Antibacterial Thermosensitive Silver-Hydrogel Nanocomposite Improves Wound Healing.

Bacterial infection and poor cell recruitment are among the main factors that prolong wound healing. To address this, a strategy is required that can prevent infection while promoting tissue repair. Here, we have created a silver nanoparticle-based hydrogel composite that is antibacterial and provides nutrients for cell growth, while filling cavities of various geometries in wounds that are difficult to reach with other dressings. Silver nanoparticles (AgNPs) were synthesized by chemical reduction and characterized using transmission electron microscopy (TEM), dynamic light scattering (DLS), and inductively coupled plasma-mass spectroscopy (ICP-MS). Using varying concentrations of AgNPs (200, 400, and 600 ppm), several collagen-based silver-hydrogel nanocomposite candidates were generated. The impact of these candidates on wound healing was assessed in a rat splinted wound model, while their ability to prevent wound infection from a contaminated surface was assessed using a rat subcutaneous infection model. Biocompatibility was assessed using the standard MTT assay and in vivo histological analyses. Synthesized AgNPs were spherical and stable, and while hydrogel alone did not have any antibacterial effect, AgNP-hydrogel composites showed significant antibacterial activity both in vitro and in vivo. Wound healing was found to be accelerated with AgNP-hydrogel composite treatment, and no negative effects were observed compared to the control group. The formulations were non-cytotoxic and did not differ significantly in hematological and biochemical factors from the control group in the in vivo study. By presenting promising antibacterial and wound healing activities, silver-hydrogel nanocomposite offers a safe therapeutic option that can be used as a functional scaffold for an acceleration of wound healing.

M-CSF-stimulated myeloid cells can convert into epithelial cells to participate in re-epithelialization and hair follicle regeneration during dermal wound healing.

Dermal wound healing is a complex process which requires the interaction of many cell types and mediators in a highly sophisticated temporal sequence. Myeloid cells which compose of a significant proportion of the inflammatory cells infiltrate to the to a wound site where they play important roles in clearance of damaged tissue and microorganisms. Myeloid cells have the capacity to be converted into fibroblast-like cells and endothelial cells during wound healing process. However, whether myeloid cells in wounds can convert into epithelial cells where they contribute to healing process is not clear. In this study, we performed double immunofluorescent staining with antibodies for hematopoietic cells and keratinocytes as well as cell tracing technique to investigate hematopoietic cell conversion. The result showed that during the healing process, some of the CD45-positive hematopoietic cells also expressed keratin 14, a marker for keratinocytes. Further, double immunofluorescent staining in dermal wounds, using CD11b and K14 antibodies indicated that CD11b-positive myeloid cells were the origin of newly generated epithelial cells. Through tracing injected labeled splenocyte-derived myeloid cells in skin, we confirmed that myeloid cells were able to convert into keratinocytes in repaired skin. Furthermore, our results from in vivo experiments provided new information on contribution of myeloid cells in hair follicle regeneration. In conclusion, this work highlights the myeloid cell contributions in wound repair and hair follicle regeneration through conversion of M-CSF-stimulated CD11b-positive myeloid cells into epithelial cells in a murine model.

Evaluating the Biocompatibility of an Injectable Wound Matrix in a Murine Model.

(1) Background: Developing a high-quality, injectable biomaterial that is labor-saving, cost-efficient, and patient-ready is highly desirable. Our research group has previously developed a collagen-based injectable scaffold for the treatment of a variety of wounds including wounds with deep and irregular beds. Here, we investigated the biocompatibility of our liquid scaffold in mice and compared the results to a commercially available injectable granular collagen-based product. (2) Methods: Scaffolds were applied in sub-dermal pockets on the dorsum of mice. To examine the interaction between the scaffolds and the host tissue, samples were harvested after 1 and 2 weeks and stained for collagen content using Masson's Trichrome staining. Immunofluorescence staining and quantification were performed to assess the type and number of cells infiltrating each scaffold. (3) Results: Histological evaluation after 1 and 2 weeks demonstrated early and efficient integration of our liquid scaffold with no evident adverse foreign body reaction. This rapid incorporation was accompanied by significant cellular infiltration of stromal and immune cells into the scaffold when compared to the commercial product (p < 0.01) and the control group (p < 0.05). Contrarily, the commercial scaffold induced a foreign body reaction as it was surrounded by a capsule-like, dense cellular layer during the 2-week period, resulting in delayed integration and hampered cellular infiltration. (4) Conclusion: Results obtained from this study demonstrate the potential use of our liquid scaffold as an advanced injectable wound matrix for the management of skin wounds with complex geometries.

Studying the in vivo application of a liquid dermal scaffold in promoting wound healing in a mouse model.

Lack of matrix deposition is one of the main factors that complicates the healing process of wounds. The aim of this study was to test the efficacy and safety of a liquid dermal scaffold, referred to as MeshFill (MF) that can fill the complex network of tunnels and cavities which are usually found in chronic wounds and hence improve the healing process. We evaluated in vitro and in vivo properties of a novel liquid dermal scaffold in a delayed murine full-thickness wound model. We also compared this scaffold with two commercially available granular collagen-based products (GCBP). Liquid dermal scaffold accelerated wound closure significantly compared with no-treated control and collagen-based injectable composites in a delayed splinted wound model. When we compared cellular composition and count between MF, no treatment and GCBP at the histology level, it was found that MF was the most analogous and consistent with the normal anatomy of the skin. These findings were matched with the clinical outcome observation. The flowable in situ forming scaffold is liquid at cold temperature and gels after application to the wound site. Therefore, it would conform to the topography of the wound when liquid and provides adequate tensile strength when solidified. This patient-ready gelling dermal scaffold also contains the nutritional ingredients and therefore supports cell growth. Applying an injectable liquid scaffold that can fill wound gaps and generate a matrix to promote keratinocytes and fibroblasts migration, can result in improvement of the healing process of complex wounds.

In Situ Forming Nutritional and Temperature Sensitive Scaffold Improves the Esthetic Outcomes of Meshed Split-Thickness Skin Grafts in a Porcine Model.

Objective: Full-thickness burn wounds require immediate coverage, and the primary clinical approaches comprise of skin allografts and autografts. The use of allografts is often temporary due to the antigenicity of allografts. In contrast, the availability of skin autografts may be limited in large burn injuries. In such cases, skin autografts can be expanded through the use of a skin mesher, creating meshed split-thickness skin grafts (MSTSGs). MSTSGs have revolutionized the treatment of large full-thickness burn injuries since the 1960s. However, contractures and poor esthetic outcomes remain a problem. We previously formulated and prepared an in situ forming skin substitute, called MeshFill (MF), which can conform to complex shapes and contours of wounds. The objective of this study was to assess the esthetic and wound healing outcomes in full-thickness wounds treated with a combination of MF and MSTSG in a porcine model. Approach: Either MSTSGs or MSTSG+MF was applied to full-thickness excisional wounds in Yorkshire pigs. Wound healing outcomes were assessed using histology, immunohistochemistry, and wound surface area analysis from day 10 to 60. Clinical evaluation of wounds were utilized to assess esthetic outcomes. Results: The results demonstrated that the combination of MSTSGs and MF improved wound healing and esthetic outcomes. Innovation: Effects of MSTSGs and reconstitutable liquid MF in a full-thickness porcine model were investigated for the first time. Conclusion: MF provides promise as a combination therapeutic regimen to improve wound healing and esthetic outcomes.

Myeloid Adherent Cells Are Involved in Hair Loss in the Alopecia Areata Mouse Model.

Alopecia areata (AA), which is defined as an autoimmune hair loss disease, has a serious impact on the quality of life for patients with AA worldwide. In this study, to our knowledge, a previously unreported method of AA induction in C3H mice has been established and validated. Using this method, we showed that dermal injection of 1-3 million of a mixture of skin cells freshly isolated from AA-affected skin induces AA in more than 80% of healthy mice. Contrary to the previous protocol, the induction of AA by this approach does not need any surgical AA skin grafting, cell manipulation, or high number of activated T cells. We also showed that dermal injection of adherent myeloid cells (mainly CD11b+) in healthy mice is as potent as a mixture of none adherent CD3+ T cells and CD19+ B cells in the induction of AA. Interestingly, most of the mice (7 out of 8) that received non-adherent cells developed AA universalis, whereas most of the mice (5 out of 7) that received adherent cells developed patchy AA. Finally, we found a high number of stage-specific embryonic antigen-expressing cells whose expression in monocytes in an inflammatory disease causes the release of inflammatory cytokines, TNF-α and IL-1ß, from these cells in AA-affected skin.

Local Expression of Indoleamine 2,3, Dioxygenase Prolongs Allogenic Skin Graft Take in a Mouse Model.

Background and Objective: Despite the effectiveness of skin autotransplantation, the high degree of immunogenicity of the skin precludes the use of allografts and systemic immunosuppression is generally inappropriate for isolated skin grafts. Indoleamine 2,3 dioxygenase (IDO) is a potent immunoregulatory factor with allo- and autoimmune suppression and tolerance induction properties. This study examines the potential use of locally expressed IDO to prolong the allogeneic skin graft take in a mouse model. Approach: Syngeneic-fibroblasts were transfected with noncompetent IDO viral vector and the level of Kynurenine (Kyn) in conditioned medium was measured as an index for IDO activity. Either 1 or 3 × 106 IDO-fibroblasts were introduced intra/hypo-dermally to the mouse skin. The expression, localization, and functionality of IDO were then evaluated. The cell-injected areas were harvested and grafted on the back of allogeneic mice. The graft survival, immune-cells infiltration, and interaction with dendritic cells were evaluated. Results: The results showed a significant improvement in allogeneic graft take injected with 1 × 106 IDO-fibroblasts (18.4 ± 3.3 days) compared with control (12.2 ± 1.9 days). This duration increased to 35.4 ± 4.7 days in grafts injected with 3 × 106 IDO-expressing cells. This observation might be due to a significantly lower T cells infiltration within the IDO-grafts. Further, the result of a flow cytometric analysis showed that the expression of PD-L1/PD-L2 on CD11c+/eFluor+ cells in the regional lymph nodes of injected skin areas was significantly higher in IDO groups compared with control. Conclusion: These data suggest that allogeneic skin graft survival outcome can be prolonged significantly by local overexpression of IDO without any systemic effect.

Split Thickness Grafts Grow From Bottom Up in Large Skin Injuries.

Autologous split thickness skin graft is necessary for the survival of patients with large burns and skin defects. It is not clear how a thin split thickness skin graft becomes remarkably thicker within a few weeks following transplantation. Here, we hypothesized that growth of split thickness graft should be from bottom up probably through conversion of immune cells into collagen producing skin cells. We tested this hypothesis in a preclinical porcine model by grafting split thickness meshed skin (0.508 mm thickness, meshed at 3:1 ratio) on full thickness wounds in pigs. New tissue formation was evaluated on days 10 and 20 postoperation through histological analysis and co-staining for immune cell markers (CD45) and type I collagen. The findings revealed that a split thickness graft grew from bottom up and reached to almost the same level as uninjured skin within 60 days postoperation. The result of immune-staining identified a large number of cells, which co-expressed immune cell marker (CD45) and collagen on day 10 postoperation. Interestingly, as the number of these cells reduced on day 20, most of these cells became positive for collagen production. In another set of experiments, we tested whether immune cells can convert to collagen producing cells in vitro. The results showed that mouse adherent immune cells started to express type 1 procollagen and α-smooth muscle actin when cultured in the presence of fibroblast conditioned media. In conclusion, the early thickening of split thickness graft is likely happening through a major contribution of infiltrated immune cells that convert into mainly collagen producing fibroblasts in large skin injuries.

Liquid Dermal Scaffold With Adipose-Derived Stem Cells Improve Tissue Quality in a Murine Model of Impaired Wound Healing.

Wound repair and regeneration is a multidisciplinary field of research with considerable potential value to the management of deep and large burn injuries. These injuries lack an appropriate tissue scaffold and pro-healing cells making them difficult to heal. An alternative to the often limited autologous skin is a therapy that would restore the essential matrix and cellular components for rapid healing. In this study, they use a novel liquid dermal scaffold capable of gelation in vivo to show that it is biocompatible with adipose-derived stem cells. Using a validated method of wound splinting in a delayed-healing murine model, we show that wounds treated with the scaffold and stem cells had a significant reduction in wound size and had accelerated healing compared with control. The wounds treated with stem cells had increased capillary formation, collagen content, epidermal thickness, and essential growth factor expression in the healed tissue compared with control and liquid scaffold alone. This liquid dermal scaffold combined with cells is a feasible treatment strategy for complex or large burn wounds that are otherwise lacking the appropriate cellular matrix necessary for healing.

Fibroblast cell-based therapy prevents induction of alopecia areata in an experimental model.

Alopecia areata (AA) is an autoimmune hair loss disease with infiltration of proinflammatory cells into hair follicles. Current therapeutic regimens are unsatisfactory mainly because of the potential for side effects and/or limited efficacy. Here we report that cultured, transduced fibroblasts, which express the immunomodulatory molecule indoleamine 2,3-dioxygenase (IDO), can be applied to prevent hair loss in an experimental AA model. A single intraperitoneal (IP) injection of IDO-expressing primary dermal fibroblasts was given to C3H/HeJ mice at the time of AA induction. While 60-70% of mice that received either control fibroblasts or vehicle injections developed extensive AA, none of the IDO-expressing fibroblast-treated mice showed new hair loss up to 20 weeks post injection. IDO cell therapy significantly reduced infiltration of CD4+ and CD8+ T cells into hair follicles and resulted in decreased expression of TNF-α, IFN-γ and IL-17 in the skin. Skin draining lymph nodes of IDO fibroblast-treated mice were significantly smaller, with more CD4+ CD25+ FoxP3+ regulatory T cells and fewer Th17 cells than those of control fibroblast and vehicle-injected mice. These findings indicate that IP injected IDO-expressing dermal fibroblasts can control inflammation and thereby prevent AA hair loss.

IDO-expressing Fibroblasts Suppress the Development of Imiquimod-induced Psoriasis-like Dermatitis.

Psoriasis is a chronic skin condition whose pathogenesis is reported to be due to the activation of the interleukin-23/interleukin-17 (IL-23/IL-17) pathway. Here, we report that indoleamine 2,3-dioxygenase (IDO)-expressing fibroblasts reduce the activity of this pathway in activated immune cells. The findings showed that intralesional injection of IDO-expressing fibroblasts in imiquimod-induced psoriasis-like dermatitis on the back and ear (Pso. ear group) in mice significantly improves the clinical lesional appearance by reducing the number of skin-infiltrated IL-17+ CD4+ T cells (1.9% ± 0.3% vs. 6.9% ± 0.6%, n = 3, P value < 0.01), IL-17+ γδ+ T cells (2.8% ± 0.3% vs. 11.6% ± 1.2%, n = 3, P value < 0.01), IL-23+ activated dendritic cells (7.6% ± 0.9% vs. 14.0% ± 0.5%, n = 3, P < 0.01), macrophages (4.3% ± 0.1% vs. 11.3% ± 1.0%, n = 3, P value < 0.01), and granulocytes (2.5% ± 0.4% vs. 4.5% ± 0.3%, n = 3, P value < 0.01) as compared to untreated psoriatic mice. This finding suggests that IDO-expressing fibroblasts, and to a lesser extent, non-IDO primary fibroblasts suppress the psoriatic-like symptoms by inhibiting the infiltration of key immune cells involved in the development of psoriasis.

Intraperitoneal injection of IDO-expressing dermal fibroblasts improves the allograft survival.

Indoleamine 2,3-dioxygenase (IDO) is an immunosuppressive enzyme with tolerogenic effects on different immune cells. Our group has previously shown that co-transplantation of IDO-expressing fibroblasts with donor tissues can delay immune rejection by inducing local immunosuppression. In this study, we have employed a systemic approach to improve allograft survival without using any immunosuppressive medication. To achieve this, 10 million lentiviral transduced IDO-expressing donor derived fibroblasts were injected into the peritoneal cavity of allograft recipients. We showed that IDO-fibroblast therapy increases the survival of both islets and skin allografts and decreases the infiltration of immune cells in subcutaneous transplanted skins. Indirect pathway of allo-reactive T cell activation was suppressed more than the direct pathway. Injected IDO-fibroblasts were found in peritoneal cavity and mesenteric lymph nodes of the recipient mice. In conclusion, IDO-expressing fibroblast therapy proved to be a novel approach in improving the allogeneic graft survival.

A new method for skin grafting in murine model.

Skin transplantation provides an excellent potential model to investigate the immunology of allograft rejection and tolerance induction. Despite the theoretical ease of performing skin transplantation, as well as the potential of directly observing the reaction to the transplanted tissue, the poor reliability of skin transplantation in the mouse has largely precluded the use of this model. Furthermore, there is controversy regarding the most appropriate skin graft donor site due to poor success of back skin transplantation, as compared with the thinner ear or tail skin. This study demonstrates a reliable method to successfully perform skin grafts in a mouse model, as well as the clinical and histologic outcome of syngeneic grafts. A total of 287 grafts were performed (in 126 mice) utilizing donor skin from the ear, tail or back. No graft failure or postoperative mortality was observed. Comparison of this technique with two previously established protocols of skin transplantation (5.0 absorbable Suture + tissue glue technique and no-suture technique) demonstrates the significant improvement in the engraftment success of the new technique. In summary, a new technique for murine skin grafting demonstrates improved reliability across donor site locations and strains, increasing the potential for investigating interventions to alter the rejection process.

Fibroblast Cell-Based Therapy for Experimental Autoimmune Diabetes.

Type 1 diabetes (T1D) results from autoimmune destruction of insulin producing ß cells of the pancreatic islets. Curbing autoimmunity at the initiation of T1D can result in recovery of residual ß cells and consequently remission of diabetes. Here we report a cell-based therapy for autoimmune diabetes in non-obese diabetic (NOD) mice using dermal fibroblasts. This was achieved by a single injection of fibroblasts, expressing the immunoregulatory molecule indoleamine 2,3 dioxygenase (IDO), into peritoneal cavity of NOD mice shortly after the onset of overt hyperglycemia. Mice were then monitored for reversal of hyperglycemia and changes in inflammatory/regulatory T cell profiles. Blood glucose levels dropped into the normal range in 82% of NOD mice after receiving IDO-expressing fibroblasts while all control mice remained diabetic. We found significantly reduced islet inflammation, increased regulatory T cells, and decreased T helper 17 cells and ß cell specific autoreactive CD8+ T cells following IDO cell therapy. We further showed that some of intraperitoneal injected fibroblasts migrated to local lymph nodes and expressed co-inhibitory molecules. These findings suggest that IDO fibroblasts therapy can reinstate self-tolerance and alleviate ß cell autoreactivity in NOD mice, resulting in remission of autoimmune diabetes.

IDO-Expressing Fibroblasts Protect Islet Beta Cells From Immunological Attack and Reverse Hyperglycemia in Non-Obese Diabetic Mice.

Indoleamine 2,3-dioxygenase (IDO) induces immunological tolerance in physiological and pathological conditions. Therefore, we used dermal fibroblasts with stable IDO expression as a cell therapy to: (i) Investigate the factors determining the efficacy of this cell therapy for autoimmune diabetes in non-obese diabetic (NOD) mice; (ii) Scrutinize the potential immunological mechanisms. Newly diabetic NOD mice were randomly injected with either 10 × 10(6) (10M) or 15 × 10(6) (15M) IDO-expressing dermal fibroblasts. Blood glucose levels (BGLs), body weight, plasma kynurenine levels, insulitis severity, islet beta cell function, autoreactive CD8(+) T cells, Th17 cells and regulatory T cells (Tregs) were then investigated in these mice. IL-1ß and cleaved caspase-3 levels were assessed in islets co-cultured with IDO-expressing fibroblasts. BGLs in 83% mice treated with 15M IDO-expressing fibroblasts recovered to normal up to 120 days. However, only 17% mice treated with 10M IDO-expressing cells were reversed to normoglycemia. A 15M IDO-expressing fibroblasts significantly reduced infiltrated immune cells in islets and recovered the functionality of remaining islet beta cells in NOD mice. Additionally, they successfully inhibited autoreactive CD8(+) T cells and Th17 cells as well as increased Tregs in different organs of NOD mice. Islet beta cells co-cultured with IDO-expressing fibroblasts had reduced IL-1ß levels and cell apoptosis. Both cell number and IDO enzymatic activity contributes to the efficiency of IDO cell therapy. Optimized IDO-expressing fibroblasts successfully reverse the progression of diabetes in NOD mice through induction of Tregs as well as inhibition of beta cell specific autoreactive CD8(+) T cells and Th17 cells. J. Cell. Physiol. 231: 1964-1973, 2016. © 2016 Wiley Periodicals, Inc.

Tolerogenic effect of mouse fibroblasts on dendritic cells.

There is controversy about the immunomodulatory effect of fibroblasts on dendritic cells (DCs). To clarify this issue, in this study, we have evaluated different features of fibroblast-primed DCs including their ability to express co-inhibitory and co-stimulatory molecules, pro-inflammatory and anti-inflammatory cytokines and their ability to induce T-cell proliferation. We also examined migratory capacity of DCs to lymphatic tissues and present fibroblast-derived antigens after encountering fibroblasts. The results of our in vitro study showed that both co-inhibitory (programmed death ligand 1 and ligand 2 and B7H4) and co-stimulatory (CD86) molecules were up-regulated when DCs were co-cultured with fibroblasts. In an animal model, we showed that intra- peritoneal injection (IP) of both syngeneic and allogeneic fibroblasts significantly increased both total DC count and expression level of co-inhibitory and co-stimulatory molecules on DCs. Priming of DCs with syngeneic and allogeneic fibroblasts reduced the proliferation of CD4(+) and CD8(+) T cells. Even activation of fibroblast- primed DCs failed to restore their ability to induce T-cell proliferation. Likewise, priming of DCs with fibroblasts blocked the ability of ovalbumin-pulsed DCs to induce proliferation of ovalbumin-specific CD4(+) T cells. Compared with non-activated DCs, fibroblast-primed DCs had significantly higher expression levels of interleukin-10 and indoleamine 2, 3 dioxygenase. Fibroblast-primed DCs had a significantly reduced interleukin-12 expression level compared with that of activated DCs. After priming with fibroblasts, DCs were able to migrate to lymphatic tissues and present fibroblast-derived antigens (ovalbumin). In conclusion, after priming with fibroblasts, DCs gain tolerogenic features. This finding suggests the potential role of fibroblasts in the maintenance of immune tolerance.

Methotrexate modulates the expression of MMP-1 and type 1 collagen in dermal fibroblast.

Methotrexate (MTX), an anti-metabolite and anti-inflammatory drug, has been used to effectively manage and prevent keloids, but its mechanism(s) of action has not been elucidated. Our study sought to evaluate the effect of MTX on the production of key extra cellular matrix components, collagen, and matrix metalloproteinase-1 (MMP-1), produced by fibroblasts and involved in development of fibrosis. The proliferation and viability of cultured human dermal fibroblasts in response to different concentrations of MTX were determined using cell counting and MTT assay, respectively. Western blot analysis was used to determine the levels of both intracellular and secreted type 1 collagen and MMP-1. The results showed no significant changes in the proliferation of fibroblasts treated with 50 ng/ml of MTX as compared to that of control. Under the same experimental conditions, the level of secreted and intracellular type I collagen was markedly reduced and, conversely, the level of MMP-1 increased in treated neonatal, adult, and hypertrophic scar fibroblasts as compared with those of controls. The possible involvement of MTX-induced extracellular signal-regulated kinase 1/2 (ERK1/2) pathway in MMP-1 production was also studied and the result showed an increase in phosphorylated ERK 1/2 in response to MTX treatment. In summary, the findings of this study revealed that MTX significantly reduced collagen production in different strains of fibroblasts derived from neonatal, adult, and hypertrophic scar tissues, while under the same experimental conditions, it increased the expression of MMP-1. As such, our findings validate and identify a potential mechanism through which MTX functions as an anti-fibrogenic factor in treating fibroproliferative disorders.

Immunoprotection and Functional Improvement of Allogeneic Islets in Diabetic Mice, Using a Stable Indoleamine 2,3-Dioxygenase Producing Scaffold.

BACKGROUND: We have previously shown that an immunomodulatory enzyme, indoleamine 2,3-dioxygenase (IDO) in dermal fibroblasts generates a tryptophan-deficient environment that selectively inhibits proliferation and induces apoptosis of bystander CD4+ and CD8+ T cells, but not pancreatic islets. Because these immune cells are involved in islet allograft rejection, we hypothesized that transplantation of islets embedded in a novel 3-dimensional composite scaffold within which stable IDO-expressing fibroblasts serve as source of local immunosuppression would lead to normoglycemia in a streptozotocin-induced diabetic mouse model. METHODS: Islet grafts were prepared by embedding stable IDO-expressing fibroblasts and allogeneic islets into a protease-resistant composite scaffold. Islets function and survival were evaluated in vitro using immunohistochemistry. Allografts were transplanted under the kidney capsule of streptozotocin-induced diabetic mice; viability, function, and criteria for graft take were evaluated. Flow cytometry was performed to determine specific intragraft, draining lymph nodes and spleen T-cell population, and splenocytes alloantigen responsiveness of graft recipients. RESULTS: The results of a series of in vitro experiments revealed that IDO-expressing fibroblasts do not compromise islet function or survival. The expression of IDO suppressed the proliferation of alloantigen-stimulated splenocytes. The in vivo experiments revealed that local IDO expression delivered by lentiviral vector prolonged islet allograft survival (51.0 ± 2.9 days) by increasing the population of FOXP3+ regulatory T cells at the graft site and graft-draining lymph nodes and preventing T-cell infiltration. CONCLUSIONS: This study shows that incorporation of islets within our novel matrix that is equipped with stable IDO-expressing fibroblasts prolongs allograft survival.

14-3-3η is a novel mediator associated with the pathogenesis of rheumatoid arthritis and joint damage.

INTRODUCTION: The aim of this study was to investigate whether 14-3-3η, a specific isoform of a family of proteins regulating processes such as cellular signalling, activates cell-signalling pathways and induces factors known to contribute to the pathophysiology of rheumatoid arthritis (RA). We also investigated whether 14-3-3η is associated with more severe disease in both early and established RA. METHODS: We investigated the effect of 14-3-3η on the activation of RA-relevant signalling cascades and induction of proinflammatory mediators that contribute to the joint damage process. 14-3-3η titres from 33 patients with early RA (mean RA duration = 1.8 months) and from 40 patients with established RA were measured in serum drawn at the 3-year time point of the Behandel Strategieën study. The relationship between 14-3-3η titres and standard clinical variables was investigated by correlation analysis. The association with radiographic damage and radiographic progression over at least a 2-year period was investigated using univariate and multivariate regression analyses. RESULTS: 14-3-3η activated selected members of the mitogen-activated protein kinase (MAPK) family, mainly extracellular regulated kinase 1/2 and c-Jun kinase, but not p38MAPK. Activation by 14-3-3η, using levels spanning the concentration range found in RA patient serum, resulted in the induction of inflammatory transcripts such as interleukin 1 (IL-1) and IL-6 and factors linked to the joint damage process, such as receptor activator of nuclear factor κB ligand and matrix metalloproteinase 1. Serum 14-3-3η correlated significantly with rheumatoid factor (RF) (r = 0.43) and anticitrullinated protein antibodies (ACPAs) (r = 0.31) in the early RA cohort, but not with C-reactive protein (CRP) or the Disease Activity Score in 28 joints in either cohort. Serum 14-3-3η concentrations were significantly higher in patients with radiographically assessed joint damage and in those who had radiographic progression. By multivariate analysis, we show that 14-3-3η complemented markers such as CRP, RF and ACPA in informing RA radiographic status and/or progression. CONCLUSIONS: Extracellular 14-3-3η activates key signalling cascades and induces factors associated with the pathogenesis of RA at concentrations found in patients with RA, and its expression is higher in patients with radiographic damage and RA progression.

Anti-scarring properties of different tryptophan derivatives.

Hypertrophic scars are associated with prolonged extracellular matrix (ECM) production, aberrant ECM degradation and high tissue cellularity. Routinely used antifibrotic strategies aim to reduce ECM deposition and enhance matrix remodeling. Our previous study investigating the antifibrotic effects of indoleamine2, 3 dioxygenase (IDO) led to the identification of kynurenine (Kyn) as an antiscarring agent. A topical antifibrogenic therapy using Kyn is very attractive; however, it is well established that Kyn passes the blood brain barrier (BBB) which causes complications including excitatory neuronal death. Here we investigated the antiscarring properties of kynurenic acid (KynA), a downstream end product of Kyn that is unlikely to pass the BBB, as an effective and safe replacement for Kyn. Our results indicated that while not having any adverse effect on dermal cell viability, KynA significantly increases the expression of matrix metalloproteinases (MMP1 and MMP3) and suppresses the production of type-I collagen and fibronectin by fibroblasts. Topical application of cream containing KynA in fibrotic rabbit ear significantly decreased scar elevation index (1.13±0.13 vs. 1.61±0.12) and tissue cellularity (221.38±21.7 vs. 314.56±8.66 cells/hpf) in KynA treated wounds compared to controls. KynA treated wounds exhibited lower levels of collagen deposition which is accompanied with a significant decrease in type-I collagen and fibronectin expression, as well as an increase in MMP1 expression compared to untreated wounds or wounds treated with cream only. The results of this study provided evidence for the first time that KynA is promising candidate antifibrogenic agent to improve healing outcome in patients at risk of hypertrophic scarring.