Bioprinted 3D Bionic Scaffolds with Pancreatic Islets as a New Therapy for Type 1 Diabetes-Analysis of the Results of Preclinical Studies on a Mouse Model.

Recently, tissue engineering, including 3D bioprinting of the pancreas, has acquired clinical significance and has become an outstanding potential method of customized treatment for type 1 diabetes mellitus. The study aimed to evaluate the function of 3D-bioprinted pancreatic petals with pancreatic islets in the murine model. A total of 60 NOD-SCID (Nonobese diabetic/severe combined immunodeficiency) mice were used in the study and divided into three groups: control group; IsletTx (porcine islets transplanted under the renal capsule); and 3D bioprint (3D-bioprinted pancreatic petals with islets transplanted under the skin, on dorsal muscles). Glucose, C-peptide concentrations, and histological analyses were performed. In the obtained results, significantly lower mean fasting glucose levels (mg/dL) were observed both in a 3D-bioprint group and in a group with islets transplanted under the renal capsule when compared with untreated animals. Differences were observed in all control points: 7th, 14th, and 28th days post-transplantation (129, 119, 118 vs. 140, 139, 140; p < 0.001). Glucose levels were lower on the 14th and 28th days in a group with bioprinted petals compared to the group with islets transplanted under the renal capsule. Immunohistochemical staining indicated the presence of secreted insulin-living pancreatic islets and neovascularization within 3D-bioprinted pancreatic petals after transplantation. In conclusion, bioprinted bionic petals significantly lowered plasma glucose concentration in studied model species.

Streptozotocin-Induced Diabetes in a Mouse Model (BALB/c) Is Not an Effective Model for Research on Transplantation Procedures in the Treatment of Type 1 Diabetes.

Type 1 diabetes (T1D) is characterized by the destruction of over 90% of the ß-cells. C-peptide is a parameter for evaluating T1D. Streptozotocin (STZ) is a standard method of inducing diabetes in animals. Eight protocols describe the administration of STZ in mice; C-peptide levels are not taken into account. The aim of the study is to determine whether the STZ protocol for the induction of beta-cell mass destruction allows for the development of a stable in vivo mouse model for research into new transplant procedures in the treatment of type 1 diabetes. Materials and methods: Forty BALB/c mice were used. The animals were divided into nine groups according to the STZ dose and a control group. The STZ doses were between 140 and 400 mg/kg of body weight. C-peptide was taken before and 2, 7, 9, 12, 14, and 21 days after STZ. Immunohistochemistry was performed. The area of the islet and insulin-/glucagon-expressing tissues was calculated. Results: Mice who received 140, 160, 2 × 100, 200, and 250 mg of STZ did not show changes in mean fasting C-peptide in comparison to the control group and to day 0. All animals with doses of 300 and 400 mg of STZ died during the experiment. The area of the islets did not show any differences between the control and STZ-treated mice in groups below 300 mg. The reduction of insulin-positive areas in STZ mice did not exceed 50%. Conclusions: Streptozotocin is not an appropriate method of inducing a diabetes model for further research on transplantation treatments of type 1 diabetes, having caused the destruction of more than 90% of the ß-cell mass in BALB/c mice.

Endometriotic Peritoneal Fluid Stimulates Recruitment of CD4+CD25highFOXP3+ Treg Cells.

Endometriosis is a common gynecological disorder characterized by the presence of endometrial-like tissue outside the uterus. The disease is associated with disturbed local and systemic immunity. It has been reported that the proportion of CD4+CD25highFOXP3+ Treg cells may be significantly increased in the peritoneal fluid of patients with endometriosis. Therefore, the aim of our study was to investigate whether the proportions of Treg cells in the peritoneal cavity of patients with endometriosis are related to the chemotactic and stimulatory activity of the local peritoneal milieu. The peritoneal fluid was collected from 13 women with ovarian endometriosis and 12 control women without the disease. T cell populations were analyzed by flow cytometry, cytokines and chemokines were evaluated using the cytometric bead kit, and cell chemotaxis was studied by cell migration assay. We confirmed that the proportions of Treg cells are increased in the peritoneal fluid of women with endometriosis as compared to the control women. Endometriosis was also associated with elevated concentrations of IL-6, IL-10, and TGF-ß1/2 as well as CCL20, CXCL8, CXCL9, and CXCL10. We did not reveal any changes in the proportion of peritoneal Th17 cells and concentrations of IL-17A. Peritoneal Treg cells positively correlated with concentrations of TGF-ß, IL-10, and CCL20. Endometriotic peritoneal fluid stimulated chemotaxis of both CD4+ and Treg cells. This chemotactic activity positively correlated with concentrations of CCL20. CCL20 stimulated the migration of Treg cells, and the chemotactic activity of the endometriotic peritoneal fluid was inhibited by neutralizing anti-CCL20 antibodies. These results imply that increased proportions of the peritoneal Treg cells in women with endometriosis may result from attraction and activation by local chemokines and cytokines, especially CCL20 and TGF-ß. Since Treg cells contribute to the immunopathogenesis of endometriosis, their chemotaxis and activation may be considered as a target for therapeutic intervention.

Peritoneal Fluid from Patients with Ovarian Endometriosis Displays Immunosuppressive Potential and Stimulates Th2 Response.

Endometriosis is a common gynaecological disorder characterized by the ectopic growth of endometrial tissue outside the uterine cavity. It is associated with chronic pelvic inflammation and autoimmune reactivity manifesting by autoantibody production and abrogated cellular immune responses. Endometriotic peritoneal fluid contains various infiltrating leucocyte populations and a bulk of proinflammatory and immunoregulatory cytokines. However, the nature and significance of the peritoneal milieu in women with endometriosis still remains obscure. Therefore, the aim of the present study was to investigate the immunoregulatory activity of the peritoneal fluid (PF) from women with endometriosis. The peritoneal fluid samples were collected during laparoscopic surgery from 30 women with and without endometriosis. Immunoregulatory cytokines (IL-2, IL-4, IL-6, IL-10, IL-17A, IFN-γ and TNF) and chemokines (CCL2, CCL5, CXCL8 and CXCL9) were evaluated in PF and culture supernatants generated by unstimulated and CD3/CD28/IL-2-stimulated CD4+ T cells cultured in the presence of PF. The effect of PF on the generation of Treg and Th17 cells in CD4+ T cell cultures, as well as the natural cytotoxic activity of peripheral blood mononuclear cells, was also investigated. Concentrations of IL-6, IL-10, CCL2, CXCL8 and CXCL9 were significantly upregulated in the PF from women with endometriosis when compared to control women, whereas concentrations of other cytokines and chemokines were unaffected. The culturing of unstimulated and CD3/CD28/IL-2-stimulated CD4+ T cells in the presence of endometriotic PF resulted in the downregulation of their IL-2, IFN-γ, IL-17A and TNF production as compared to culture medium alone. On the other side, endometriotic PF significantly stimulated the production of IL-4 and IL-10. Endometriotic PF also stimulated the release of CCL2 and CXCL8, whereas the production of CCL5 and CXCL9 was downregulated. Endometriotic PF stimulated the generation of Treg cells and had an inhibitory effect on the generation of Th17 cells in cultures of CD4+ T cells. It also inhibited the NK cell cytotoxic activity of the peripheral blood lymphocytes. These results strongly imply that the PF from patients with endometriosis has immunoregulatory/immunosuppressive activity and shifts the Th1/Th2 cytokine balance toward the Th2 response, which may account for deviation of local and systemic immune responses. However, a similar trend, albeit not a statistically significant one, was also observed in case of PF from women without endometriosis, thus suggesting that peritoneal milieu may in general display some immunoregulatory/immunosuppressive properties. It should be stressed, however, that our present observations were made on a relatively small number of PF samples and further studies are needed to reveal possible mechanism(s) responsible for this phenomenon.

A Role of NKR-P1A (CD161) and Lectin-like Transcript 1 in Natural Cytotoxicity against Human Articular Chondrocytes.

Normal cartilage cells are susceptible to lysis by NK cells. This phenomenon may play a role in immune cartilage destruction; however, the mechanisms of chondrocyte recognition by NK cells remain poorly understood. Therefore, the aim of this study was to reveal a possible role of NKR-P1A/lectin-like transcript 1 (LLT1) interaction in NK cell-mediated cytotoxicity against normal human articular chondrocytes. Chondrocytes were isolated from articular cartilage obtained during talonavicular joint surgery. PBMC or polyclonal NK cells isolated from normal donors served as effector cells. Cell-mediated cytotoxicity against chondrocytes was evaluated by means of 18-h 51Cr-release assay. Specific mRNA expression was evaluated by classical and quantitative RT-PCR, and proteins were detected by Western blot analysis. We found that lysis of articular chondrocytes by PBMC or polyclonal NK cells was potentiated by stimulation with IL-2. Stimulation of effector cells with IL-2 downregulated mRNA expression of inhibitory NKR-P1A NK cell receptor, and blocking of NKR-P1A with specific mAbs resulted in increased chondrocyte killing. Chondrocytes constitutively expressed LLT1, a ligand of NKR-P1A. LLT1 expression by chondrocytes could be upregulated by IL-1α and TNF. Chondrocyte treatment with IL-1α resulted in their increased resistance to killing by natural cytotoxic cells. This could be reversed by blocking of NKR-P1A. These results show that susceptibility of normal articular chondrocytes to lysis by NK cells is modulated by NKR-P1A/LLT1 interactions. Thus, NKR-P1A/LLT1 interaction might provide some novel target for therapeutic interventions in the course of pathological cartilage injury.

Uncompleted polymerization and cytotoxicity of dental restorative materials as potential health risk factors.

INTRODUCTION: Composite materials used in dentistry indicate adverse biological effects in laboratory conditions. One reason for this activity is incomplete conversion of their polymer matrix, favoring chemical instability and release of biologically harmful components to the external environment. AIM: The aim of the study was to assess the degree of conversion of restorative materials commonly available on the European market and to examine the cytotoxic effects of their eluates in vitro. MATERIAL AND METHODS: Using the Fournier transform infrared spectroscopy (FTIR) technique of analysis, the degree of polymer matrix conversion of 6 restorative materials was examined: Gradia Direct, Arkon, Filtek Z550, Herculite XRV, Tetric Evo Ceram, Charisma, polymerized with LED light. In order to assess the cytotoxicity of eluates of the studied materials obtained after 1 hour , 24 hours and 7 days, the MTT assay was used in cultured 3T3 cells. The results were statistically analyzed at significance level of p=0.05. RESULTS: The conversion degree of the assessed polymers ranged from 31.56% for Tetric Evo Ceram to 75.84% for Arcon. The strongest (p=0.05) cytotoxic effect was demonstrated after 7-day observation of Tetric Evo Ceram eluates, reducing the metabolic activity of cells down to 56%. A positive correlation (r(x, y) = 0.62) between the degree of conversion of composite materials and cytotoxic effects of their eluates on cell cultures was confirmed. CONCLUSIONS: 1. Restorative dental materials are chemically unstable in the conditions of the present study. 2. Polymer-based restorative dental materials available on the European market demonstrate cytotoxic properties constituting a potential threat to the patients' health.

CD4⁺ CD25⁺ FOXP3⁺ regulatory T cells in peripheral blood and peritoneal fluid of patients with endometriosis.

STUDY QUESTION: Is endometriosis associated with changes in CD4⁺ CD25⁺ FOXP3⁺ regulatory T cells (Treg cells)? SUMMARY ANSWER: Endometriosis is associated with disturbed compartmentalization of CD25(high) FOXP3⁺ Treg cells. WHAT IS KNOWN ALREADY: Endometriosis is associated with an abrogated immune response and displays some features of an autoimmune disorder. Treg cells play a part in the development of autoimmune reactions; however, their role in pathogenesis of endometriosis is still poorly recognized. STUDY DESIGN, SIZE AND DURATION: Case-control study comparing 17 women with laparoscopically and histopathologically confirmed ovarian endometriosis with 15 control women without visible endometriosis foci, pelvic inflammation or related pathology who were subjected to laparoscopic surgery between 2010 and 2011. PARTICIPANTS/MATERIALS, SETTING AND METHODS: Peripheral blood and peritoneal fluid were collected during laparoscopy and T cell subpopulations were analysed by flow cytometry using specific monoclonal antibodies recognizing CD4⁺, CD25⁺ and FOXP3⁺ markers. MAIN RESULTS: The percentage of CD25(high) FOXP3⁺ Treg cells was significantly decreased in the peripheral blood of women with ovarian endometriosis compared with control women. On the other hand, the proportion of these cells was significantly increased in the peritoneal fluid of women with endometriosis. LIMITATIONS, REASONS FOR CAUTION: The present study is limited to patients with ovarian endometrioma and further investigations are needed, including patients with lower grade of endometriosis. WIDER IMPLICATIONS OF THE FINDINGS: The present results suggest that Treg cells may play a part in immunopathogenesis of endometriosis being responsible for abrogated local cellular immune responses and facilitation and development of autoimmune reactions. Treg cells may be thus a potential target in the treatment of endometriosis. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by 1M15/N/2011 and NK1W grants from the I Faculty of Medicine, Warsaw Medical University. None of the authors has any competing interests to declare.

An evaluation of sterilisation processes.

A sterile environment is one of the basic elements of in vitro cell culture. When choosing an appropriate sterilisation method, the possibility that the physical and chemical properties of the sterilised material could be altered by the sterilisation process itself, should be considered. Avoiding any potential problems of toxicity arising as a consequence of the sterilisation process is essential, not only in in vitro cell culture procedures, but especially in the case of the sterilisation of medical devices which come into contact with human tissue (e.g. catheters, surgical tools, and containers used for transplant preparation and storage). As it is not possible to predict the potential effects of every combination of test material and sterilisation process, we have designed a simple test, which can be easily performed to ensure the absence of cytotoxicity. The test involves the culturing of a non-adherent cell line in direct contact with the test material, in micro-wells attached to the surface of the test device. By using this novel test method, three sterilisation procedures were compared for each material. The results indicated that, neither ionising irradiation nor ethylene oxide left toxic residues on the surface of polystyrene; and that, in the case of steel, neither steam sterilisation nor ethylene oxide left toxic residues on the metal. The cold plasma system, which left toxic residues on the surface of both materials, required a post-sterilisation period of 24 hours in the case of steel, and 10 days in the case of polystyrene, in order to eliminate toxic residues prior to their use.