Severe Combined Immunodeficient Mice Research Articles

The Effect of a Novel Serine Protease Inhibitor on Inflammation and Intestinal Permeability in a Murine Colitis Transfer Model.

Background: A protease/antiprotease disbalance is observed in inflammatory bowel diseases (IBD). We therefore studied the effect of the novel serine protease inhibitor UAMC-00050 on intestinal inflammation and permeability in a chronic colitis T cell transfer mouse model to get further insight into the regulation of T cell-mediated immunopathology. Methods: Colitis was induced in severe combined immunodeficient (SCID) mice, by the adoptive transfer of CD4+CD25−CD62L+ T cells. Animals were treated intraperitoneally (i.p.) 2x/day with vehicle or UAMC-00050 (5 mg/kg) from week 2 onwards. Colonic inflammation was assessed by clinical parameters, colonoscopy, macroscopy, microscopy, myeloperoxidase activity and cytokine expression levels. At week 4, 4 kDa FITC-dextran intestinal permeability was evaluated and T helper transcription factors, protease-activated receptors and junctional proteins were quantified by RT-qPCR. Results: Adoptive transfer of CD4+CD25−CD62L+ T cells resulted in colonic inflammation and an altered intestinal permeability. The serine protease inhibitor UAMC-00050 ameliorated both the inflammatory parameters and the intestinal barrier function. Furthermore, a decrease in colonic mRNA expression of Tbet and PAR4 was observed in colitis mice after UAMC-00050 treatment. Conclusion: The beneficial effect of UAMC-00050 on inflammation was apparent via a reduction of Tbet, IFN-γ, TNF-α, IL-1β and IL-6. Based on these results, we hypothesize a pivotal effect of serine protease inhibition on the Th1 inflammatory profile potentially mediated via PAR4.

Preparing Adipogenic Hydrogel with Neo-Mechanical Isolated Adipose-Derived Extracellular Vesicles for Adipose Tissue Engineering.

Subcutaneous transplantation of decellularized adipose tissue was capable of recellularization during soft tissue repair. However, further improvements are required to promote angiogenesis and adipogenesis. Here, the authors proposed a neo-mechanical protocol to isolate adipose tissue-derived extracellular vesicles (ATEVs) through lipoaspirate as a mediator for both angiogenesis and adipogenesis, and prepared ATEV-rich decellularized adipose tissue hydrogel for adipose tissue engineering. Adipose liquid extract and lipid-devoid adipose tissue were extracted through homogenization and repeated freeze and thaw cycles. ATEVs were isolated from adipose liquid extract by ultracentrifugation. Decellularized adipose tissue hydrogel was prepared by optimized decellularization of lipid-devoid adipose tissue. The optimum dose of ATEVs for angiogenesis and adipogenesis was estimated by co-culturing with vascular endothelial cells and 3T3-L1 cells, then mixed with the hydrogel. ATEV-enriched hydrogel was injected subcutaneously into the back of severe combined immunodeficiency mice, and then subjected to supplementary injection of ATEVs on postoperative day 14. ATEV-free decellularized adipose tissue hydrogel was injected as control. The newly formed tissue samples were harvested at postoperative weeks 2, 4, and 8 and subjected to volume measurement, hematoxylin and eosin staining, and immunofluorescence (CD31 and perilipin) staining. The optimum dose of ATEVs for promoting angiogenesis and adipogenesis was 50 μg/ml. The newly formed tissue mediated by ATEV-enriched hydrogel had increased volume well as improved angiogenesis and adipogenesis at postoperative week 4 and 8. ATEV-enriched adipogenic hydrogel promotes enhanced angiogenesis and adipogenesis and could serve as a promising biomaterial for adipose tissue engineering.

Isolation and in vitro cultivation of Babesia venatorum (Apicomplexa: Babesiidae), a zoonotic hemoprotozoan from Ixodes persulcatus ticks in China

The emerging Babesia venatorum upsurges as a potential health threat occurring in China and other endemic countries. Few attempts to isolate and culture the Babesia species had been conducted in vitro. We collected the questing Ixodes persulcatus from identified endemic areas and allowed them to feed on the severe combined immunodeficiency (SCID) mice. The positive mice were chosen to provide positive erythrocytes with asexual B. venatorum for continuous culture in mouse or human erythrocytes in vitro, with RPMI 1640 medium and appropriate serum. With B. venatorum in the SCID mice, erythrocytes were cultured in vitro for confirmation by morphological observations with transmission electron microscopes. Sequences of B. venatorum were then identified by way of conventional PCR amplification. Parasitemia counts monitored the growth of B. venatorum on thin blood smears and real-time quantitative PCR in parallel. As expected, B. venatorum positive mice were harvested by sufficient attacks of I. persulcatus ticks. The erythrocytes of the infected mice were then inoculated and successfully cultured in donated erythrocytes from humans and mice in RPMI 1640 culture medium. Active growth of B. venatorum was well demonstrated in human erythrocytes with 3.3 times parasite-load when compared with a mouse under similar conditions. With the increased subcultures, a prolonged period of detectable parasitemia with much higher peak parasitemia and shorter time to reach peak parasitemia were observed in the following subcultures. A new strategy for isolation and in vitro culture of B. venatorum has been provided with a continuous supply of sufficient pathogens to satisfy human babesiosis's testings and clinical therapies.

Interleukin-10 Producing T Lymphocytes Attenuate Dermal Scarring.

Objective:Demonstrate the impact of IL-10 producing T lymphocytes on mediating dermal scarring.Summary Background Data:We demonstrated that CD4+ cells are essential to improving postinjury wound healing and preventing fibrosis. CD4+ subsets secrete differential cytokine and growth factor profiles, though their role in fibrosis is not known. IL-10, a key anti-inflammatory cytokine shown to promote regenerative wound healing, is secreted by some CD4+ subsets. We, therefore, hypothesize that IL-10 producing CD4+ T lymphocyte subsets selectively attenuate dermal wound fibrosis.Methods:IL-10-/- and wild-type murine splenocytes were enriched for CD4+ lymphocytes and adoptively transferred into severe combined immunodeficient (SCID) mice that received full-thickness wounds which were analyzed at days 7 and 28 for inflammation and collagen content. We then sorted CD4+CD44int/lowFoxP3-CD62L+ T cells (Tnaive) or CD4+CD44HiFoxP3- type 1 regulatory (Tr1) T cell subsets from 10BiT murine splenocytes, activated them, and transferred them into wounds. In vitro, dermal fibroblasts were cocultured with Tnaive or Tr1 and the effect on extracellular matrix (ECM) regulation was analyzed.Results:The anti-inflammatory and antifibrotic effects of CD4+ cells on SCID wounds were lost with cells from IL-10-/- mice. Adoptive transfer of Tr1 into SCID mice resulted in accelerated wound closure at d7 with reduced fibrosis at d28, with Tr1 favoring hyaluronan production by fibroblasts, an ECM molecule implicated in IL-10-induced regenerative healing.Conclusions:IL-10 producing T-lymphocytes, specifically Tr1, regulate inflammatory cell cytokine expression to promote HA-rich ECM deposition and attenuate fibrosis. Promoting IL-10 producing lymphocytes in wounds may be a therapeutic target to promote regenerative wound healing.

Hyperbaric oxygen suppressed tumor progression through the improvement of tumor hypoxia and induction of tumor apoptosis in A549-cell-transferred lung cancer

Tumor cells have long been recognized as a relative contraindication to hyperbaric oxygen treatment (HBOT) since HBOT might enhance progressive cancer growth. However, in an oxygen deficit condition, tumor cells are more progressive and can be metastatic. HBOT increasing in oxygen partial pressure may benefit tumor suppression. In this study, we investigated the effects of HBOT on solid tumors, such as lung cancer. Non-small cell human lung carcinoma A549-cell-transferred severe combined immunodeficiency mice (SCID) mice were selected as an in vivo model to detect the potential mechanism of HBOT in lung tumors. HBOT not only improved tumor hypoxia but also suppressed tumor growth in murine xenograft tumor models. Platelet endothelial cell adhesion molecule (PECAM-1/CD31) was significantly increased after HBOT. Immunostaining of cleaved caspase-3 was demonstrated and apoptotic tumor cells with nuclear debris were aggregated starting on the 14th-day after HBOT. In vitro, HBOT suppressed the growth of A549 cells in a time-dependent manner and immediately downregulated the expression of p53 protein after HBOT in A549 cells. Furthermore, HBOT-reduced p53 protein could be rescued by a proteasome degradation inhibitor, but not an autophagy inhibitor in A549 cells. Our results demonstrated that HBOT improved tissue angiogenesis, tumor hypoxia and increased tumor apoptosis to lung cancer cells in murine xenograft tumor models, through modifying the tumor hypoxic microenvironment. HBOT will merit further cancer therapy as an adjuvant treatment for solid tumors, such as lung cancer.

HBO1 overexpression is important for hepatocellular carcinoma cell growth

Hepatocellular carcinoma (HCC) is a common primary liver malignancy lacking effective molecularly-targeted therapies. HBO1 (lysine acetyltransferase 7/KAT7) is a member of MYST histone acetyltransferase family. Its expression and potential function in HCC are studied. We show that HBO1 mRNA and protein expression is elevated in human HCC tissues and HCC cells. HBO1 expression is however low in cancer-surrounding normal liver tissues and hepatocytes. In HepG2 and primary human HCC cells, shRNA-induced HBO1 silencing or CRISPR/Cas9-induced HBO1 knockout potently inhibited cell viability, proliferation, migration, and invasion, while provoking mitochondrial depolarization and apoptosis induction. Conversely, ectopic overexpression of HBO1 by a lentiviral construct augmented HCC cell proliferation, migration and invasion. In vivo, xenografts-bearing HBO1-KO HCC cells grew significantly slower than xenografts with control HCC cells in severe combined immunodeficient mice. These results suggest HBO1 overexpression is important for HCC cell progression.

CD52 is a novel target for the treatment of FLT3-ITD-mutated myeloid leukemia

Internal tandem duplication (ITD) of FMS-like tyrosine kinase 3 (FLT3) confers poor prognosis and is found in approximately 25% of cases of acute myeloid leukemia (AML). Although FLT3 inhibitors have shown clinical benefit in patients with AML harboring FLT3-ITD, the therapeutic effect is limited. Here, to explore alternative therapeutics, we established a cellular model of monoallelic FLT3ITD/WT cells using the CRISPR-Cas9 system in a human myeloid leukemia cell line, K562. cDNA microarray analysis revealed elevated CD52 expression in K562–FLT3ITD/WT cells compared to K562–FLT3WT/WT cells, an observation that was further confirmed by quantitative real-time-PCR and flow cytometric analyses. The elevated expression of CD52 in K562–FLT3ITD/WT cells was decreased in wild-type FLT3 (FLT3-WT) knock-in K562–FLT3ITD/WT cells. In K562–FLT3ITD/WT cells, a STAT5 inhibitor, pimozide, downregulated CD52 protein expression while an AKT inhibitor, afuresertib, did not affect CD52 expression. Notably, an anti-CD52 antibody, alemtuzumab, induced significant antibody-dependent cell-mediated cytotoxicity (ADCC) in K562-FLT3ITD/WT cells compared to K562–FLT3WT/WT cells. Furthermore, alemtuzumab significantly suppressed the xenograft tumor growth of K562–FLT3ITD/WT cells in severe combined immunodeficiency (SCID) mice. Taken together, our data suggested that genetically modified FLT3-ITD knock-in human myeloid leukemia K562 cells upregulated CD52 expression via activation of STAT5, and alemtuzumab showed an antitumor effect via induction of ADCC in K562–FLT3ITD/WT cells. Our findings may allow establishment of a new therapeutic option, alemtuzumab, to treat leukemia with the FLT3-ITD mutation.

Lentinan inhibited colon cancer growth by inducing endoplasmic reticulum stress-mediated autophagic cell death and apoptosis

Lentinan (SLNT) has been shown to be directly cytotoxic to cancer cells. However, this direct antitumour effect has not been thoroughly investigated in vivo, and the mechanism remains unclear. We aimed to examine the direct antitumour effect of SLNT on human colon cancer and the mechanism in vivo and in vitro. SLNT significantly inhibited tumour growth and induced autophagy and endoplasmic reticulum stress (ERS) in HT-29 cells and tumour-bearing nonobese diabetic (NOD)/severe combined immunodeficiency (SCID) mice. Experiments with the autophagy inhibitors chloroquine (CQ) and 3-methyladenine (3-MA) showed that autophagy facilitated the antitumour effect of SLNT. Moreover, ERS was identified as the common upstream regulator of SLNT-induced increases in Ca2+concentrations, autophagy and apoptosis by using ERS inhibitors. In summary, our study demonstrated that SLNT exerted direct antitumour effects on human colon cancer via ERS-mediated autophagy and apoptosis, providing a novel understanding of SLNT as an anti-colon cancer therapy.

Combining Mobilizing Agents with Busulfan to Reduce Chemotherapy-Based Conditioning for Hematopoietic Stem Cell Transplantation.

In the context of hematopoietic stem cell (HSC) transplantation, conditioning with myelo- and immune-ablative agents is used to eradicate the patient’s diseased cells, generate space in the marrow and suppress immune reactions prior to the infusion of donor HSCs. While conditioning is required for effective and long-lasting HSC engraftment, currently used regimens are also associated with short and long-term side effects on extramedullary tissues and even mortality. Particularly in patients with severe combined immunodeficiency (SCID), who are generally less than 1-year old at the time of transplantation and often suffer from existing comorbidities. There is a pressing need for development of alternative, less toxic conditioning regimens. Hence, we here aimed to improve efficacy of currently used myeloablative protocols by combining busulfan with stem-cell niche-directed therapeutic agents (G-CSF or plerixafor) that are approved for clinical use in stem cell mobilization. T, B and myeloid cell recovery was analyzed in humanized NSG mice after different conditioning regimens. Increasing levels of human leukocyte chimerism were observed in a busulfan dose-dependent manner, showing comparable immune recovery as with total body irradiation in CD34-transplanted NSG mice. Notably, a better T cell reconstitution compared to TBI was observed after busulfan conditioning not only in NSG mice but also in SCID mouse models. Direct effects of reducing the stem cell compartment in the bone marrow were observed after G-CSF and plerixafor administration, as well as in combination with low doses of busulfan. Unfortunately, these direct effects on the stem population in the bone marrow were not reflected in increased human chimerism or immune recovery after CD34 transplantation in NSG mice. These results indicate moderate potential of reduced conditioning regimens for clinical use relevant for all allogeneic transplants.

Transcriptional Profiling of Porcine HCC Xenografts Provides Insights Into Tumor Cell Microenvironment Signaling.

Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related death worldwide, representing the most common form of liver cancer. As HCC incidence and mortality continue to increase, there is a growing need for improved translational animal models to bridge the gap between basic HCC research and clinical practice to improve early detection and treatment strategies for this deadly disease. Recently the Oncopig cancer model—a novel transgenic swine model that recapitulates human cancer through Cre recombinase induced expression of KRASG12D and TP53R167H driver mutations—has been validated as a large animal translational model for human HCC. Due to the similar size, anatomy, physiology, immunology, genetics, and epigenetics between pigs and humans, the Oncopig has the potential to improve translation of novel diagnostic and therapeutic modalities into clinical practice. Recent studies have demonstrated the importance of tumor cells in shaping its surrounding microenvironment into one that is more proliferative, invasive, and metastatic; however, little is known about the impact of microenvironment signaling on HCC tumor biology and differential gene expression between HCC tumors and its tumor microenvironment (TME). In this study, transcriptional profiling was performed on Oncopig HCC xenograft tumors (n = 3) produced via subcutaneous injection of Oncopig HCC cells into severe combined immunodeficiency (SCID) mice. To differentiate between gene expression in the tumor and surrounding tumor microenvironment, RNA-seq reads originating from porcine (HCC tumor) and murine (microenvironment) cells were bioinformatically separated using Xenome. Principle component analysis (PCA) demonstrated clustering by group based on the expression of orthologous genes. Genes contributing to each principal component were extracted and subjected to functional analysis to identify alterations in pathway signaling between HCC cells and the microenvironment. Altered expression of genes associated with hepatic fibrosis deposition, immune response, and neo angiogenesis were observed. The results of this study provide insights into the interplay between HCC and microenvironment signaling in vivo, improving our understanding of the interplay between HCC tumor cells, the surrounding tumor microenvironment, and the impact on HCC development and progression.

Low-dose decitabine modulates T-cell homeostasis and restores immune tolerance in immune thrombocytopenia

AbstractOur previous clinical study showed that low-dose decitabine exhibited sustained responses in nearly half of patients with refractory immune thrombocytopenia (ITP). The long-term efficacy of decitabine in ITP is not likely due to its simple role in increasing platelet production. Whether decitabine has the potential to restore immune tolerance in ITP is unknown. In this study, we analyzed the effect of decitabine on T-cell subpopulations in ITP in vitro and in vivo. We found that low-dose decitabine promoted the generation and differentiation of regulatory T (Treg) cells and augmented their immunosuppressive function. Splenocytes from CD61 knockout mice immunized with CD61+ platelets were transferred into severe combined immunodeficient mouse recipients to induce a murine model of ITP. Low-dose decitabine alleviated thrombocytopenia and restored the balance between Treg and helper T (Th) cells in active ITP mice. Treg deletion and depletion offset the effect of decitabine in restoring CD4+ T-cell subpopulations in ITP mice. For patients who received low-dose decitabine, the quantity and function of Treg cells were substantially improved, whereas Th1 and Th17 cells were suppressed compared with the pretreatment levels. Next-generation RNA-sequencing and cytokine analysis showed that low-dose decitabine rebalanced T-cell homeostasis, decreased proinflammatory cytokines, and downregulated phosphorylated STAT3 in patients with ITP. STAT3 inhibition analysis suggested that low-dose decitabine might restore Treg cells by inhibiting STAT3 activation. In conclusion, our data indicate that the immunomodulatory effect of decitabine provides one possible mechanistic explanation for the sustained response achieved by low-dose decitabine in ITP.

α-Tocopherol Attenuates Oxidative Phosphorylation of CD34+ Cells, Enhances Their G0 Phase Fraction and Promotes Hematopoietic Stem and Primitive Progenitor Cell Maintenance.

Alpha tocopherol acetate (αTOA) is an analogue of alpha tocopherol (αTOC) that exists in the form of an injectable drug. In the context of the metabolic hypothesis of stem cells, we studied the impact of αTOA on the metabolic energetic profile and functional properties of hematopoietic stem and progenitor cells. In ex vivo experiments performed on cord blood CD34+ cells, we found that αTOA effectively attenuates oxidative phosphorylation without affecting the glycolysis rate. This effect concerns complex I and complex II of the mitochondrial respiratory chain and is related to the relatively late increase (3 days) in ROS (Reactive Oxygen Species). The most interesting effect was the inhibition of Hypoxia-Inducible Factor (HIF)-2α (Hexpression, which is a determinant of the most pronounced biological effect—the accumulation of CD34+ cells in the G0 phase of the cell cycle. In parallel, better maintenance of the primitive stem cell activity was revealed by the expansion seen in secondary cultures (higher production of colony forming cells (CFC) and Severe Combined Immunodeficiency-mice (scid)-repopulating cells (SRC)). While the presence of αTOA enhanced the maintenance of Hematopoietic Stem Cells (HSC) and contained their proliferation ex vivo, whether it could play the same role in vivo remained unknown. Creating αTOC deficiency via a vitamin E-free diet in mice, we found an accelerated proliferation of CFC and an expanded compartment of LSK (lineagenegative Sca-1+cKit+) and SLAM (cells expressing Signaling Lymphocytic Activation Molecule family receptors) bone marrow cell populations whose in vivo repopulating capacity was decreased. These in vivo data are in favor of our hypothesis that αTOC may have a physiological role in the maintenance of stem cells. Taking into account that αTOC also exhibits an effect on proliferative capacity, it may also be relevant for the ex vivo manipulation of hematopoietic stem cells. For this purpose, low non-toxic doses of αTOA should be used.

Analysis of DnaK Expression from a Strain of Mycoplasma fermentans in Infected HCT116 Human Colon Carcinoma Cells.

Several species of mycoplasmas, including Mycoplasma fermentans, are associated with certain human cancers. We previously isolated and characterized in our laboratory a strain of human mycoplasma M. fermentans subtype incognitus (MF-I1) able to induce lymphoma in a Severe Combined Immuno-Deficient (SCID) mouse model, and we demonstrated that its chaperone protein, DnaK, binds and reduces functions of human poly-ADP ribose polymerase-1 (PARP1) and ubiquitin carboxyl-terminal hydrolase protein-10 (USP10), which are required for efficient DNA repair and proper p53 activities, respectively. We also showed that other bacteria associated with human cancers (including Mycoplasma pneumoniae, Helicobacter pylori, Fusobacterium nucleatum, Chlamydia thrachomatis, and Chlamydia pneumoniae) have closely related DnaK proteins, indicating a potential common mechanism of cellular transformation. Here, we quantify dnaK mRNA copy number by RT-qPCR analysis in different cellular compartments following intracellular MF-I1 infection of HCT116 human colon carcinoma cells. DnaK protein expression in infected cells was also detected and quantified by Western blot. The amount of viable intracellular mycoplasma reached a steady state after an initial phase of growth and was mostly localized in the cytoplasm of the invaded cells, while we detected a logarithmically increased number of viable extracellular bacteria. Our data indicate that, after invasion, MF-I1 is able to establish a chronic intracellular infection. Extracellular replication was more efficient while MF-I1 cultured in cell-free axenic medium showed a markedly reduced growth rate. We also identified modifications of important regulatory regions and heterogeneous lengths of dnaK mRNA transcripts isolated from intracellular and extracellular MF-I1. Both characteristics were less evident in dnaK mRNA transcripts isolated from MF-I1 grown in cell-free axenic media. Taken together, our data indicate that MF-I1, after establishing a chronic infection in eukaryotic cells, accumulates different forms of dnaK with efficient RNA turnover.

Bioluminescent Tumor Signal Is Mouse Strain and Pelt Color Dependent: Experience in a Disseminated Lymphoma Model.

Many preclinical cancer studies use mice with varied phenotypes to monitor tumor treatment. We compared survival and optical imaging characteristics of strains with varied coat colors harboring luciferase-expressing disseminated lymphoma. Luciferase-expressing lymphoma cells (Raji-luc) were injected via tail vein into severe combined immunodeficient (SCID) and Rag2-IL2rg (R2G2) mice, and survival was tracked. Tumor signals were obtained by imaging ventral and dorsal aspects of mice. Signal attenuation by isolated mouse pelts was measured in vitro. R2G2 mice had decreased survival compared to SCID mice (17 vs. 32 days, p<0.001) despite similar bioluminescence signal when mice were imaged dorsally (p=0.37). However, signal was 17.3-fold higher in R2G2 mice compared to SCID (p<0.001) when imaged ventrally. Isolated dark R2G2 dorsal pelts attenuated signal more than ventral pelts when placed over cells in vitro. Mouse pelt color and imaging aspect are critical considerations for quantifying bioluminescent tumor signal, and the R2G2 mouse strain may prove useful for preclinical targeted therapy studies.

Effects of rhBMP-2 on Bone Formation Capacity of Rat Dental Stem/Progenitor Cells from Dental Follicle and Alveolar Bone Marrow.

Dental stem/progenitor cells are a promising cell sources for alveolar bone (AB) regeneration because of their same embryonic origin and superior osteogenic potential. However, their molecular processes during osteogenic differentiation remain unclear. The objective of this study was to identify the responsiveness of dental follicle cells (DFCs) and AB marrow-derived mesenchymal stem cells (ABM-MSCs) to recombinant human bone morphogenetic protein-2 (rhBMP-2). These cells expressed vimentin and MSC markers and did not express cytokeratin and hematopoietic stem cell markers and showed multilineage differentiation potential under specific culture conditions. DFCs exhibited higher proliferation and colony-forming unit-fibroblast efficiency than ABM-MSCs; rhBMP-2 induced DFCs to differentiate toward a cementoblast/osteoblast phenotype and ABM-MSCs to differentiate only toward a osteoblast phenotype; and rhBMP-2-induced DFCs exhibited higher osteogenic differentiation potential than ABM-MSCs. These cells adhered, grew, and produced extracellular matrix on nanohydroxyapatite/collagen/poly(l-lactide) (nHAC/PLA). During a 14-day culture on nHAC/PLA, the extracellular alkaline phosphatase (ALP) activity of DFCs decreased gradually and that of ABM-MSCs increased gradually; rhBMP-2 enhanced their extracellular ALP activity, intracellular osteocalcin (OCN), and osteopontin (OPN) protein expression; and DFCs exhibited higher extracellular ALP activity and intracellular OCN protein expression than ABM-MSCs. When implanted subcutaneously in severe combined immunodeficient mice for 3 months, DFCs+nHAC/PLA+rhBMP-2 obtained higher percentage of bone formation area, OCN, and cementum attachment protein expression and lower OPN expression than ABM-MSCs+nHAC/PLA+rhBMP-2. These results showed that DFCs possessed superior proliferation and osteogenic differentiation potential in vitro, and formed higher quantity and quality bones in vivo. It suggested that DFCs might exhibit a more sensitive responsiveness to rhBMP-2, so that DFCs enter a relatively mature stage of osteogenic differentiation earlier than ABM-MSCs after rhBMP-2 induction. The findings imply that these dental stem/progenitor cells are alternative sources for AB engineering in regenerative medicine, and developing dental tissue may provide better source for stem/progenitor cells.

Anticancer Activity of Triazolo-Thiadiazole Derivatives and Inhibition of AKT1 and AKT2 Activation

The fusion of 1,2,4-triazole and 1,3,4-thiadiazole rings results in a class of heterocycles compounds with an extensive range of pharmacological properties. A series of 1,2,4-triazolo[3,4-b]-1,2,4-thiadiazoles was synthesized and tested for its enzyme inhibition potential and anticancer activity. The results show that 1,2,4-triazolo[3,4-b]-1,2,4-thiadiazoles display potent anticancer properties in vitro against a panel of cancer cells and in vivo efficacy in HT-29 human colon tumor xenograft in CB17 severe combined immunodeficient (SCID) mice. Preliminary mechanistic studies revealed that KA25 and KA39 exhibit time- and concentration-dependent inhibition of Akt Ser-473 phosphorylation. Molecular modeling experiments indicated that 1,2,4-triazolo[3,4-b]-1,2,4-thiadiazoles bind well to the ATP binding site in Akt1 and Akt2. The low acute toxicity combined with in vitro and in vivo anticancer activity render triazolo[3,4-b]thiadiazoles KA25, KA26, and KA39 promising cancer therapeutic agents.

Preclinical safety of TG-C – Persistence and distribution of TG-C in rat joints, tumorigenicity in nude athymic and severe combined immunodeficient mice (SCID) and multiple dose safety in goats

Purpose: TG-C is a cell-mediated gene therapy that is in clinical development for the treatment of knee osteoarthritis (OA). It consists of a 3:1 mixture of non-irradiated allogeneic human chondrocytes and irradiated transduced human GP2-293 cells that serve as a protein production platform over-expressing transforming growth factor- β1 [TGF-β1]). The safety and efficacy of a single intra-articular injection of TG-C into the knee joint have been evaluated in a Phase 1, multicenter, single-blind, randomized, placebo-controlled, dose-escalation trial, a Phase 2, multicenter, double-blind, randomized, placebo-controlled, fixed-dose trial, and an ongoing Phase 3, multicenter, double-blind, randomized, placebo-controlled trial.

Xenografts derived from patients with head and neck cancer recapitulate patient tumour properties.

Rodent models mimic the heterogeneity of head and neck cancer (HNC) malignancies and are used to investigate HNC-associated biomarkers and evaluate drug responses. To assess the utility of patient-derived xenografts (PDXs) as an HNC model, 18 tumour samples were obtained from surgical specimens of patients with HNC and implanted into non-obese diabetic severe combined immunodeficient mice. The histological features of PDXs and corresponding patient samples were compared. Furthermore, the present study investigated how PDX responses to anticancer drugs mimic patient clinical responses, as well as the expression of adenosine triphosphate-binding cassette transporters through chemotherapy in an HNC-PDX model. A total of five PDXs from patients with HNC exhibiting high correspondence with histopathological features of the original patient samples were established (establishment rate, 28%). The responses of three PDXs to cisplatin were associated with clinical responses of the patients. ABC transporter expression was augmented in one PDX model after anticancer drug treatment, but not in PBS-treated passaged PDXs. PDX models exhibited similar biological and chemosensitive characteristics to those of the primary tumours. PDXs could be a useful preclinical tool to test novel therapeutic agents and identify novel targets and biomarkers in HNC.

MAFG-driven osteosarcoma cell progression is inhibited by a novel miRNA miR-4660

Osteosarcoma (OS) is the most common primary bone malignancy in the adolescent population. MAFG (v-maf avian musculoaponeurotic fibrosarcoma oncogene homolog G) forms a heterodimer with Nrf2 (NF-E2-related factor 2), binding to antioxidant response element (ARE), which is required for Nrf2 signaling activation. We found that MAFG mRNA and protein expression is significantly elevated in human OS tissues as well as in established and primary human OS cells. In human OS cells, MAGF silencing or knockout (KO) largely inhibited OS cell growth, proliferation, and migration, simultaneously inducing oxidative injury and apoptosis activation. Conversely, ectopic overexpression of MAFG augmented OS cell progression in vitro. MicroRNA-4660 (miR-4660) directly binds the 3′ untranslated region (UTR) of MAFG mRNA in the cytoplasm of OS cells. MAFG 3′ UTR luciferase activity and expression as well as OS cell growth were largely inhibited with forced miR-4660 overexpression but augmented with miR-4660 inhibition. In vivo, MAGF short hairpin RNA (shRNA) or forced overexpression of miR-4660 inhibited subcutaneous OS xenograft growth in severe combined immunodeficient mice. Furthermore, MAFG silencing or miR-4660 overexpression inhibited OS xenograft in situ growth in proximal tibia of the nude mice. In summary, MAFG overexpression-driven OS cell progression is inhibited by miR-4660. The miR-4660-MAFG axis could be novel therapeutic target for human OS.

Promoter usage regulating the surface density of CAR molecules may modulate the kinetics of CAR-T cells in vivo

Although chimeric antigen receptor T (CAR-T) cell therapy achieves high remission rates, challenges (e.g., toxicity management and relapse prevention) remain. The major risks are cytokine release syndrome and related neurological toxicity. The influence of the CAR surface density on the efficacy/safety of CAR-T cell therapy and the factors determining CAR density were not elucidated comprehensively. Here, we discovered that the use of the MND promoter increased the transduction rate and reduced the CAR surface density. Additionally, MND-driven CAR-T cells had prolonged antileukemia activity in a mouse model. In an initial dual-armed anti-CD19 CAR-T cell pilot study (ClinicalTrials.gov: NCT03840317), eight and six subjects were infused with MND and EF1α promoter-driven autologous CAR-T cells (3 × 105 CAR-T cells/kg), respectively. MND subjects developed mild fever and lower interferon gamma (IFN-γ) concentrations than in the EF1A19 group. All but one subject in each cohort reached minimal residual disease (MRD)-negative complete remission after the first month of evaluation. These results represent the first comprehensive study on the promoter-driven modulation of CAR-T cell functionality. These findings encourage further evaluation of the potential of the MND promoter to drive CAR-T cells as a broadly applicable cellular product for anticancer immunotherapy.