Betulin Stimulates Osteogenic Differentiation of Human Osteoblasts-Loaded Alginate-Gelatin Microbeads.

Osteoporosis, a terminal illness, has emerged as a global public health problem in recent years. The long-term use of bone anabolic drugs to treat osteoporosis causes multi-morbidity in elderly patients. Alternative therapies, such as allogenic and autogenic tissue grafts, face important issues, such as a limited source of allogenic grafts and tissue rejection in autogenic grafts. However, stem cell therapy has been shown to increase bone regeneration and decrease osteoporotic bone formation. Stem cell therapy combined with betulin (BET) supplementation might be adequate for bone remodeling and new bone tissue generation. In this study, the effect of BET on the viability and osteogenic differentiation of hFOB 1.19 cells was investigated. The cells were encapsulated in alginate-gelatin (AlGel) microbeads. In vitro tests were conducted during the 12 d of incubation. While BET showed cytotoxic activity (>1 µM) toward non-encapsulated hFOB 1.19 cells, encapsulated cells retained their functionality for up to 12 days, even at 5 µM BET. Moreover, the expression of osteogenic markers indicates an enhanced osteo-inductive effect of betulin on encapsulated hFOB 1.19, compared to the non-encapsulated cell culture. The 3D micro-environment of the AlGel microcapsules successfully protects the hFOB 1.19 cells against BET cytotoxicity, allowing BET to improve the mineralization and differentiation of osteoblast cells.

Preparation of Cell-Loaded Microbeads as Stable and Injectable Delivery Platforms for Tissue Engineering.

Cell transplants in therapeutic studies do not preserve their long-term function inside the donor body. In mesenchymal stem cell (MSC) transplants, transplanted cells disperse through the body and are prone to degradation by immune cells after the transplant process. Various strategies, such as usage of the immunosuppressive drugs to eliminate allograft rejection, are designed to increase the efficiency of cell therapy. Another strategy is the construction of biomimetic encapsulates using polymeric materials, which isolate stem cells and protect them from environmental effects. In this study, fibroblasts (L929) and MSCs were investigated for their improved viability and functionality once encapsulated inside the alginate microbeads under in vitro conditions for up to 12 days of incubation. Thus, uniform and injectable (<200 µm) cell-loaded microbeads were constructed by the electrostatically assisted spraying technique. Results showed that both L929 and MSCs cells continue their metabolic activity inside the microbeads during the incubation periods. Glucose consumption and lactic acid production levels of both cell lines were consistently observed. The released cell number on day 12 was found to be increased compared to day 0. Protein expression levels of both groups increased every day with the expected doubling rate. Hence, this strategy with a simple yet clever design to encapsulate either MSCs or L929 cells might outstand as a potential cell delivery platform for cell therapy-based tissue engineering.

Lentiviral Micro-dystrophin Gene Treatment into Late-stage mdx Mice for Duchenne Muscular Dystrophy Disease.

AIM: Duchenne Muscular Dystrophy (DMD) results in a deficiency of dystrophin expression in patient muscle fibers, leading to progressive muscle degeneration. Treatment of DMD has undertaken current transformation with the advancement of novel gene therapy and molecular biology techniques, which are secure, well-tolerated, and effective therapeutic approaches. INTRODUCTION: DMD gene therapies have mainly focused on young DMD patients as in vivo animal model trials have been performed in 0-1-month DMD mice. However, it has not yet been answered how micro-dystrophin encoding lentiviral treatment affects Dystrophin expression and DMD symptoms in 10-month mdx mice. METHODS: We planned to integrate the micro-Dystrophin gene sequence into the muscle cells by viral transfer, using micro-Dystrophin-encoding lentivirus to reduce the dystrophic pathology in late-stage dmd mice. The histopathological and physiological-functional regeneration activities of the lentiviralmicro- Dystrophin gene therapy methods were compared, along with changes in temporal Dystrophin expression and their functionality, toxicity, and gene expression level. RESULTS: Here, we showed that the micro-dystrophin transgene transfers intramuscularly and intraperitoneally in late-stage dmd-mdx-4cv mice restored dystrophin expression in the skeletal and cardiac muscle (p <0.001). Furthermore, motor performance analysis, including hanging and tracking tests, improved statistically significantly after the treatment (p <0.05). CONCLUSION: Consequently, this study suggests that patients in the late stages of muscular dystrophy can benefit from lentiviral micro-dystrophin gene therapies to present an improvement in dystrophic muscle pathology.

In Vitro FVIII-Encoding Transgenic Mesenchymal Stem Cells Maintain Successful Coagulation in FVIII-Deficient Plasma Mimicking Hemophilia A

Objective: Hemophilia A is an X-linked recessive bleeding disorder caused by a deficiency of plasma coagulation factor VIII (FVIII), and it accounts for about 80%-85% of all cases of hemophilia. Plasma-derived therapies or recombinant FVIII concentrates are used to prevent and treat the bleeding symptoms along with FVIII-mimicking antibodies. Recently, the European Medicines Agency granted conditional marketing approval for the first gene therapy for hemophilia A. The aim of this study was to determine the effectiveness of coagulation in correcting FVIII deficiency with FVIII-secreting transgenic mesenchymal stem cells (MSCs). Materials and Methods: A lentiviral vector encoding a B domain-deleted FVIII cDNA sequence with CD45R0 truncated (CD45R0t) surface marker was designed to develop a transgenic FVIII-expressing primary cell line by transducing MSCs. The efficacy and functionality of the FVIII secreted from the MSCs was assessed with anti-FVIII ELISA, CD45R0t flow cytometry, FVIII western blot, and mixing test analysis in vitro. Results: The findings of this study showed that the transgenic MSCs maintained persistent FVIII secretion. There was no significant difference in FVIII secretion over time, suggesting stable FVIII expression from the MSCs. The functionality of the FVIII protein secreted in the MSC supernatant was demonstrated by applying a mixing test in coagulation analysis. In the mixing test analysis, FVIII-deficient human plasma products were mixed with either a saline control or FVIII-secreted MSC supernatant. The mean FVIII level of the saline control group was 0.41±0.03 IU/dL, whereas the mean level was 25.41±33.38 IU/dL in the FVIII-secreting MSC supernatant mixed group (p<0.01). The mean activated partial thromboplastin time (aPTT) of the saline control group was 92.69±11.38 s, while in the FVIII-secreting MSC supernatant mixed group, the mean aPTT level decreased to 38.60±13.38 s (p<0.001). Conclusion: The findings of this in vitro study suggest that the new method presented here is promising as a possible treatment for hemophilia A. Accordingly, a study of FVIII-secreting transgenic MSCs will next be initiated in a FVIII-knockout animal model.

The Therapeutic Significance of Mesenchymal Stem Cells in COVID-19 Acute Pulmonary Respiratory Disease.

The coronavirus disease 2019 pandemic caused by severe acute respiratory syndrome-related coronavirus-2 continues its effects around the world with its new variants. Coronavirus disease 2019 infection may continue with a post-coronavirus disease period, which is characterized by high morbidity apart from the acute and subacute phases. Host immune response quality and inflammasome-induced uncontrollable inflammatory response take a role together in the pathogenesis of severe acute respiratory syndrome-related corona- virus-2 infection. Therefore, treatment of severe acute respiratory syndrome-related coronavirus-2 infection should basically include 3 measures: Viral replication, inflammation, and tissue damage control. Today, there is no effective therapy to control these points. At this point, preclinical studies have shown that mesenchymal stem cells can control inflammatory reactions and lung damage through both immune regulation and inflammasome control. Subsequently, controlled clinical studies on severe acute respiratory syndrome-related coronavirus-2 infection confirm their ability, indicating that mesenchymal stem cells may be a safe treatment option while reducing severe acute respiratory syndrome-related coronavirus-2-related morbidity and mortality. On the other hand, post-coronavirus syndrome is as important as acute coronavirus syndrome, it is a picture that can cause morbidity and mortality. Mesenchymal stem cell application can prevent the development of post-coronavirus syndrome through the mechanism of an inflammasome. However, there is no study that analyzes the effects of current treatments using mesenchymal stem cells in the post-coronavirus disease period, and that tests the use of mesenchymal stem cells when post-coronavirus syndrome develops. In this respect, studies that test the efficacy of mesenchymal stem cells in the post-coronavirus disease period are certainly needed.

The effects of thymoquinone on pancreatic cancer and immune cells.

OBJECTIVES: Black cumin is widely used as a spice and as a traditional treatment. The active ingredient in black cumin seeds is thymoquinone. Thymoquinone has shown anticancer effects in some cancers. We planned to investigate its anticancer effect on pancreatic cancer cell lines. METHODS: Thymoquinone chemical component in various doses was prepared and inoculated on pancreatic cancer cell culture, healthy mesenchymal stem cells, and peripheral blood mononuclear cell culture. IC50 values were calculated by absorbance data and measuring cell viability by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide staining of cells incubated with thymoquinone at 24, 48, and 72 h. RESULTS: There was dose-related cytotoxicity. Maximal cytotoxicity was observed at 24 h and 100 µM thymoquinone concentrations in pancreatic cancer cell culture and mesenchymal stem cells. Any concentration of thymoquinone was not cytotoxic to peripheral blood mononuclear cell. Thymoquinone even caused proliferation at a concentration of 6.25 µM. CONCLUSIONS: Since the cytotoxic concentration of thymoquinone on pancreatic cancer cell culture and mesenchymal stem cells is the same, it is not appropriate to use thymoquinone to achieve cytotoxicity in pancreatic cancer. However, since thymoquinone provides proliferation in peripheral blood mononuclear cell at a noncytotoxic dose, it may have an immune activator effect. Therefore, in vivo studies are needed to investigate the effect of thymoquinone on the immune system.

microRNA 1307 Is a Potential Target for SARS-CoV-2 Infection: An in Vitro Model.

microRNAs (miRs) are proposed as critical molecular targets in SARS-CoV-2 infection. Our recent in silico studies identified seven SARS-CoV-2 specific miR-like sequences, which are highly conserved with humans, including miR-1307-3p, with critical roles in COVID-19. In this current study, Vero cells were infected with SARS-CoV-2, and miR expression profiles were thereafter confirmed by qRT-PCR. miR-1307-3p was the most highly expressed miR in the infected cells; we, therefore, transiently inhibited its expression in both infected and uninfected cells. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) cell proliferation assay assessed cell viability following SARS-CoV-2 infection, identifying that miR-1307 expression is inversely correlated with cell viability. Lastly, changes in miR-1307-dependent pathways were analyzed through a detailed miRNOME and associated in silico analysis. In addition to our previously identified miRs, including miR-1307-3p, the upregulation of miR-193a-5p, miR-5100, and miR-23a-5p and downregulation of miR-130b-5p, miR34a-5p, miR-505-3p, miR181a-2-3p, miR-1271-5p, miR-598-3p, miR-34c-3p, and miR-129-5p were also established in Vero cells related to general lung disease-related genes following SARS-CoV-2 infection. Targeted anti-miR-1307-3p treatment rescued cell viability in infection when compared to SARS CoV-2 mediated cell cytotoxicity only. We furthermore identified by in silico analysis that miR-1307-3p is conserved in all SARS-CoV-2 sequences/strains, except in the BA.2 variant, possibly contributing to the lower disease severity of this variant, which warrants further investigation. Small RNA seq analysis was next used to evaluate alterations in the miRNOME, following miR-1307-3p manipulation, identifying critical pathobiological pathways linked to SARS-CoV-2 infection-mediated upregulation of this miR. On the basis of our findings, miRNAs like miR-1307-3p play a critical role in SARS-CoV-2 infection, including via effects on disease progression and severity.

Preliminary Report of the Academic CAR-T (ISIKOK-19) Cell Clinical Trial in Turkey: Characterization of Product and Outcomes of Clinical Application

Objective: Chimeric antigen receptor T (CAR-T) cell therapies have already made an impact on the treatment of B-cell malignancies. Although CAR-T cell therapies are promising, there are concerns about commercial products regarding their affordability and sustainability. In this preliminary study, the results of the first production and clinical data of an academic CAR-T cell (ISIKOK-19) trial in Turkey are presented. Materials and Methods: A pilot clinical trial (NCT04206943) designed to assess the safety and feasibility of ISIKOK-19 T-cell therapy for patients with relapsed and refractory CD19+ tumors was conducted and participating patients received ISIKOK-19 infusions between October 2019 and July 2021. The production data of the first 8 patients and the clinical outcome of 7 patients who received ISIKOK-19 cell infusions are presented in this study. Results: Nine patients were enrolled in the trial [5 with acute lymphoblastic leukemia (ALL) and 4 with non-Hodgkin lymphoma (NHL)], but only 7 patients could receive treatment. Two of the 3 participating ALL patients and 3 of the 4 NHL patients had complete/partial response (overall response rate: 72%). Four patients (57%) had CAR-T-related toxicities (cytokine release syndrome, CAR-T-related encephalopathy syndrome, and pancytopenia). Two patients were unresponsive and had progressive disease following CAR-T therapy. Two patients with partial response had progressive disease during follow-up. Conclusion: Production efficacy and fulfillment of the criteria of quality control were satisfactory for academic production. Response rates and toxicity profiles were also acceptable for this heavily pretreated/refractory patient group. ISIKOK-19 cells appear to be a safe, economical, and efficient treatment option for CD19+ tumors. However, the findings of this study need to be supported by the currently ongoing ISIKOK-19 clinical trial.

Mutational landscape of SARS-CoV-2 genome in Turkey and impact of mutations on spike protein structure.

The Coronavirus Disease 2019 (COVID-19) was declared a pandemic in March 2020 by the World Health Organization (WHO). As of May 25th, 2021 there were 2.059.941 SARS-COV2 genome sequences that have been submitted to the GISAID database, with numerous variations. Here, we aim to analyze the SARS-CoV-2 genome data submitted to the GISAID database from Turkey and to determine the variant and clade distributions by the end of May 2021, in accordance with their appearance timeline. We compared these findings to USA, Europe, and Asia data as well. We have also evaluated the effects of spike protein variations, detected in a group of genome sequences of 13 patients who applied to our clinic, by using 3D modeling algorithms. For this purpose, we analyzed 4607 SARS-CoV-2 genome sequences submitted by different lab centers from Turkey to the GISAID database between March 2020 and May 2021. Described mutations were also introduced in silico to the spike protein structure to analyze their isolated impacts on the protein structure. The most abundant clade was GR followed by G, GH, and GRY and we did not detect any V clade. The most common variant was B.1, followed by B.1.1, and the UK variant, B.1.1.7. Our results clearly show a concordance between the variant distributions, the number of cases, and the timelines of different variant accumulations in Turkey. The 3D simulations indicate an increase in the surface hydrophilicity of the reference spike protein and the detected mutations. There was less surface hydrophilicity increase in the Asp614Gly mutation, which exhibits a more compact conformation around the ACE-2 receptor binding domain region, rendering the structure in a "down" conformation. Our genomic findings can help to model vaccination programs and protein modeling may lead to different approaches for COVID-19 treatment strategies.

Preclinical Assessment of Efficacy and Safety Analysis of CAR-T Cells (ISIKOK-19) Targeting CD19-Expressing B-Cells for the First Turkish Academic Clinical Trial with Relapsed/Refractory ALL and NHL Patients

Objective: Relapsed and refractory CD19-positive B-cell acute lymphoblastic leukemia (ALL) and non-Hodgkin lymphoma (NHL) are the focus of studies on hematological cancers. Treatment of these malignancies has undergone recent transformation with the development of new gene therapy and molecular biology techniques, which are safer and well-tolerated therapeutic approaches. The CD19 antigen is the most studied therapeutic target in these hematological cancers. This study reports the results of clinical-grade production, quality control, and in vivo efficacy processes of ISIKOK-19 cells as the first academic clinical trial of CAR-T cells targeting CD19-expressing B cells in relapsed/refractory ALL and NHL patients in Turkey. Materials and Methods: We used a lentiviral vector encoding the CD19 antigen-specific antibody head (FMC63) conjugated with the CD8-CD28-CD3ζ sequence as a chimeric antigen receptor (CAR) along with a truncated form of EGFR (EGFRt) on human T-lymphocytes (CAR-T). We preclinically assessed the efficacy and safety of the manufactured CAR-T cells, namely ISIKOK-19, from both healthy donors' and ALL/NHL patients' peripheral blood mononuclear cells. Results: We showed significant enhancement of CAR lentivirus transduction efficacy in T-cells using BX-795, an inhibitor of the signaling molecule TBK1/IKKƐ, in order to cut the cost of CAR-T cell production. In addition, ISIKOK-19 cells demonstrated a significantly high level of cytotoxicity specifically against a CD19+ B-lymphocyte cancer model, RAJI cells, in NOD/SCID mice. Conclusion: This is the first report of preclinical assessment of efficacy and safety analysis of CAR-T cells (ISIKOK-19) targeting CD19-expressing B cells in relapsed/refractory ALL and NHL patients in Turkey.