A Cell-Penetrant Peptide Disrupting the Transcription Factor CP2c Complexes Induces Cancer-Specific Synthetic Lethality.

Despite advances in precision oncology, cancer remains a global public health issue. In this report, proof-of-principle evidence is presented that a cell-penetrable peptide (ACP52C) dissociates transcription factor CP2c complexes and induces apoptosis in most CP2c oncogene-addicted cancer cells through transcription activity-independent mechanisms. CP2cs dissociated from complexes directly interact with and degrade YY1, leading to apoptosis via the MDM2-p53 pathway. The liberated CP2cs also inhibit TDP2, causing intrinsic genome-wide DNA strand breaks and subsequent catastrophic DNA damage responses. These two mechanisms are independent of cancer driver mutations but are hindered by high MDM2 p60 expression. However, resistance to ACP52C mediated by MDM2 p60 can be sensitized by CASP2 inhibition. Additionally, derivatives of ACP52C conjugated with fatty acid alone or with a CASP2 inhibiting peptide show improved pharmacokinetics and reduced cancer burden, even in ACP52C-resistant cancers. This study enhances the understanding of ACP52C-induced cancer-specific apoptosis induction and supports the use of ACP52C in anticancer drug development.

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.

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.

Current status of red blood cell manufacturing in 3D culture and bioreactors.

Owing to donor-related issues, blood shortages and transfusion-related adverse reactions have become global issues of grave concern. In vitro manufactured red blood cells (RBCs) are promising substitutes for blood donation. In the United Kingdom, a clinical trial for allogeneic mini transfusion of cultured RBCs derived from primary hematopoietic stem cells has recently begun. However, current production quantities are limited and need improved before clinical use. New methods to enhance manufacturing efficiencies have been explored, including different cell sources, bioreactors, and 3-dimensional (3D) materials; however, further research is required. In this review, we discuss various cell sources for blood cell production, recent advances in bioreactor manufacturing processes, and the clinical applications of cultured blood.

The inactivation and destruction of viruses by reactive oxygen species generated through physical and cold atmospheric plasma techniques: Current status and perspectives.

BACKGROUND: Outbreaks of airborne viral infections, such as COVID-19, can cause panic regarding other severe respiratory syndrome diseases that may develop and affect public health. It is therefore necessary to develop control methods that offer protection against such viruses. AIM OF REVIEW: To identify a feasible solution for virus deactivation, we critically reviewed methods of generating reactive oxygen species (ROS), which can attack a wide range of molecular targets to induce antiviral activity, accounting for their flexibility in facilitating host defense mechanisms against a comprehensive range of pathogens. Recently, the role of ROS in microbial decontamination has been critically investigated as a major topic in infectious diseases. ROS can eradicate pathogens directly by inducing oxidative stress or indirectly by promoting pathogen removal through numerous non-oxidative mechanisms, including autophagy, T-cell responses, and pattern recognition receptor signaling. KEY SCIENTIFIC CONCEPTS OF REVIEW: In this article, we reviewed possible methods for the in vitro generation of ROS with antiviral activity. Furthermore, we discuss, in detail, the novel and environmentally friendly cold plasma delivery system in the destruction of viruses. This review highlights the potential of ROS as therapeutic mediators to modernize current techniques and improvement on the efficiency of inactivating SARS-CoV2 and other viruses.

Red cell manufacturing using parallel stirred-tank bioreactors at the final stages of differentiation enhances reticulocyte maturation.

The aim of this study was to develop a robust, quality controlled, and reproducible erythroid culture system to obtain high numbers of mature erythroblasts and red blood cells (RBCs). This was achieved using a fully controlled stirred-tank bioreactor by the design of experiments (DOE) methods in the serum-free medium by defining the appropriate culture parameters. Human cord blood CD34+ cells were first cultured in static flasks and then inoculated to stirred-tank bioreactors. Cell diameter was gradually decreased and final RBC yields were significantly higher when cells were inoculated at sizes smaller than 12 µm. The larger immature cells in the basophilic stage did not survive, while smaller mature erythroid cells were successfully expanded at high agitation speeds, demonstrating that appropriate seeding timing is critical. A high inoculation cell density of 5 × 106 cells/ml was achieved reaching 1.5 × 107 cells/ml. By using DOE analysis fitted to precise stages of erythropoiesis, we were able to acquire the optimal culture parameters for pH (7.5), temperature (37°C), dissolved oxygen, agitation speed (500 rpm), inoculation timing (cell diameter 12-13 µm), media feeding regimen, and cell seeding density (5 × 106 cells/ml). The final pure RBCs showed appropriate functions compared with fresh donor RBCs, confirming that manufacturing mature RBCs with reproducibility is possible.

Role of Plasma Gelsolin Protein in the Final Stage of Erythropoiesis and in Correction of Erythroid Dysplasia In Vitro.

Gelsolin, an actin-remodeling protein, is involved in cell motility, cytoskeletal remodeling, and cytokinesis and is abnormally expressed in many cancers. Recently, human recombinant plasma gelsolin protein (pGSN) was reported to have important roles in cell cycle and maturation of primary erythroblasts. However, the role of human plasma gelsolin in late stage erythroblasts prior to enucleation and putative clinical relevance in patients with myelodysplastic syndrome (MDS) and hemato-oncologic diseases have not been reported. Polychromatic and orthochromatic erythroblasts differentiated from human cord blood CD34+ cells, and human bone marrow (BM) cells derived from patients with MDS, were cultured in serum-free medium containing pGSN. Effects of pGSN on mitochondria, erythroid dysplasia, and enucleation were assessed in cellular and transcriptional levels. With pGSN treatment, terminal maturation at the stage of poly- and ortho-chromatic erythroblasts was enhanced, with higher numbers of orthochromatic erythroblasts and enucleated red blood cells (RBCs). pGSN also significantly decreased dysplastic features of cell morphology. Moreover, we found that patients with MDS with multi-lineage dysplasia or with excess blasts-1 showed significantly decreased expression of gelsolin mRNA (GSN) in their peripheral blood. When BM erythroblasts of MDS patients were cultured with pGSN, levels of mRNA transcripts related to terminal erythropoiesis and enucleation were markedly increased, with significantly decreased erythroid dysplasia. Moreover, pGSN treatment enhanced mitochondrial transmembrane potential that is unregulated in MDS and cultured cells. Our findings demonstrate a key role for plasma gelsolin in erythropoiesis and in gelsolin-depleted MDS patients, and raises the possibility that pGSN administration may promote erythropoiesis in erythroid dysplasia.

Improvement of Red Blood Cell Maturation In Vitro by Serum-Free Medium Optimization.

Despite recent studies on media additives, the low viability and dysplastic features of terminally mature erythroid cells are still major problems in enhancing erythroid cell yields in vitro. Moreover, research on enhancing terminal erythropoiesis has been focused on the immature stage of erythroid cells such as burst forming unit-erythroid and colony forming unit-erythroid. Here, we tested many commercially available serum-free culture media and developed a superior culture media formulation compared with the conventional control for higher expansion-fold, higher viability, and therefore enhanced red cell productivity. The addition of the specific medium to the previously known best media at a specific ratio, whose effects were not dose-dependent, enabled the generation of significantly higher erythrocyte products with over 1.3 million-fold proliferation of erythroid cells after maintenance for 21 days throughout the maturation stages from CD34+ cells. The cells cultured in this condition expressed maturation markers and were significantly superior in differentiation and enucleation. Comparative mRNA profiling revealed that erythroid cells in this medium showed more efficient maturation in mRNA levels. The cultured cells showed comparable erythroblast survival and also restored better red blood cell (RBC) functions of oxygen saturation profile with expression of adult globin up to 99%. However, to develop chemically defined media, the well-known supplements including hormones, cytokines, and serum-replacement reagents were not sufficient to replace the optimized media in producing mature RBCs. Taken together, our optimized medium formulation under serum-free culture conditions produced the best reproducible results on productivity and maturation in erythroid cells with economic benefits. These culture conditions may thus serve as a useful platform for further investigation of in vitro erythropoiesis and to develop defined serum-free media for clinical trials.

The effects of plasma gelsolin on human erythroblast maturation for erythrocyte production.

Gelsolin is an actin binding protein present in blood plasma and in cytoplasm of cells including macrophages. Gelsolin has important functions in cell cycle regulation, apoptotic regulation, and morphogenesis. Even though bone marrow macrophages and serum factors are critical for regulating erythropoiesis, the role of gelsolin on human erythroblasts has not been studied. Here, we investigated the effects of human recombinant plasma gelsolin (pGSN) on human immature erythroblasts. CD34+ cells isolated from cord blood were differentiated into erythroid cells in serum-free medium. When pGSN was applied to the culture medium, it accelerated basophilic and polychromatic erythroblast maturation and increased the enucleation rate with highly expressed erythropoiesis-related mRNAs. Also, pGSN was effective in reducing dysplastic changes caused by vincristine, suggesting its role in cell cycle progression at G2/M checkpoints. Also, pGSN activated caspase-3 during maturation stages in which caspase-3 functions as a non-apoptotic maturational signal or a pro-apoptotic signal depending on maturation stages. Our results suggest that pGSN has a pivotal role in maturation of erythroblasts and this factor might be one of the way how bone marrow macrophages and previously unknown serum factors work to control erythropoiesis. pGSN might be used as additive for in vitro production of erythrocytes.

Mbd2-CP2c loop drives adult-type globin gene expression and definitive erythropoiesis.

During hematopoiesis, red blood cells originate from the hematopoietic stem cell reservoir. Although the regulation of erythropoiesis and globin expression has been intensively investigated, the underlining mechanisms are not fully understood, including the interplay between transcription factors and epigenetic factors. Here, we uncover that the Mbd2-free NuRD chromatin remodeling complex potentiates erythroid differentiation of proerythroblasts via managing functions of the CP2c complexes. We found that both Mbd2 and Mbd3 expression is downregulated during differentiation of MEL cells in vitro and in normal erythropoiesis in mouse bone marrow, and Mbd2 downregulation is crucial for erythropoiesis. In uninduced MEL cells, the Mbd2-NuRD complex is recruited to the promoter via Gata1/Fog1, and, via direct binding through p66α, it acts as a transcriptional inhibitor of the CP2c complexes, preventing their DNA binding and promoting degradation of the CP2c family proteins to suppress globin gene expression. Conversely, during erythropoiesis in vitro and in vivo, the Mbd2-free NuRD does not dissociate from the chromatin and acts as a transcriptional coactivator aiding the recruitment of the CP2c complexes to chromatin, and thereby leading to the induction of the active hemoglobin synthesis and erythroid differentiation. Our study highlights the regulation of erythroid differentiation by the Mbd2-CP2c loop.

Red blood cell generation by three-dimensional aggregate cultivation of late erythroblasts.

Stem cell-derived erythroid cells hold great potential for the treatment of blood-loss anemia and for erythropoiesis research; however, cultures using conventional flat plates or bioreactors have failed to show promising results. By mimicking the in vivo bone marrow (BM) environment in which most erythroid cells are physically aggregated, we show that a three-dimensional (3D) aggregate culture system facilitates erythroid cell maturation and red blood cell (RBC) production more effectively than two-dimensional high-density cell cultivation. Late erythroblasts (polychromatic or orthochromatic erythroblasts) were differentiated from cord blood CD34(+) cells over 15 days and then allowed to form tight aggregates at a minimum density of 1×10(7) cells/mL for 2-3 days. To scale up the cell culture and to make the media supply efficient throughout the cell aggregates, several macroporous microcarriers and porous scaffolds were applied to the 3D culture system. In comparison to control culture conditions, erythroid cells in 3D aggregates were significantly more differentiated toward RBCs with significantly reduced nuclear dysplasia. When 3D culture was performed inside macroporous microcarriers, the cell culture scale was increased and cells exhibited enhanced differentiation and enucleation. Microcarriers with a pore diameter of approximately 400 µm produced more mature cells than those with a smaller pore diameter. In addition, this aggregate culture method minimized the culture space and media volume required. In conclusion, a 3D aggregate culture system can be used to generate transfusable human erythrocytes at the terminal maturation stage, mimicking the in vivo BM microenvironment. Porous structures can efficiently maximize the culture scale, enabling large-scale production of RBCs. These results enhance our understanding of the importance of physical contact among late erythroblasts for their final maturation into RBCs.

De novo hairy cell leukemia with a major BCR/ABL1 rearrangement: a case report with a literature review.

Hairy cell leukemia (HCL) is a very rare mature B-cell neoplasm and its simultaneous occurrence with chronic myeloid leukemia has been reported in only three cases. The pathogenesis and relationship of the two diseases are not clear. Here we report a case of HCL expressing a BCR/ABL1 clone, which showed molecular remission of the fusion clones and achieved partial remission over nine months of cladribine therapy. After a thorough analysis of previous studies and the results of this patient, we speculate that a subclone evolved to have an additional genetic BCR/ABL1 rearrangement. We also review all published literature on HCL with BCR/ABL1 rearrangement and discuss the pathophysiology of these unusual cases.

The RNA in reticulocytes is not just debris: it is necessary for the final stages of erythrocyte formation.

Reticulocytes contain both RNA and micro-organelles and represent the last stage of erythropoiesis before full maturation to red blood cells (RBCs). Even though there is continuing synthesis of hemoglobin and membrane-bound proteins in reticulocytes, the small amount of RNA that they contain has been regarded as non-functional residual material. Here we show that this residual RNA is both functional and essential for further reticulocyte maturation. Reticulocytes from which the remnant RNA had been removed by exposure to RNase did not survive or mature into RBCs in either humans or mice. Conversely, reticulocytes treated with an RNase Inhibitor were able to form normal biconcave cells. Similarly, poor survival was also seen in reticulocytes in which protein synthesis had been blocked. To identify the signaling pathways involved we isolated RNAs in reticulocytes versus those present in fully matured erythroblasts cultured from hematopoietic stem cells. RNAs found in erythroblasts were related to exocytosis, metabolism, and signal transduction all of which are critical for maturation through reticulocyte and into a fully mature, biconcave erythrocyte. Our results suggest that the mRNA in reticulocytes has to be translated into novel proteins that act to preserve mitochondria and maintain cell membrane integrity as reticulocytes mature. These results enhance our understanding of the final stage of erythropoiesis and may clarify why in vitro-generated reticulocytes for transfusion purposes survive poorly.

Randomized controlled trials in endourology: a quality assessment.

PURPOSE: To analyze the quality of studies reporting randomized clinical trials (RCTs) in the field of endourology. MATERIALS AND METHODS: RCTs published in the Journal of Endourology from 1993 until 2011 were identified. The Jadad scale, van Tulder scale, and Cochrane Collaboration Risk of Bias Tool (CCRBT) were used to assess the quality of the studies. The review period was divided into early (1993-1999), mid (2000-2005), and late (2006-2011) terms. Studies were categorized by country of origin, subject matter, single- vs multicenter setting, Institutional Review Board (IRB) approval and funding support, and blinding vs nonblinding. RESULTS: In total, 3339 articles had been published during the defined review period, of which 165 articles were reporting a RCT. There was a significant increase in the number of RCTs published over time, with 18 (2.81%), 43 (4.88%), and 104 (5.72%) studies identified in the early, mid, and late term, respectively (P=0.009). Nevertheless, there was no difference in terms of quality of reporting, as assessed with the Jadad scale, van Tulder scale, or CCRBT, between the three study terms. On the other hand, significant differences were found in both the number of high qualitative RCTs that used blinding methodology and those that had IRB review, when comparing the early, mid, and late terms. CONCLUSION: There has been a growing number of Journal of Endourology publications reporting on RTC over the last two decades. The quality of reporting for these studies remains suboptimal, however. Researchers should focus on a more appropriate description of key features of any given RCT, such as randomization and allocation methods, as well as disclosure of IRB review and financial support.

Autonomous control of terminal erythropoiesis via physical interactions among erythroid cells.

In vitro erythropoiesis has been studied extensively for its application in the manufacture of transfusable erythrocytes. Unfortunately, culture conditions have not been as effective as in vivo growth conditions, where bone marrow macrophages are known to be a key regulator of erythropoiesis. This study focused on the fact that some erythroblasts are detached from macrophages and only contact other erythroblasts. We hypothesized that additional factors regulate erythroblasts, likely through either physical contact or secreted factors. To further elucidate these critical factors, human erythroblasts derived from cord blood were cultured at high density to mimic marrow conditions. This growth condition resulted in a significantly increased erythroid enucleation rate and viability. We found several novel contact-related signals in erythroblasts: intercellular adhesion molecule-4 (ICAM-4) and deleted in liver cancer-1 (DLC-1). DLC-1, a Rho-GTPase-activating protein, has not previously been reported in erythroid cells, but its interaction with ICAM-4 was demonstrated here. We further confirmed the presence of a secreted form of human ICAM-4 for the first time. When soluble ICAM-4 was added to media, cell viability and enucleation increased with decreased nuclear dysplasia, suggesting that ICAM-4 is a key factor in contact between cells. These results highlight potential new mechanisms for autonomous control of erythropoiesis. The application of these procedures to erythrocyte manufacturing could enhance in vitro erythrocyte production for clinical use.

Topical treatment of the buccal mucosa and wounded skin in rats with a triamcinolone acetonide-loaded hydrogel prepared using an electron beam.

In this study, a triamcinolone acetonide-loaded hydrogel was prepared by electron beam irradiation and evaluated for use as a buccal mucoadhesive drug delivery system. A poloxamer was modified to have vinyl end groups for preparation of the hydrogel via an irradiation cross-linking reaction. Carbopol was introduced to improve the mucoadhesive properties of the hydrogel. The in vitro release of triamcinolone acetonide from the hydrogel was examined at 37 °C. To investigate the topical therapeutic effect of triamcinolone acetonide on wounded rat skin and buccal mucosa, the appearance and histological changes were evaluated for 15 days after treatment with saline, triamcinolone acetonide solution, triamcinolone acetonide hydrogel, and blank hydrogel, respectively. Triamcinolone acetonide was released constantly from the gel formulation at 37 °C and reach 100% at about 48 h. After 15 days, in the skin of the group treated with the triamcinolone acetonide-loaded hydrogel, the wound was almost completely free of crust and a number of skin appendages, including hair follicles, had formed at the margins of the tissue. Moreover, the inflammatory response in the buccal mucosa was milder than that in the other groups, and the wound surface was completely covered with regenerating, hyperkeratotic, thickened epithelial cells. Our results indicate that the triamcinolone-acetonide hydrogel showed sustained drug release behavior, while causing no significant histopathological changes in buccal and skin tissues. Therefore, this hydrogel system may be a powerful means of drug delivery for buccal administration with controlled release and no tissue irritation.