Irradiation is not sufficient to eradicate residual immortalized erythroid cells in in vitro-generated red blood cell products.

BACKGROUND: The generation of immortalized erythroid progenitor cell lines capable of producing enough red blood cells (RBCs) for blood transfusion typically requires the overexpression of oncogenes in stem cells or progenitor cells to permanently proliferate immature cells. It is essential that any live oncogene-expressing cells are eliminated from the final RBC products for clinical use. STUDY DESIGN AND METHODS: It is believed that safety issues may be resolved by using a leukoreduction filter or by irradiating the final products, as is conventionally done in blood banks; however, this has never been proven to be effective. Therefore, to investigate whether immortalized erythroblasts can be completely removed using γ-ray irradiation, we irradiated the erythroblast cell line, HiDEP, and the erythroleukemic cell line, K562 that overexpress HPV16 E6/E7. We then analyzed the extent of cell death using flow cytometry and polymerase chain reaction (PCR). The cells were also subjected to leukoreduction filters. RESULTS: Using γ-ray irradiation at 25 Gy, 90.4% of HiDEP cells, 91.6% of K562-HPV16 E6/E7 cells, and 93.5% of non-transduced K562 cells were dead. In addition, 5.58 × 107 HiDEP cells were passed through a leukoreduction filter, and 38 intact cells were harvested, revealing a filter removal efficiency of 99.9999%. However, both intact cells and oncogene DNA were still detected. DISCUSSION: Irradiation cannot induce total cell death of oncogene-expressing erythroblasts and leukocyte filter efficiency is not 100%. Therefore, our findings imply that for clinical applications, safer methods should be developed to completely remove residual nucleated cells from cell line-derived RBC products.

Pancreatic endocrine-like cells differentiated from human umbilical cords Wharton's jelly mesenchymal stem cells using small molecules.

Following success of pancreatic islet transplantation in the treatment of Type I diabetes mellitus, there is a growing interest in using cell-based treatment approaches. However, severe shortage of donor islets-pancreas impeded the growth, and made researchers to search for an alternative treatment approaches. In this context, recently, stem cell-based therapy has gained more attention. The current study demonstrated that epigenetic modification improves the in vitro differentiation of Wharton's jelly mesenchymal stem cells (WJMSCs) into pancreatic endocrine-like cells. Here we used two histone deacetylase (HDAC) inhibitors namely trichostatin A (TSA) and TMP269. TSA inhibits both class I and II HDACs whereas TMP269 inhibits only class IIa HDACs. WJMSCs were differentiated using a multistep protocol in a serum-free condition with or without TSA pretreatment. A marginal improvement in differentiation was observed after TSA pretreatment though it was not significant. However, exposing endocrine precursor-like cells derived from WJMSCs to TMP269 alone has significantly improved the differentiation toward insulin-producing cells. Further, increase in the expression of paired box 4 (PAX4), insulin, somatostatin, glucose transporter 2 (GLUT2), MAF bZIP transcription factor A (MAFA), pancreatic duodenal homeobox 1 (PDX-1), and NKX6.1 was observed both at messenger RNA and protein levels. Nevertheless, TMP269-treated cells secreted higher insulin upon glucose challenge, and demonstrated increased dithizone staining. These findings suggest that TMP269 may improve the in vitro differentiation of WJMSCs into insulin-producing cells.

Prosthodontic treatment of a retrognathic edentulous maxilla demonstrating limited interarch distance: 3.5-year results with fixed and removable implant prostheses.

The prosthodontic treatment of patients with a retrognathic edentulous maxilla should consider the restoration of the lower facial profile and access for oral hygiene. This clinical report describes prosthodontic treatments of a patient with edentulism who presented with repeated fractures of the denture teeth of a maxillary implant-supported complete fixed dental prosthesis (ICFDP) and a mandibular implant-supported overdenture. Considerable plaque accumulation was noted on the ICFDP, which was replaced with an open palatal design implant-supported overdenture. However, the patient experienced difficulty managing the 2 removable prostheses. The patient's mandible was eventually restored with a milled titanium alloy framework ICFDP with metal occlusal surfaces. This combined approach of fixed and removable prostheses was stable at 3.5-year follow-up appointment, without compromising the patient's oral hygiene or comfort.

Cardiomyogenic Differentiation of Human Dental Follicle-derived Stem Cells by Suberoylanilide Hydroxamic Acid and Their In Vivo Homing Property.

The purpose of the present study was to investigate the in vitro cardiomyogenic differentiation potential of human dental follicle-derived stem cells (DFCs) under the influence of suberoylanilide hydroxamic acid (SAHA), a member of the histone deacetylase inhibitor family, and analyze the in vivo homing capacity of induced cardiomyocytes (iCMs) when transplanted systemically. DFCs from extracted wisdom teeth showed mesenchymal stem cell (MSC) characteristics such as plate adherent growing, expression of MSC markers (CD44, CD90, and CD105), and mesenchymal lineage-specific differentiation potential. Adding SAHA to the culture medium induced the successful in vitro differentiation of DFCs into cardiomyocytes. These iCMs expressed cardiomyogenic markers, including alpha-smooth muscle actin (α-SMA), cardiac muscle troponin T (TNNT2), Desmin, and cardiac muscle alpha actin (ACTC1), at both the mRNA and protein level. For the assessment of homing capacity, PKH26 labeled iCMs were intraperitoneally injected (1×106 cells in 100 µL of PBS) into the experimental mice, and the ratios of PKH26 positive cells to the total number of injected cells, in multiple organs were determined. The calculated homing ratios, 14 days after systemic cell transplantation, were 5.6 ± 1.0%, 3.6 ± 1.1%, and 11.6 ± 2.7% in heart, liver, and kidney respectively. There was no difference in the serum levels of interleukin-2 and interleukin-10 at 14 days after transplantation, between the experimental (iCM injected) and control (no injection or PBS injection) groups. These results demonstrate that DFCs can be an excellent source for cardiomyocyte differentiation and regeneration. Moreover, the iCMs can be delivered into heart muscle via systemic administration without eliciting inflammatory or immune response. This can serve as the pilot study for further investigations into the in vitro cardiomyogenic differentiation potential of DFCs under the influence of SAHA and the in vivo homing capacity of the iCMs into the heart muscle, when injected systemically.

Impact of implant support on mandibular free-end base removable partial denture: theoretical study.

OBJECTIVES: This study investigated the impact of implant support on the development of shear force and bending moment in mandibular free-end base removable partial dentures (RPDs). MATERIAL AND METHODS: Three theoretical test models of unilateral mandibular free-end base RPDs were constructed to represent the base of tooth replacement, as follows: Model 1: first and second molars (M1 and M2); Model 2: second premolar (P2), M1, and M2; and Model 3: first premolar (P1), P2, M1, and M2. The implant support located either at M1 or M2 sites. The occlusal loading was concentrated at each replacement tooth to calculate the stress resultants developed in the RPD models using the free-body diagrams of shear force and bending moment. RESULTS: There was a trend of reduction in the peak shear force and bending moment when the base was supported by implant. However, the degree of reduction varied with the location of implant support. The moment reduced by 76% in Model 1, 58% in Model 2, and 42% in Model 3, when the implant location shifted from M1 to M2 sites. CONCLUSIONS: The shear forces and bending moments subjected to mandibular free-end base RPDs were found to decrease with the addition of implant support. However, the impact of implant support varied with the location of implant in this theoretical study.

Expedited Record Base Fabrication Using an Irreversible Hydrocolloid Cast.

The registration of a maxillomandibular relationship requires additional clinical and laboratory procedures when the mouth presents with loss of occlusal support. This procedure can be a challenge for a patient who needs urgent care or resides in a remote area. This article describes a procedure for expediting the mounting of a master cast for the fabrication of a maxillary immediate complete denture. The technique presented describes the use of a silicone record base made on an irreversible hydrocolloid cast generated from the final impression.

Indirect method of base adaptation against supporting element of tooth root for a partial overdenture prosthesis.

The base of a partial overdenture prosthesis should be fitted intraorally against the supporting element of a tooth root. Chairside relining is a common method; however, an autopolymerizing acrylic resin presents high porosity when polymerized intraorally. This article describes an indirect method where an impression is made with a silicone occlusion registration material to create a replica of the supporting elements of the residual ridge and the tooth root in a high-viscosity polyvinyl siloxane impression material.

Current status of platelet manufacturing in 3D or bioreactors.

Blood shortages for transfusion are global issues of grave concern. As in vitro manufactured platelets are promising substitutes for blood donation, recent research has shown progresses including different cell sources, different bioreactors, and three-dimensional materials. The first-in-human clinical trial of cultured platelets using induced pluripotent stem cell-derived platelets began in Japan and demonstrated its quality, safety, and efficacy. A novel bioreactor with fluid motion for platelet production has been reported. Herein, we discuss various cell sources for blood cell production, recent advances in manufacturing processes, and clinical applications of cultured blood.

Simple method of record base fabrication for a bar-retained implant overdenture.

A record base should be stable and accurately transferable from the cast to the mouth. This article describes a simple and practical method of fabricating a record base for mounting a master cast used to fabricate an implant connecting bar for an implant-retained overdenture.

Removable partial denture assisted by implant-retained fixed prosthesis opposing implant-retained overdenture.

Restoring an edentulous mouth is a challenge when the patient has both high esthetic expectations and financial limitations. This case report describes the prosthodontic management of an elderly edentulous patient with a maxillary anterior implant-supported fixed partial denture in conjunction with a distal extension-base removable partial denture opposing a mandibular implant-retained overdenture. The patient's clinical outcome after 30 months is presented.

Temsirolimus Enhances Anti-Cancer Immunity by Inducing Autophagy-Mediated Degradation of the Secretion of Small Extracellular Vesicle PD-L1.

Tumor-derived small extracellular vesicle (sEV) programmed death-ligand 1 (PD-L1) contributes to the low reactivity of cells to immune checkpoint blockade therapy (ICBT), because sEV PD-L1 binds to programmed death 1 (PD-1) in immune cells. However, there are no commercially available anti-cancer drugs that activate immune cells by inhibiting tumor-derived sEV PD-L1 secretion and cellular PD-L1. Here, we aimed to investigate if temsirolimus (TEM) inhibits both sEV PD-L1 and cellular PD-L1 levels in MDA-MB-231 cells. In cancer cell autophagy activated by TEM, multivesicular bodies (MVBs) associated with the secretion of sEV are degraded through colocalization with autophagosomes or lysosomes. TEM promotes CD8+ T cell-mediated anti-cancer immunity in co-cultures of CD8+ T cells and tumor cells. Furthermore, the combination therapy of TEM and anti-PD-L1 antibodies enhanced anti-cancer immunity by increasing both the number and activity of CD4+ and CD8+ T cells in the tumor and draining lymph nodes (DLNs) of breast cancer-bearing immunocompetent mice. In contrast, the anti-cancer effect of the combination therapy with TEM and anti-PD-L1 antibodies was reversed by the injection of exogenous sEV PD-L1. These findings suggest that TEM, previously known as a targeted anti-cancer drug, can overcome the low reactivity of ICBT by inhibiting sEV PD-L1 and cellular PD-L1 levels.

Atorvastatin Enhances the Efficacy of Immune Checkpoint Therapy and Suppresses the Cellular and Extracellular Vesicle PD-L1.

According to clinical studies, statins improve the efficacy of programmed death-1/programmed death-ligand 1 (PD-1/PD-L1) blockade therapy for breast cancer; however, the underlying mechanisms are unclear. Herein, we showed that atorvastatin (ATO) decreased the content of PD-L1 in extracellular vesicles (EVs) by reducing cellular PD-L1 expression and inhibiting EV secretion in breast cancer cells, thereby enhancing the efficacy of anti-PD-L1 therapy. ATO reduced EV secretion by regulating the Rab proteins involved in EV biogenesis and secretion. ATO-mediated inhibition of the Ras-activated MAPK signaling pathway downregulated PD-L1 expression. In addition, ATO strongly promoted antitumor efficacy by inducing T cell-mediated tumor destruction when combined with an anti-PD-L1 antibody. Moreover, suppression of EV PD-L1 by ATO improved the reactivity of anti-PD-L1 therapy by enhancing T-cell activity in draining lymph nodes of EMT6-bearing immunocompetent mice. Therefore, ATO is a potential therapeutic drug that improves antitumor immunity by inhibiting EV PD-L1, particularly in response to immune escape during cancer.

Macitentan improves antitumor immune responses by inhibiting the secretion of tumor-derived extracellular vesicle PD-L1.

Extracellular vesicles (EVs) carrying tumor cell-derived programmed death-ligand 1 (PD-L1) interact with programmed death 1 (PD-1)-producing T cells, thus significantly lowering a patient's response to immune checkpoint blockade drugs. No drug that reinvigorates CD8+ T cells by suppressing EV PD-L1 has been approved for clinical usage. Here we have identified macitentan (MAC), an FDA-approved oral drug, as a robust booster of antitumor responses in CD8+ T cells by suppressing tumor cell-derived EV PD-L1. Methods: EV was analyzed by the data from nanoparticle tracking, immunoblotting analyses, and nano-flow cytometry. Antitumor immunity was evaluated by luciferase assay and immune phenotyping using flow cytometry. Clinical relevance was analyzed using the cancer genome atlas database. Results: MAC inhibited secretion of tumor-derived EV PD-L1 by targeting the endothelin receptor A (ETA) in breast cancer cells and xenograft models. MAC enhanced CD8+ T cell-mediated tumor killing by decreasing the binding of PD-1 to the EV PD-L1 and thus synergizing the effects of the anti-PD-L1 antibody. MAC also showed an anticancer effect in triple-negative breast cancer (TNBC)-bearing immunocompetent mice but not in nude mice. The combination therapy of MAC and anti-PD-L1 antibody significantly improved antitumor efficacy by increasing CD8+ T cell number and activity with decreasing Treg number in the tumors and draining lymph nodes in TNBC, colon, and lung syngeneic tumor models. The antitumor effect of MAC was reversed by injecting exogenous EV PD-L1. Notably, ETA level was strongly associated with the innate anti-PD-1 resistance gene signature and the low response to the PD-1/PD-L1 blockade. Conclusion: These findings strongly demonstrate that MAC, already approved for clinical applications, can be used to improve and/or overcome the inadequate response to PD-1/PD-L1 blockade therapy.

Sulfisoxazole Elicits Robust Antitumour Immune Response Along with Immune Checkpoint Therapy by Inhibiting Exosomal PD-L1.

Despite their potent antitumor activity, clinical application of immune checkpoint inhibitors has been significantly limited by their poor response rates (<30%) in cancer patients, primarily due to immunosuppressive tumor microenvironments. As a representative immune escape mechanism, cancer-derived exosomes have recently been demonstrated to exhaust CD8+ cytotoxic T cells. Here, it is reported that sulfisoxazole, a sulfonamide antibacterial, significantly decreases the exosomal PD-L1 level in blood when orally administered to the tumor-bearing mice. Consequently, sulfisoxazole effectively reinvigorates exhausted T cells, thereby eliciting robust antitumor effects in combination with anti-PD-1 antibody. Overall, sulfisoxazole regulates immunosuppression through the inhibition of exosomal PD-L1, implying its potential to improve the response rate of anti-PD-1 antibodies.

Mammalian and Fish Gelatin Methacryloyl-Alginate Interpenetrating Polymer Network Hydrogels for Tissue Engineering.

Gelatin methacryloyl (GelMA) has been widely studied as a biomaterial for tissue engineering. Most studies focus on mammalian gelatin, but certain factors, such as mammalian diseases and diet restrictions, limit the use of mammalian gelatin. Thus, fish gelatin has received much attention as a substitute material in recent years. To develop a broadly applicable hydrogel with excellent properties, an interpenetrating polymer network (IPN) hydrogel was synthesized, since IPN hydrogels consist of at least two different hydrogel components to combine their advantages. In this study, we prepared GelMA using type A and fish gelatin and then synthesized IPN hydrogels using GelMA with alginate. GelMA single-network hydrogels were used as a control group. The favorable mechanical properties of type A and fish hydrogels improved after the synthesis of the IPN hydrogels. Type A and fish IPN hydrogels showed different mechanical properties (mechanical strength, swelling ratio, and degradation rate) and different cross-sectional morphologies, since the degree of mechanical enhancement in fish IPN hydrogels was less than that in type A; however, the cell biocompatibilities were not significantly different. Therefore, these findings could serve as a reference for future studies when selecting GelMA as a biological material for tissue engineering.