Journal of immunology and regenerative medicine最新文献

As a treatment for type I diabetes, clinical islet transplantation (CIT) in which donor islets of Langerhans are transplanted intrahepatically has become a viable option for patients. However, the success of this procedure is limited by factors including ischemia, host immunological factors, and delayed vascularization of the hypoxia-sensitive islets. One solution would be to use a synthetic polymer scaffold as a carrier for the transplanted islets, as it would allow for their transplantation into a more favorable environment and could protect the cells from host immune reactions. To realize this potential solution, it is important that the synthetic polymer used does not interfere with the functionality and survival of the islets. In order to determine which synthetic polymers best meet this requirement, we examined the interactions of human islets from six donors with four clinically approved materials: polyetheretherketone (PEEK), polyvinylidene fluoride (PVDF), polyphenylsulfone (PPSU) and polysulfone (PSU) in vitro. Human islet morphology, viability, insulin secretion, functionality and gene expression were investigated to assess the suitability of these synthetic polymers as a carrier for transplanted islets. We found three of the synthetic synthetic polymers (PEEK, PPSU and PVDF) showed promise based on their overall performance, while the glucose responsiveness of islets cultured on PSU resulted in significantly reduced insulin secretion from five of six donors. Our findings demonstrate that close examination of human islets and their interaction with synthetic polymers is an important factor to consider when selecting synthetic polymers for engineering islet replacement devices.

Type 1 diabetes (T1D) is characterized by hyperglycemia due to autoimmune destruction of the insulin-producing beta-cells in the pancreas. The reason for this occurring is still unknown and effective therapies to halt the autoimmune response are lacking, but several promising concepts are being trialed. Regenerative medicine approaches in expanding the remaining beta cell pool, or transplanting beta cells derived from stem cells are other options trialed. Lessons learned from the study of immune regulation in T1D could be applied to immunosuppression in transplantation of beta cells derived from stem cell sources to avoid recurring autoimmunity. In this review, immunological issues in beta cell replacement therapies are discussed along possible treatment avenues such as genetically modified grafts to evade immune responses, novel immunosuppressive protocols, and the harnessing of endogenous pools of regulatory immune cell subsets. Looking into promising treatments for T1D may lead to effective immunosuppressive regimens also for beta-cell grafts from stem cells where recurring autoimmunity is a major issue to address.

Hematopoietic stem cells (HSCs) are essential in the production and maintenance of red blood and immune cells. Small molecules that target HSC modulators may aid in the proliferation and expansion of HSCs. To that end, we investigated the effect of two small molecules on HSC expansion: pluripotin (an ERK1 and RasGAP inhibitor) and CHIR-99021 (a GSK-3 inhibitor). After 7 days of treatment, both Pluripotin and CHIR-99021 resulted in a 3-fold increase in the murine pool of HSCs in a dose-dependent manner. Furthermore, we looked into the effect of Pluripotin on the ex vivo expansion of human umbilical cord blood and bone marrow mononuclear cells. Pluripotin treatment, in particular, increased human CD34⁺ and ALDHbr HSC content up to threefold when compared to the control. Furthermore, Pluripotin treatment increased the number of human CD133+ HSC cells by a factor of five. Intriguingly, Pluripotin treatment reduces bone marrow-derived mesenchymal stem cell (MSC) proliferation and fibroblast growth while having no effect on adipose-derived MSCs. CHIR-99021 treatment had no effect on MSC or fibroblast proliferation. In conclusion, pluripotin-induced stem cell expansion is unique to HSCs and can be used to expand HSCs while suppressing unwanted fibroblast or MSC growth in primary ex vivo cultures.

Introduction

The aim of this study was to investigate the ability of osteoarthritic human chondrocytes to produce articular cartilage (AC) tissues with a reduced inflammatory environment in response to 4 anti-inflammatory nutraceuticals: alpha-tocopherol (Alpha), gallic acid (G), ascorbic acid (AA), and catechin hydrate (C).

Methods

Chondrocytes isolated from patients who underwent total knee arthroplasty surgeries were divided into groups (9 male; mean age, 66.2 ± 3.5 years and 11 female; mean age, 64.2 ± 3.1 years). Cells were cultured based on sex and supplemented with either a negative control (NC) medium or NC plus one of the nutraceuticals at a concentration of 50 μM. At day 21, cultures were characterized histologically, biochemically, and for gene expression of vital markers.

Results

At day 21, 62.3% and 66.2% reduction in nitric oxide (NO) content was evident for female and male cells, respectively. G-treatment of female cells resulted in the lowest expression of nitric oxide synthase-2 (NOS2), matrix metalloproteinase-13 (MMP13), and collagen type-10 (COL10). Alpha-treatment of male cells resulted in the lowest expression of NOS2, bone morphogenic protein-2, MMP13, COL10 and tumor necrosis factor alpha induced protein-6 (TNFAIP6) relative to NC. AA and Alpha treatment resulted in the highest glycosaminoglycan (GAG) content for female and male cultures, respectively.

Conclusion

A sex-dependent response of osteoarthritic chondrocytes to nutraceutical treatment was evident. Our results suggest the use of G for female cells and Alpha for male cells in OA applications seems to be favorable in reducing inflammation and enhancing chondrocytes’ ability to form AC tissues.

Objectives

‘Universal’ blood typically refers to blood components that can be donated to most individuals. Allogenic platelet refractoriness is often caused by incompatibility of human leukocyte antigens (HLA) and antibodies in the recipient binding donor epitopes. HLA-matched platelets are not always readily available for transfusions, hindered by a general rise in platelet demand, logistical challenges of HLA-selected platelet provisions and the limited HLA repertoire of current stocks. Several groups have therefore progressed towards making in vitro platelets lacking HLA class I molecules, to avoid immunogenic reactions. This review will outline the progress to date in establishing these ‘HLA-universal platelets,’ taking a comparative view of the gene-editing technologies used for their creation while also evaluating the resulting products and the immunogenic properties of HLA-deficient cells.

Key findings

Universal platelets have been differentiated from HLA class I knockdown or knockout progenitors, generated mainly through gene modification techniques targeting B2M, which encodes the β2m-light chain component of HLA class I antigens. B2M-knockdown through RNA interference technology reduces but does not completely deplete HLA class I expression, whereas B2M-knockout via endonucleases (TALENs or CRISPR/Cas9 systems) creates HLA-negative cells. Cultured HLA-silenced or ablated megakaryocytes and platelets are functional and can circulate in mouse models, while evading immune detection. HLA class I proteins serve as ligands for Natural Killer (NK) cell inhibitory receptors. A key concern for patient welfare, using HLA-universal cells, is the triggering of immune reactions including NK cytotoxicity; therefore, a possible approach to circumvent this has been to either retain or co-express alternative inhibitory or immunoregulatory molecules.

Conclusions

The collection of studies brought together and reviewed here show proof-of-concept that functional HLA-universal platelets, inert to immune responses, can be produced using powerful gene-editing techniques. CRISPR/Cas9, in particular, offers versatility to co-engineer multiple specific gene knockouts in combination with options to knock in immunomodulating proteins or platelet factors, to correspondingly further enhance immune tolerance or improve platelet functions. Together these data demonstrate the rapid development and feasibility of moving HLA-universal platelets towards the clinic.

The number of preclinical studies that use drugs or endogenous factors for various targetable sites aiming to improve islet function and engraftment has recently gained more interest. Several molecules have been proposed to support and augment the functionality of islets. Here, we review five different classes of beta cell protection factors, classified as antioxidants, anti-inflammatory, anti-apoptotic, immunomodulatory, vascularization enhancers, and promoter of beta cell proliferation and function and their effect on islet grafting and function after transplantation in animal models of type 1 diabetes.

The thymus plays a crucial role in adaptive immunity through the regulation of T cell development. Thymus dysfunction can have a number of implications, such as increased predisposition to infection and autoimmunity, age-related reduced response to vaccines, and increased risk of cancer development. This review describes several strategies to treat thymic impairment, including endogenous regeneration by growth factor administration, cellular and stem cell therapy, gene therapy and combinations thereof.

Objectives

Islet transplantation is a treatment option for patients with type 1 diabetes and recurrent life-threatening hypoglycaemia. Pancreatic tissue is dissociated, and islets are purified from exocrine tissue of a donor pancreas through enzymatic and mechanical separation followed by short term culture and transplantation into the portal vein of the recipient. Despite improvements to isolation and transplantation protocols, insulin independence is often not sustained demonstrating stress towards the islets and impaired transplantation outcomes. These stressors include loss of the specific microenvironment and exposure to hypoxia following disconnection from the blood supply. Tissue engineering approaches will be investigated to minimize these stressors.

Key findings

Different tissue engineering strategies are available to improve islet health and function and therefore outcomes of islet transplantation. Strategies for the replacement of extracellular matrix in the microenvironment of isolated islets should provide cell-matrix contacts and a three-dimensional microenvironment but avoid cyto-toxic components. Strategies for immune protection should shield islets from the immune system whilst enabling sufficient oxygen and mass transfer. Strategies for improved oxygenation of islets should consider in vitro and/or in vivo oxygen requirements. Finally, alternative cell sources of β-cells may provide a standardised and less stressed product, but efficiency, safety, and costs require further improvement.

Conclusion

This review summarises the development and composition of islets and their microenvironment in adult pancreata. The impact of peri-transplant stressors including islet isolation and transplantation are explored as well as strategies to minimize these towards enhanced tissue engineered β-cell replacement.

The pancreatic islet of Langerhans is a multicellular system that relies on cell–cell interaction and communication for its function. The most abundant cells in the islets are alpha, beta and endothelial cells, all of which have been shown to positively support each other by generating direct cell–cell interactions, extracellular matrix proteins, or through the secretion of soluble factors into the extracellular space. Knowing how these cell types assemble and support each other to improve viability, migration, and function (glucose responsiveness) is important for the aim of re-establishing beta cell mass with a de novo cell source derived from human cells.

Introduction

Macrophages are capable of extreme plasticity and their activation state has been strongly associated with solid tumor growth progression and regression. Although the macrophage response to extracellular matrix (ECM) isolated from normal tissue is reasonably well understood, there is a relative dearth of information regarding their response to ECM isolated from chronically inflamed tissues, pre-neoplastic tissues, and neoplastic tissues. Esophageal adenocarcinoma (EAC) is a type of neoplasia driven by chronic inflammation in the distal esophagus, and the length of the esophagus provides the opportunity to investigate macrophage behavior in the presence of ECM isolated from a range of disease states within the same organ. Methods. Normal, metaplastic, and neoplastic ECM hydrogels were prepared from decellularized EAC tissue. The hydrogels were evaluated for their nanofibrous structure (SEM), biochemical profile (targeted and global proteomics), and direct effect upon macrophage (THP-1 cell) activation state (qPCR, ELISA, immunolabeling) and indirect effect upon epithelial cell (Het-1A) migration (Boyden chamber). Results. Nanofibrous ECM hydrogels from the three tissue types could be formed, and normal and neoplastic ECM showed distinctive protein profiles by targeted and global mass spectroscopy. ECM proteins functionally related to cancer and tumorigenesis were identified in the neoplastic esophageal ECM including collagen alpha-1(VIII) chain (COL8A1), lumican, and elastin. Metaplastic and neoplastic esophageal ECM induce distinctive effects upon THP-1 macrophage signaling compared to normal esophageal ECM. These effects include activation of pro-inflammatory IFNγ and TNFα gene expression and anti-inflammatory IL1RN gene expression. Most notably, neoplastic ECM robustly increased macrophage TNFα protein expression. The secretome of macrophages pre-treated with metaplastic and neoplastic ECM increases the migration of normal esophageal epithelial cells, similar behavior to that shown by tumor cells. Metaplastic ECM shows similar but less pronounced effects than neoplastic ECM suggesting the abnormal signals also exist within the pre-cancerous state. Conclusion. A progressively diseased ECM, as exists within the esophagus exposed to chronic gastric reflux, can provide insights into novel biomarkers of early disease and identify potential therapeutic targets.