Tff3 Deficiency Protects against Hepatic Fat Accumulation after Prolonged High-Fat Diet.

Trefoil factor 3 (Tff3) protein is a small secretory protein expressed on various mucosal surfaces and is involved in proper mucosal function and recovery via various mechanisms, including immune response. However, Tff3 is also found in the bloodstream and in various other tissues, including the liver. Its complete attenuation was observed as the most prominent event in the early phase of diabetes in the polygenic Tally Ho mouse model of diabesity. Since then, its role in metabolic processes has emerged. To elucidate the complex role of Tff3, we used a new Tff3-deficient mouse model without additional metabolically relevant mutations (Tff3-/-/C57BL/6NCrl) and exposed it to a high-fat diet (HFD) for a prolonged period (8 months). The effect was observed in male and female mice compared to wild-type (WT) counter groups (n = 10 animals per group). We monitored the animals' general metabolic parameters, liver morphology, ultrastructure and molecular genes in relevant lipid and inflammatory pathways. Tff3-deficient male mice had reduced body weight and better glucose utilization after 17 weeks of HFD, but longer HFD exposure (32 weeks) resulted in no such change. We found a strong reduction in lipid accumulation in male Tff3-/-/C57BL/6NCrl mice and a less prominent reduction in female mice. This was associated with downregulated peroxisome proliferator-activated receptor gamma (Pparγ) and upregulated interleukin-6 (Il-6) gene expression, although protein level difference did not reach statistical significance due to higher individual variations. Tff3-/-/C57Bl6N mice of both sex had reduced liver steatosis, without major fatty acid content perturbations. Our research shows that Tff3 protein is clearly involved in complex metabolic pathways. Tff3 deficiency in C57Bl6N genetic background caused reduced lipid accumulation in the liver; further research is needed to elucidate its precise role in metabolism-related events.

Production and Secretion of Gelsolin by Both Human Macrophage- and Fibroblast-like Synoviocytes and GSN Modulation in the Synovial Fluid of Patients with Various Forms of Arthritis.

Gelsolin (GSN) is an actin-binding protein involved in cell formation, metabolism and wound closure processes. Since this protein is known to play a role in arthritis, here we investigate how the synovial membrane with its specific synoviocytes contributes to the expression of GSN and how the amount of GSN expressed is modulated by different types of arthritis. Synovial membranes from adult healthy subjects and patients with rheumatoid arthritis (RA) and osteoarthritis (OA) are analyzed by immunofluorescence, Western blot and ELISA. Macrophage-like synoviocytes (MLS) and fibroblast-like synoviocytes (FLS) were isolated, cultured and analyzed for their potential to produce and secrete GSN. In addition, the GSN concentrations in the synovial fluid of various forms of arthritis are determined by ELISA. GSN is produced by the healthy and arthritic synovial membranes. Both forms of synoviocytes (MLS and FLS) release GSN. The results show that there is a significant reduction in GSN in the synovial fluid in adult patients with OA. This reduction is also detectable in adult patients with RA but is not as evident. In juvenile arthritis, there is a slight increase in GSN concentration in the synovial fluid. This study shows that primary MLS and FLS express GSN and that these cells, in addition to articular chondrocytes, contribute to GSN levels in synovial fluid. Furthermore, GSN concentrations are modulated in different types of arthritis. Further studies are needed to fully understand how GSN is involved in joint homeostasis.

Anti-inflammatory effects of an autologous gold-based serum therapy in osteoarthritis patients.

Osteoarthritis (OA) involves activation and recruitment of immune cells to affected joints, including the production of pro-inflammatory cytokines. Here, a gold-based autologous serum therapy is investigated for its effect on peripheral blood cell composition and cytokine levels in OA patients. From six OA patients serum and blood samples were collected before and after second therapy treatment for analysis of peripheral blood cell composition as well as cytokine levels compared to control samples. This therapy significantly downregulates CD4+ T cells and B cells in OA patients after second treatment compared to healthy controls. Monocytes are significantly upregulated in patients after second treatment Serum IL-9 and TNF-α levels are downregulated in patients after second treatment compared to healthy control serum. The activation status of immune cells was modulated after therapy in patients. Anti-inflammatory effects of the peripheral blood cell composition in OA patients can be seen after therapy treatment. After two treatments IL-9 and TNF-α are significantly downregulated in patient serum. Here, primary data of a new autologous therapy for OA treatment and its modulatory effects on cytokines are presented.

GPR40 Activation Abolishes Diabetes-Induced Painful Neuropathy by Suppressing VEGF-A Expression.

G-protein-coupled receptor 40 (GPR40) is a promising target to support glucose-induced insulin release in patients with type 2 diabetes. We studied the role of GPR40 in the regulation of blood-nerve barrier integrity and its involvement in diabetes-induced neuropathies. Because GPR40 modulates insulin release, we used the streptozotocin model for type 1 diabetes, in which GPR40 functions can be investigated independently of its effects on insulin release. Diabetic wild-type mice exhibited increased vascular endothelial permeability and showed epineural microlesions in sciatic nerves, which were also observed in naïve GPR40-/- mice. Fittingly, expression of vascular endothelial growth factor-A (VEGF-A), an inducer of vascular permeability, was increased in diabetic wild-type and naïve GPR40-/- mice. GPR40 antagonists increased VEGF-A expression in murine and human endothelial cells as well as permeability of transendothelial barriers. In contrast, GPR40 agonists suppressed VEGF-A release and mRNA expression. The VEGF receptor inhibitor axitinib prevented diabetes-induced hypersensitivities and reduced endothelial and epineural permeability. Importantly, the GPR40 agonist GW9508 reverted established diabetes-induced hypersensitivity, an effect that was blocked by VEGF-A administration. Thus, GPR40 activation suppresses VEGF-A expression, thereby reducing diabetes-induced blood-nerve barrier permeability and reverting diabetes-induced hypersensitivities.

Gold-based blood serum treatment promotes wound closure of corneal epithelial cell defects in primary in vitro experiments.

BACKGROUND: Wound healing disorders characterised by impaired or delayed reepithelialisation are a serious medical problem. In the present study, we show that gold-based blood serum therapy is a suitable therapeutic approach and shows a supportive effect in wound closure of human corneal epithelial cells (HCE) in primary in vitro experiments. METHODS: For this purpose, blood from healthy individuals was incubated without (S.Ctrl) or with gold-microparticles (S.Therapy) for 24 h. Prior to human epithelial cell stimulation (HCE), the gold particles were removed and the serum was diluted in DMEM (10 % or 30 %). Both groups of serum were compared after injury. HCE were cultured and injured (corneal in vitro wound model) and then stimulated with S.Ctrl or S. RESULTS: Treatment with serum from a gold-based serum therapy (S.Therapy) shows a supportive effect on wound healing in HCE cells in vitro. In addition, gold therapy supports the secretion of important cytokines normally associated with ocular surface wound healing (IL-1ß, IL-6, TNF-α and TGF-ß) in HCE cells. CONCLUSIONS: Therapy with gold-based blood serum significantly promotes the secretion and expression of cytokines and growth factors in HCE cells in vitro. Further preclinical experiments are necessary to demonstrate the influence of this therapy on HCE cells for possible clinical application on the human ocular surface and to prove its function also in poorly healing corneal lesions.

The distribution of conjunctival goblet cells in mice.

PURPOSE: To evaluate the density and distribution of conjunctival goblet cells in mice without clinical evidence of ocular surface diseases. METHODS: Immediately after euthanasia of C57BL/6 wild-type mice, the eyes including eyelids were removed and fixed in paraformaldehyde. Entire eyeballs and eyelids were cut in series along the sagittal axis from nasal to temporal on a microtome and then stained with Periodic Acid-Schiff acid to visualize the goblet cells. At each section stained in this way, the conjunctival goblet cells of the entire upper and lower lid conjunctiva were counted by light microscopy. Additional (transmission electron microscopy) (TEM)-Analysis on ultrathin sections was performed to evaluate morphological differences. RESULTS: The total number of conjunctival goblet cells differs markedly between individual animals. Categorisation into upper eyelid (UL) and lower eyelid (LL) and into regions (nasal, middle, temporal) revealed a significant increase of goblet cells from nasal to temporal in the UL and a significant decrease in the LL. CONCLUSION: The distribution of conjunctival goblet cells in mice differs considerably from humans and between individual animals. Therefore, precise selection of sampling and methods are needed to obtain comparable data. We recommend to use the middle region of the conjunctiva of UL/LL for goblet cell studies in mice. These findings are of particular interest for dry eye mouse models as well as pharmacological studies on mice with influence on their goblet cells.

Recombinant human gelsolin promotes the migration of human articular cartilage chondrocytes by regulating gene expression in vitro.

Objective: It is known that recombinant human gelsolin (rhuGSN) supports wound closure and migration processes in avascular tissue. Since articular cartilage degradation plays an important role in osteoarthritis (OA), we are investigating how rhuGSN affects regeneration processes in human articular cartilage and represents a promising new therapeutic approach for the treatment of OA. Methods: Primary human chondrocytes (phCs) from articular knee cartilage were cultured with different concentrations of rhuGSN to analyse its direct effect in vitro. In addition, phCs were stimulated with 10 ng/mL IL-1ß or TNF-α to simulate osteoarthritis in vitro and treated with different concentrations of rhuGSN to investigate the beneficial effect in disease treatment. Cytokine secretion and gene expression as well as wound assays were performed. Results: GSN significantly promotes wound closure in phCs after 60 h compared to untreated cells. After 24 h treatment with 30 µg/mL rhuGSN, TGF-ß secretion increases significantly in the in vitro osteoarthritis model. Gene expression of MMP1 as well as SPARC is reduced in chondrocytes due to treatment with GSN in the OA model. At the same time, CXCR4 expression increases significantly after 24 h treatment with 3 µg/mL GSN. Conclusion: In the in vitro model of osteoarthritis, rhuGSN promotes wound closure of chondrocytes by a supported migration as well as expression of reconstructive and down regulated expression of deconstructive genes concentration dependently. Further experiments are needed to fully understand the beneficial effect of gelsolin on human chondrocytes and to verify this promising approach for a pharmacological treatment of osteoarthritis.

Effect of Tff3 Deficiency and ER Stress in the Liver.

Endoplasmic reticulum (ER) stress, a cellular condition caused by the accumulation of unfolded proteins inside the ER, has been recognized as a major pathological mechanism in a variety of conditions, including cancer, metabolic and neurodegenerative diseases. Trefoil factor family (TFFs) peptides are present in different epithelial organs, blood supply, neural tissues, as well as in the liver, and their deficiency has been linked to the ER function. Complete ablation of Tff3 expression is observed in steatosis, and as the most prominent change in the early phase of diabetes in multigenic mouse models of diabesity. To elucidate the role of Tff3 deficiency on different pathologically relevant pathways, we have developed a new congenic mouse model Tff3-/-/C57BL6/N from a mixed background strain (C57BL6/N /SV129) by using a speed congenics approach. Acute ER stress was evoked by tunicamycin treatment, and mice were sacrificed after 24 h. Afterwards the effect of Tff3 deficiency was evaluated with regard to the expression of relevant oxidative and ER stress genes, relevant proinflammatory cytokines/chemokines, and the global protein content. The most dramatic change was noticed at the level of inflammation-related genes, while markers for unfolded protein response were not significantly affected. Ultrastructural analysis confirmed that the size of lipid vacuoles was affected as well. Since the liver acts as an important metabolic and immunological organ, the influence of Tff3 deficiency and physiological function possibly reflects on the whole organism.

Structure, regulation and related diseases of the actin-binding protein gelsolin.

Gelsolin (GSN), one of the most abundant actin-binding proteins, is involved in cell motility, shape and metabolism. As a member of the GSN superfamily, GSN is a highly structured protein in eukaryotic cells that can be regulated by calcium concentration, intracellular pH, temperature and phosphatidylinositol-4,5-bisphosphate. GSN plays an important role in cellular mechanisms as well as in different cellular interactions. Because of its participation in immunologic processes and its interaction with different cells of the immune system, GSN is a potential candidate for various therapeutic applications. In this review, we summarise the structure of GSN as well as its regulating and functional roles, focusing on distinct diseases such as Alzheimer's disease, rheumatoid arthritis and cancer. A short overview of GSN as a therapeutic target in today's medicine is also provided.