Ulrike Hampel

Chemokine and cytokine levels in osteoarthritis and rheumatoid arthritis synovial fluid.

To develop a method of the assay of chemokine and cytokine signaling in synovial fluid from patients suffering from osteoarthritis (OA) or rheumatoid arthritis (RA) and evaluate the effect of heterophilic antibodies on the reliability of the data. 21 synovial fluid samples from OA and 16 synovial fluid samples from RA patients were analyzed using a unique 2 step dot sandwich ELISA based micro-well protein array designed to detect heterophilic antibody signaling in the presence or absence of an effective heterophilic blocking reagent with assays carried out for Eotaxin, hGROa, interleukin (IL)-8, IP10, MCP-1, MCP-2, MIG, RANTES, TARC and IL-6. Array analysis reveals that the selective presence of heterophilic antibodies interferes with the accurate assay of synovial fluid samples from a minority of RA patients but not OA synovia. Using a commercial blocking diluent OA and RA synovial fluids reveal significant differences in chemokine content (IL-6, Eotaxin, hGROa, MCP-2, MIG, TARC, IL-8, RANTES). Using a two-step assay protocol it is possible to readily detect inappropriate antibody signaling due to heterophilic antibodies and devise a protocol designed to eliminate this problem thereby more accurately quantify cytokines and chemokines specific to both RA and OA fluids.

Trefoil factor family peptide 3 (TFF3) is upregulated under experimental conditions similar to dry eye disease and supports corneal wound healing effects in vitro.

PURPOSEnTo elucidate the role of trefoil family peptide (TFF) 3 at the ocular surface under conditions similar to dry eye disease (DED) and in tears of patients suffering from DED.nnnMETHODSnTrefoil family peptide 3 levels in tear samples from non-Sjögrens DED patients with moderate dry eye were analyzed by ELISA and compared with tears from healthy volunteers. A human corneal epithelial (HCE) cell line was treated with proinflammatory cytokines IL-1β and TNF-α, hyperosmolar medium, or scratching for up to 24 hours. Trefoil family peptide 3 gene expression and protein biosynthesis were analyzed by RT-PCR, immunofluorescence, and ELISA. Migration and proliferation of HCE cells under recombinant (r) human (h) trefoil factor family peptide 3 (TFF3) stimulation were investigated by scratching and bromodeoxyuridine (BrdU) proliferation assays.nnnRESULTSnTears of patients suffering from DED contained significantly higher TFF3 levels than tears from healthy volunteers. Stimulation of HCE cells with proinflammatory cytokines, culture under hyperosmolar conditions, or scratching resulted, with the exception of hyperosmolar conditions, in an increase in TFF3 expression and elevated secretion level of TFF3. Cell proliferation decreased and cell migration increased after 24-hours stimulation with rhTFF3.nnnCONCLUSIONSnThese results suggest that inflammatory factors or ocular surface damage as they occur in DED, lead to an increase of TFF3 tear film concentration, whereas hyperosmolarity does not. Our data underline a potential role for TFF3 as a candidate therapeutic for the ocular surface damage observed in DED.

Fresh and cryopreserved amniotic membrane secrete the trefoil factor family peptide 3 that is well known to promote wound healing

Amniotic membrane (AM) is often used for the treatment of ocular surface ulcerations and other corneal defects. Trefoil factor family (TFF) peptide 3 is produced by conjunctival goblet cells, participates in tear film physiology and has also been shown to be involved in ocular surface restitution after corneal injury. In the present study, we questioned whether AM also might be a source of TFF3 and if yes whether the secretion rate of TFF3 is changed by proinflammatory cytokines or by cryoconservation of AM. By means of RT-PCR, the mRNA expression of all three known TFF peptides could be detected in AM. Immunohistochemistry on paraffin-embedded sections localized TFF3 protein and also TFF2 in AM cells and Western blot analysis revealed TFF3 protein in AM. Stimulation experiments with proinflammatory cytokines and subsequent TFF3 ELISA measurements revealed that the secretion rate of fresh or cryoconserved AM was not significantly changed. The results indicate that TFF peptides are produced by AM. TFF3 may contribute to ocular surface wound healing after AM transplantation, but its production by AM is not further inducible by proinflammatory stimuli. Cryopreservation has no effect on the secretion rate of TFF3 supporting the use of cryopreserved AM for transplantation.

In vitro effects of sex hormones in human meibomian gland epithelial cells.

Meibomian gland dysfunction (MGD) is considered the most common cause of dry eye disease (DED). Sex hormones seem to play a role in the pathogenesis of MGD although their involvement is not completely understood. Therefore, in the present study we evaluated the effect of dihydrotestosteron (DHT) and estradiol (β-Est) on an immortalized human meibomian gland epithelial cell line (HMGEC). Protein expression of sex hormone receptors in HMGEC was investigated by western blot. Ultrastructural morphology, Sudan III lipid staining, cell proliferation as well as vitality assays were performed. Furthermore, expression of MGD-associated markers for keratinization (hornerin, involucrin and CK6), proliferation (CK5 and CK14) and lipid synthesis (fatty acid synthase and stearoyl-CoA desaturase) were analyzed by real time RT-PCR. Western blot revealed presence of androgen receptor (AR), estrogen receptors α and -β (ERα, ERβ) and progesterone receptor (PR) in HMGEC. PR, ERα and ERβ expression was significantly induced under cultivation with serum, whereas sex hormones stimulation showed no further effect on protein expression of PR, ERα and ERβ. Our results showed no impact of MGD-associated sex hormones to cellular morphology and lipid accumulation in HMGEC. Cell proliferation was slightly induced through application of sex hormones and supplementation of calcium. However, both sex hormones and calcium altered gene expression of MGD-associated markers. Especially keratinization genes hornerin (HRNR) and cornulin (COR) were induced after application of sex hormones and calcium in serum-free cultivated HMGEC. This may promote keratinization processes that are associated with MGD. Further investigations are necessary to analyze the (hyper)keratinization processes that occur during MGD and using HMGEC as an inxa0vitro model.

In vitro effects of docosahexaenoic and eicosapentaenoic acid on human meibomian gland epithelial cells

To investigate the effect of ω-3 fatty acids on human meibomian gland epithelial cells (HMGECs, cell line) inxa0vitro. HMGECs were stimulated with docosahexaenoic acid (DHA) or combinations with eicosapentaenoic acid (EPA) and acetyl sialic acid (ASA). Sudan III fat staining, viability and proliferation assays, electric cell-substrate impedance sensing, real-time PCR for gene expression of cyclooxygenase-2 and 15-lipoxygenase and ELISAs for resolvin D1 (RvD1), IFNγ, TNFα and IL-6 were applied. Lipid droplet accumulation and viability was increased by 100xa0μM DHA in the presence or absence of EPA in serum cultured HMGECs. In contrast, HMGECs cultured with DHA and EPA under serum-free conditions showed minimal lipid accumulation, decreased proliferation and viability. Normalized impedance was significantly reduced in serum-free cultured HMGECs when stimulated with DHA and EPA. HMGECs cultured in serum containing medium showed increased normalized impedance under DHA and EPA stimulation compared to DHA or EPA alone or controls. IL-6 and IFNγ were downregulated in HMGECs treated for 72xa0h with DHA and EPA. In general, TNFα, IFNγ and IL-6 levels were decreased after 72xa0h compared to 24xa0h in serum containing medium with or without DHA or EPA. The concentration of RvD1 was elevated 2-fold after DHA treatment. Cyclooxygenase-2 gene expression decreased compared to controls during DHA stimulation after 72xa0h. Treatment with DHA and ASA revealed a decreased 15-lipoxygenase gene expression which was reduced after three days of DHA incubation. DHA and EPA supplementation affected HMGECs inxa0vitro and supported anti-inflammatory effects by influencing cytokine levels, decreasing COX-2 expression and increasing the production of RvD1.

Relaxin 2 is functional at the ocular surface and promotes corneal wound healing.

PURPOSEnWe aimed to determine if the insulin-like peptide hormone relaxin 2 (RLN2) is expressed at the ocular surface and in tears and if RLN2 influences wound healing at the ocular surface, which is associated with extracellular matrix (ECM) remodeling.nnnMETHODSnWe analyzed transcript levels of human RLN2 and its cognate relaxin-like receptors RXFP1 and RXFP2 in tissues of the ocular surface, lacrimal apparatus, and human corneal (HCE), conjunctival (HCjE) and sebaceous (SC) cell lines. We analyzed effects of human RLN2 on cell proliferation and migration and quantified mRNA expression of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in HCE, HCjE, and SC. Using an alkali-induced corneal wounding model, we analyzed the wound healing rate in C57BL/6 mice eyes after topically applied RLN2.nnnRESULTSnThe presence of RLN2, RXFP1, and RXFP2 transcripts was detected in lacrimal gland, eyelid, conjunctiva, cornea, primary corneal fibroblasts, nasolacrimal ducts, and all three cell lines. ELISA revealed RLN2 protein in all ocular surface tissues analyzed and in human tears. Stimulation of HCE, HCjE, and SC with RLN2 significantly increased cell proliferation and migration. Relative mRNA expression levels of MMP2, MMP9, TIMP1, and TIMP2 were significantly influenced by RLN2 in all three cell lines at different time points studied. The local application of RLN2 onto denuded corneal surface resulted in significantly elevated corneal wound healing.nnnCONCLUSIONSnOur data support a novel role for the RLN2 ligand-receptor system at the ocular surface and in the lacrimal apparatus as a potential future therapeutic during wound healing at the ocular surface.

Morphological features of the porcine lacrimal gland and its compatibility for human lacrimal gland xenografting.

In this study, we present first data concerning the anatomical structure, blood supply and location of the lacrimal gland of the pig. Our data indicate that the porcine lacrimal gland may serve as a potential xenograft candidate in humans or as an animal model for engineering of a bioartificial lacrimal gland tissue construct for clinical application. For this purpose, we used different macroscopic preparation techniques and digital reconstruction of the histological gland morphology to gain new insights and important information concerning the feasibility of a lacrimal gland transplantation from pig to humans in general. Our results show that the lacrimal gland of the pig reveals a lot of morphological similarities to the analogous human lacrimal gland and thus might be regarded as a xenograft in the future. This is true for a similar anatomical location within the orbit as well as for the feeding artery supply to the organ. Functional differences concerning the composition of the tear fluid, due to a different secretory unit distribution within the gland tissue will, however, be a challenge in future investigations.

Characterization of the mucocutaneous junction of the human eyelid margin and meibomian glands with different biomarkers

PURPOSEnTo investigate the morphology of the human eyelid margin and the presence of different cytokeratins, mucins and stem cell markers within the skin epithelium, mucocutaneous junction (MCJ) and palpebral conjunctiva.nnnMETHODSnEyelids of body donors were investigated histologically and ultrastructurally as well as by immunohistochemical methods using antibodies to cytokeratins 1, 4, 7, 8, 10, 13, 14, 15, and 19; mucins MUC1, MUC4, and MUC5AC and potential stem cell markers K15, BCRP/ABCG2, integrin β1, and N-cadherin.nnnRESULTSnThe expression pattern of cytokeratins, mucins and stem cell markers varied across the different epithelia of the human eyelid. Within the MCJ, CK7, 15 and 19 were absent, whereas the epithelium reacted positive to antibodies to CK1, 4, 8, 10, 13 and 14. Reactivity was also observed for MUC1 and MUC4, but not for MUC5AC. No reactivity was determined for K15, BCRP/ABCG2 and integrin β1 in the area of the MCJ epithelium but a strong reactivity was present for N-cadherin.nnnCONCLUSIONSnThe present immunohistochemical findings lead to a better characterization of the MCJ. Additionally, the knowledge of distribution of biomarkers like cytokeratins, mucins and stem cells can be useful in the investigation of MCJ disturbances which occur in several disorders of the meibomian glands and the lid epithelium in the course of dry eye syndrome and especially meibomian gland dysfunction.

Serum-induced keratinization processes in an immortalized human meibomian gland epithelial cell line.

Purpose The aim of this study was to evaluate a human meibomian gland epithelial cell line (HMGEC) as a model for meibomian gland (patho)physiology in vitro. Methods HMGEC were cultured in the absence or presence of serum. Sudan III lipid staining, ultrastructural analysis and lipidomic analyses were performed. Impedance sensing, desmoplakin 1/2 mRNA and cytokeratin (CK) 1, 5, 6, 14 levels were evaluated. Serum containing medium supplemented with higher serum, glucose, an omega-3 lipid cocktail, eicosapentaenoic acid or sebomed medium were investigated for lipid accumulation and ultrastructural morphology. Results Lipid droplet accumulation in HMGEC was induced by serum containing media after 1 day, but decreased over time. Cultivation in serum induced desmosome and cytokeratin filament formation. Desmoplakin 1/2 gene levels were significantly upregulated after 1d of serum treatment. Furthermore, the normalized impedance increased significantly. Lipidome analysis revealed high levels of phospholipids (over 50%), but very low levels of wax ester and cholesteryl esters (under 1%). Stimulation with eicosapentaenoic acid increased lipid accumulation after one day. Conclusion Serum treatment of HMGEC caused lipid droplet formation to some extent but also induced keratinization. The cells did not produce typical meibum lipids under these growth conditions. HMGEC are well suited to study (hyper)keratinization processes of meibomian gland epithelial cells in vitro.

Insulin-like Factor 3 Promotes Wound Healing at the Ocular Surface

Tear fluid is known to contain many different hormones with relevance for ocular surface homeostasis. We studied the presence and functional role of insulin-like factor 3 (INSL3) and its cognate receptor RXFP2 (relaxin/insulin-like family peptide receptor 2) at the ocular surface and in tears. Expression of human INSL3 and RXFP2 was determined in tissues of the ocular surface and lacrimal apparatus; in human corneal (HCE), conjunctival (HCjE), and sebaceous (SC) epithelial cell lines; and in human tears by RT-PCR and ELISA. We investigated effects of human recombinant INSL3 (hrINSL3) on cell proliferation and cell migration and the influence of hrINSL3 on the expression of MMP2, -9, and -13 and TIMP1 and -2 was quantified by real-time PCR and ELISA in HCE, HCjE, and SC cells. We used a C57BL/6 mouse corneal defect model to elucidate the effect of topical application of hrINSL3 on corneal wound healing. INSL3 and RXFP2 transcripts and INSL3 protein were detected in all tissues and cell lines investigated. Significantly higher concentrations of INSL3 were detected in tears from male vs. female volunteers. Stimulation of HCE, HCjE, and SC with hrINSL3 significantly increased cell proliferation in HCjE and SC and migration of HCjE. Treatment with hrINSL3 for 24 hours regulated MMP2, TIMP1, and TIMP2 expression. The local application of hrINSL3 onto denuded corneal surface resulted in significantly accelerated corneal wound healing in mice. These findings suggest a novel and gender-specific role for INSL3 and cognate receptor RXFP2 signaling in ocular surface homeostasis and determined a novel role for hrINSL3 in corneal wound healing.