and 30% AMH (p = 0.001) inhibited HCEC proliferation significantly. Conclusion: With unchanged HCEC viability and proliferation and increased HCEC migration, 15% and 30% AMS application seems to be the most appropriate method to support epithelial healing.