New strategies for culturing and co-culturing of the main types of urinary bladder cells are essential for successful establishment of biomimetic in vitro models, which could be applied for research into, and management of, diverse urological disorders. Porcine normal urothelial cells are available in nearly unlimited amounts and have many properties equivalent to human urothelial cells. In the present study, we established normal differentiated porcine urothelial cells in co-cultures with porcine urinary bladder normal fibroblasts and/or smooth muscle cells. The optimal culture medium for establishment of differentiated urothelial cells, demonstrated by positive immunofluorescence of uroplakins, cytokeratins (CK 7, CK 20), zonula occludens 1 (ZO-1), claudin 4, claudin 8, and E-cadherin, was the medium composed of equal parts of Advanced Dulbecco's modified Eagle's medium (A-DMEM) and MCDB 153 medium with physiological calcium concentration of 2.5 mM and without fetal bovine serum, named UroM (+Ca - S). This medium was also proven to be suitable for culturing of bladder fibroblasts and smooth muscle cells and co-culturing of urothelial cells with these mesenchymal cells. Urothelial cell differentiation was optimal in UroM (+Ca - S) medium in all co-culture conditions and when compared to all conditioned-media combinations. To summarize, these strategies for culturing and co-culturing of urinary bladder urothelial cells with mesenchymal cells could be used as new in vitro models for future basic and applicable research of the urinary bladder and thus potentially also for translational tissue engineering studies.

Introduction Influenza may cause severe complications in patients with autoimmune inflammatory rheumatic disease (AIRD), to whom vaccinations are especially recommended. However, AIRD patients require cautious scrutiny of immunogenicity as they might exhibit poor antibody response to vaccination, especially when taking immunomodulatory medications. Aim The aim was to determine immunogenicity of seasonal and pandemic influenza vaccine in AIRD patients, its timeline/ persistence, and influence of medications on immune response. Methods One hundred and thirty-seven AIRD and 54 healthy controls were vaccinated with trivalent seasonal influenza. After 3-5 weeks, 15 healthy controls and 93 AIRD were vaccinated with pandemic influenza vaccine, and 63 of patients were vaccinated a second time after 3-5 weeks. Sera were collected before vaccination, 18-90 days after each vaccination, and more than 180 days after the last vaccination. The immune response was measured using hemagglutination inhibition (HI) assay and IgG/IgA antibodies against influenza A/B with ELISA. Results Our findings indicate that following vaccination with seasonal influenza vaccine, seroprotection, seroresponse, and change in geometric mean titers (GMT) in AIRD patients was not compromised compared to healthy. Similarly, we report for pandemic influenza vaccination little added benefit of the second dose. We confirm lowest increase in HI titer in rituximab-treated AIRD compared to other medications. Vaccination largely tilts the balance from negative ELISA A IgG and IgA titers to positive titers in seasonal H1N1 seroresponsive AIRD patients and controls. A significant decrease in HI GMT and seroprotection was observed only in AIRD at > 180 days after vaccination highlighting an absent persistence of immunogenic response in AIRD patients. Due to high initial HI titers for influenza vaccine, we foresee their benefit in personalized medicine in the future. Conclusion Influenza vaccination is immunologically active for AIRD, with little value of the second dose of the pandemic vaccine and further scrutiny on persistence of immune response to vaccine in AIRD is needed.

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