Increase In Total Cell Number Research Articles

Anti-pneumoconiosis effect of schisantherin A in PMA-induced A549 cells and SiO2/TiO2nanoparticles-induced acute pulmonary injury in mice

There has been significant global interest in respiratory health driven by the coronavirus disease (COVID-19) and severe environmental pollution. This study explored the potential of schisantherin A (SchA), a compound derived from Schisandra chinensis, to protect against acute pneumoconiosis. We assessed the effects of SchA on phorbol 12-myristate 13-acetate (PMA)-stimulated A549 alveolar epithelial cells and SiO2/TiO2-induced pulmonary injury in mice. In A549 cells, SchA significantly decreased pro-inflammatory mediators such as inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and interleukin (IL)-8 levels. SchA-mediated reduction in inflammatory mediators was associated with the downregulation of PMA-stimulated nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and mitogen-activated protein kinase (MAPK) signaling activation. In SiO2/TiO2-induced lung-injured mice, SchA administration significantly reduced MUC5AC production in lung tissue. SchA administration significantly downregulated the overexpression of NK-κB and the subsequent production of COX-2, iNOS, and NOD-like receptor pyrin domain-containing protein 3 (NLRP3) inflammasomes. It significantly suppressed expected increases in total cell numbers and pro-inflammatory cytokines, including tumor necrosis factor-alpha (TNF-α) and IL-1β in the bronchoalveolar lavage fluid (BALF) in SiO2/TiO2-stimulated mice. In contrast, the SiO2/TiO2-mediated decrease in IL-10 levels was significantly improved by SchA treatment. These fundamental results can be used to develop potential treatments involving SchA for acute pneumoconiosis.

Enhancement of porcine in vitro embryonic development through luteolin-mediated activation of the Nrf2/Keap1 signaling pathway

BackgroundOxidative stress, caused by an imbalance in the production and elimination of intracellular reactive oxygen species (ROS), has been recognized for its detrimental effects on mammalian embryonic development. Luteolin (Lut) has been documented for its protective effects against oxidative stress in various studies. However, its specific role in embryonic development remains unexplored. This study aims to investigate the influence of Lut on porcine embryonic development and to elucidate the underlying mechanism.ResultsAfter undergoing parthenogenetic activation (PA) or in vitro fertilization, embryos supplemented with 0.5 µmol/L Lut displayed a significant enhancement in cleavage and blastocyst formation rates, with an increase in total cell numbers and a decrease in the apoptosis rate compared to the control. Measurements on D2 and D6 revealed that embryos with Lut supplementation had lower ROS levels and higher glutathione levels compared to the control. Moreover, Lut supplementation significantly augmented mitochondrial content and membrane potential. Intriguingly, activation of the Nrf2/Keap1 signaling pathway was observed in embryos supplemented with Lut, leading to the upregulation of antioxidant-related gene transcription levels. To further validate the relationship between the Nrf2/Keap1 signaling pathway and effects of Lut in porcine embryonic development, we cultured PA embryos in a medium supplemented with brusatol, with or without the inclusion of Lut. The positive effects of Lut on developmental competence were negated by brusatol treatment.ConclusionsOur findings indicate that Lut-mediated activation of the Nrf2/Keap1 signaling pathway contributes to the enhanced production of porcine embryos with high developmental competence, and offers insight into the mechanisms regulating early embryonic development.

Identification and Functional Validation of Neoantigen-Specific T Cells in Pediatric Patients with Fusion-Derived Acute Leukemias

Leukemia emerges from the accumulation of genetic mutations in hematopoietic cells, resulting in formation of altered proteins. Some of these mutant proteins (neoantigens) are consequently processed and presented as neoepitopes through major histocompatibility complex (MHC) molecules expressed on the surface of leukemia cells, permitting tumor-specific immune targeting. While adoptive T cell therapies against high mutation burden tumors have shown promising results, less is known about leveraging tumor-specific T cells as an effective approach in pediatric patients, including high-risk leukemia. In this study, peripheral blood or bone marrow samples were obtained from 34 leukemia patients aged 0 -14 years old. The patient tumors had founder fusion genes, including 15 patients with acute lymphoblastic leukemia (ALL), 15 patients with acute myeloid leukemia (AML), and 4 patients with mixed phenotype acute leukemia (MPAL). Samples were collected at the time of diagnosis or relapse. While leukemia blasts were the most abundant cellular component in these samples, the frequency of lymphocytes ranged between 1%- 25%, with a median of 3.5%. KMT2A rearrangements were expressed with either AFF1, MLLT1, MLLT3, MLLT4, MLLT6, or MLLT10 fusion partner in 73.5% of the patients' samples in this study. This included (14/15) ALL, (10/15) AML, and (1/4) MPAL patients . T cells were enriched and expanded for a total of 14 days using a rapid expansion protocol culture system. This approach was successful on all tested samples irrespective of their mutation backgrounds or initial lymphocyte count, with a range of 133-4957fold increases in total cell number and average frequencies of 59.7% and 26.1% for CD4+ and CD8+ T cell, respectively. We further screened the T cell response by examining the expression of 4-1BB, OX40, and IFN-g upon coculture with their autologous leukemia cells, and additionally validated the T cell killing activity against the blasts by a cell killing assay using standard protocol. We found that the expanded T cells exhibited a robust immune activation when cocultured with the autologous leukemia cells from ALL patients with KMT2A rearrangements, indicating an endogenous cell response in all patients in the ALL cohort (100%). However, we observed a considerably reduced reactivity in the samples from the AML and MPAL patients. Interestingly, we observed that such diminished reactivity was associated with a marked downregulation in the expression of MHC class I and II molecules, and that pretreating the leukemia blasts with IFN-γ prior to coculturing with the expanded T cells was sufficient to induce the expression of MHC molecules and subsequent activation of the expanded T cells. Single-cell TCR sequencing of the sorted, tumor-reactive T cells identified the major clonotypes among these reactive T cell clones and the top 4 - 6 clonotypes were selected from 5 patients to evaluate reactivity against the leukemia blasts and examine their specificity against the corresponding driving fusions. We further generated TCR-expressing clones and subsequently cocultured each clone with leukemia blasts to validate their antigen recognition and reactivity by cytotoxicity assay. Interestingly, and in line with the findings from investigations on solid tumors, the leukemia- reactive T cells were consistently characterized by higher expression of both PD1 and CD39 (PD1 hi CD39 +), indicating the capability of utilizing both markers for the enrichment of T cell prior to expansion and subsequent implementation. Despite the high-risk prognosis and low mutation burden in our patient cohort, our findings demonstrate that patient T cells contain endogenous neoantigen-specific clones which can be therapeutically leveraged for targeting leukemia blasts. These results support the utilization of adoptive T cell therapy in the context of hematological malignancies especially given the less restrictive spatial segregation between tumor-specific T cells and leukemia cells compared to solid tumors, where T cell infiltration is a prerequisite for antitumor immunity. Overall, these findings suggest the possibility of leveraging leukemia-associated lymphocytes, fusion oncogene vaccines, and TCR-based therapy as novel immunotherapeutic approaches for mitigating pediatric leukemia, particularly in high-risk patients.

Tunisian Horehound (Marrubium vulgare) Aqueous Extract Improves Treatment of Bleomycin-Induced Lung Fibrosis in Rat.

Pulmonary fibrosis (PF) remains one of the most serious pneumopathies whose diagnosis and physiopathogenesis are still poorly understood and no treatment has been shown to be effective. Recently, many studies have shown a renewed interest in plants thanks to their pharmacological potentials, like horehound, known, for its anti-inflammatory and antioxidant activities. The present study investigated the effects of the aqueous extract of horehound (Mae) on bleomycin (BLM)-induced PF in rats. Thirty rats were divided into three groups. The control group received no treatment, the BLM group received only intratracheally BLM (2 mg/kg), and the Mae group underwent administration of BLM+ Mae (2 mL/kg) daily for 20 days. Obtained results showed that Mae, rich in polyphenols, could significantly improve the damage caused by BLM by reducing the inflammatory index and the fibrosis score, bringing the lung structure of fibrotic rats close to that of control rats. As well, Mae obviously acted on the BLM inflammatory reaction, and the counting of bronchoalveolar lavage fluid (Balf) cells showed an increase in total cell number and a decrease in the infiltration of inflammatory cells in the bronchoalveolar space. In addition, the BLM instillation was accompanied by oxidative stress in the lung, liver, and kidney tissues, proven by an increase in lipid peroxidation, as well as through depletion of superoxide dismutase (SOD) and catalase (CAT). The Mae treatment reversed all disturbances of BLM-induced oxidative stress parameters promoting antioxidant and anti-inflammatory of the latter. These findings point to Mae as a promising candidate for the treatment of pulmonary fibrosis.

Conductive Supramolecular Polymer Nanocomposites with Tunable Properties to Manipulate Cell Growth and Functions.

Synthetic bioactive nanocomposites show great promise in biomedicine for use in tissue growth, wound healing and the potential for bioengineered skin substitutes. Hydrogen-bonded supramolecular polymers (3A-PCL) can be combined with graphite crystals to form graphite/3A-PCL composites with tunable physical properties. When used as a bioactive substrate for cell culture, graphite/3A-PCL composites have an extremely low cytotoxic activity on normal cells and a high structural stability in a medium with red blood cells. A series of in vitro studies demonstrated that the resulting composite substrates can efficiently interact with cell surfaces to promote the adhesion, migration, and proliferation of adherent cells, as well as rapid wound healing ability at the damaged cellular surface. Importantly, placing these substrates under an indirect current electric field at only 0.1 V leads to a marked acceleration in cell growth, a significant increase in total cell numbers, and a remarkable alteration in cell morphology. These results reveal a newly created system with great potential to provide an efficient route for the development of multifunctional bioactive substrates with unique electro-responsiveness to manipulate cell growth and functions.

Recombinant Pyrin Domain Protein Attenuates Airway Inflammation and Alleviates Epithelial-Mesenchymal Transition by Inhibiting Crosstalk Between TGFβ1 and Notch1 Signaling in Chronic Asthmatic Mice.

This article aims to investigate the effects of recombinant pyrin domain (RPYD) on airway inflammation and remodeling in mice with chronic asthma. The chronic asthma BALB/c mouse model was first sensitized by ovalbumin (OVA) and then challenged by OVA nebulization. RPYD or dexamethasone was given before OVA challenge. Our results showed that RPYD significantly inhibited the increase of total cell number, eosinophils, neutrophils and lymphocytes in bronchoalveolar lavage fluid (BALF) induced by OVA, and reduced the infiltration of inflammatory cells, the proliferation of goblet cells and collagen deposition. In addition, RPYD inhibited the mRNA and protein levels of α-smooth muscle actin (α-SMA), transforming growth factor (TGF)-β1, Jagged1, Notch1, Hes1 and Smad3, as well as Smad3 phosphorylation. TGFβ1 down-regulated the level of E-cadherin and promoted the expression of α-SMA, thus inducing epithelial-mesenchymal transition (EMT) in bronchial epithelial cells. We found that RPYD reduced EMT by inhibiting TGFβ1/smad3 and Jagged1/Notch1 signaling pathways. Further overexpression of NICD showed that under the stimulation of TGFβ1, NICD enhanced the phosphorylated Smad3 and nuclear Smad3, accompanied by the increased expression of Notch1 target gene Hes1. In contrast, after treatment with smad3 siRNA, the expression of Hes1 was down regulated as the decrease of Smad3, which indicates that there is crosstalk between smad3 and NICD on Hes1 expression. In conclusion, RPYD reduces airway inflammation, improves airway remodeling and reduces EMT in chronic asthmatic mice by inhibiting the crosstalk between TGFβ1/smad3 and Jagged1/Notch1 signaling pathways.

Advancing the robust manufacture of t-cell therapies through the application of stirred tank bioreactors

Background & Aim With over 90 active trials in the United States with many more in pre-clinical phases T-Cell immunotherapies represent a large share of the expansion we are observing in the regenerative medicine industry. While CAR-T therapies have dominated the headlines, other technologies such as TCR, TIL, Treg, CAR-NK, γδ T-Cells have shown impressive results the oncologic space and elsewhere, however a robust manufacturing process is required to realize these solutions in the clinic. A main hurdle in establishing a robust manufacturing process for these therapies is a lengthy expansion period is typically necessary to reach a relevant dose of therapeutic cells. This only becomes more critical as allogeneic cell therapies become a reality. The issues with current culture methods present with a high cost of goods, increased production timelines, and highly variant expansion rates. Methods, Results & Conclusion At Hitachi Chemical Advanced Therapeutic Solutions, we investigated stirred tank bioreactors as an alternative to standard static and rocking motion culture methods using the ambr15 microbioreactor system and confirming those results in a two liter stirred tank bioreactors. Because of the extensive history stirred tank bioreactors have in cell culture we have demonstrated that a power input of 7.5 W/m3 does not create undo shear stress on T-Cells and were able to directly translate that parameter from the 15mL scale to the 2L. This work focused on defining the specific parameters that would facilitate stirred tank bioreactors as a unique solution for the manufacture of both autologous and allogeneic T-cell therapies with high rates of mass and gas transfer and established process control methods. Given these advantages, a comparison study of T-cells expanded in the stirred tank bioreactor and batch fed static cultures resulted in a three-fold increase in total cell number with the bioreactors. Further development of stirred tank culture methods has demonstrated five population doublings over an eight-day culture period. These outcomes indicate at a T-cell expansion method that could expand the potential of the revolutionary therapies through reduced media requirements, shortened culture periods, and high yields. By further utilizing established techniques in cell culture, T-cell therapies become increasingly viable treatments for a range of indications.

SAT-LB053 Investigating the Effects of Growth Hormone on Tumor-derived Exosome Output and Content

Exosomes, the extracellular vesicles originating from endosomes, modulate their target cell responses systematically by the nature of exosomal cargoes and their source tissues. The exosomes thus occupy a new-found critical role in the diagnoses of multiple pathologies especially cancer as well as in anti-cancer therapy. Growth hormone (GH) produced centrally by the pituitary gland or peripherally by several tissues, contributes to the regulation of growth and metabolism. We and others have shown that GH significantly promotes cancer progression, metastasis, and drug resistance- processes also regulated by exosomes from tumor tissues. In this report, we show for the first time that GH increases cancer cell exosome secretion as well as content which in turn potentially amplify tumor invasion and metastases. Our data show that GH treatment, significantly increases levels of exosomal markers in human melanoma SK-MEL-30 (skin cancer), PANC1 (pancreatic cancer) and H1299 (lung cancer) cell lines without a comparable increase in total cell numbers, indicating an increased exosome output. In addition, GH treatment induced changes in the tumor exosomal cargoes including Matrix Metalloproteinases (MMPs) and Transforming Growth Factor beta (TGFβ). Also, GHR as a hitherto unknown exosomal cargo was found in exosomes from H1299 and PANC1 cell lines. Exosomes are a hotbed of current biological research and have diverse potential as an indicator in diagnosis, as a drug target, or the drug itself. Our study identifies the previously unknown effects of GH on exosome secretion from cancer cells and the nature of the respective exosomal cargoes. Acknowledgement This work was supported in part by the State of Ohio’s Eminent Scholar Program that includes a gift from Milton and Lawrence Goll, by the AMVETS, and the Edison Biotechnology Institute at Ohio University. Unless otherwise noted, all abstracts presented at ENDO are embargoed until the date and time of presentation. For oral presentations, the abstracts are embargoed until the session begins. s presented at a news conference are embargoed until the date and time of the news conference. The Endocrine Society reserves the right to lift the embargo on specific abstracts that are selected for promotion prior to or during ENDO.

Generation of Fanconi Anemia iPSC Clones By Addition of a Small Molecule Inhibitor of p53 during Reprogramming

Fanconi anemia (FA) is an inherited bone marrow failure syndrome characterized by loss of function mutations in any of at least 22 FANC genes that are critical for DNA repair. Progressive loss of hematopoietic stem/progenitor cells (HSPCs) in FA patients has been attributed in part to a heightened p53/p21 axis that restricts the cell cycle to G0/G1 in response to accumulation of unresolved DNA damage and replicative stress. Silencing of p53 was previously shown to rescue HSPC defects in FA models in vitro and in vivo. Due to insufficient numbers of primary HSPCs in FA for disease modeling and cell therapies, the concept of generating induced pluripotent stem cells (iPSCs) from an individual patient, and differentiating the FA-iPSCs into a theoretically infinite supply of autologous HSPCs arose. However, reprogramming FA cells has proven to be challenging and mostly dependent on hypoxic culture conditions or genetic complementation of the defect by integrating lentiviral vectors expressing a normal FANC gene. A recent study reported low-efficiency generation of integration-free FA-iPSCs by electroporation of fibroblasts with episomal plasmid vectors expressing both reprogramming factors and p53 shRNA. Here we sought to develop an improved and streamlined reprogramming strategy for FA by transduction of readily accessible FA mononuclear cells (MNCs) with commercially available non-integrating reprogramming Sendai Viruses (SeV2), combined with a more adaptable inhibition of the p53 pathway by addition of variable concentrations of a small molecule during reprogramming. Approximately 100,000 MNCs from an FA patient with confirmed biallelic mutations in FANCA were cultured for 10 days under hypoxic conditions (5% O2) favoring either myeloid or erythroid expansion. While myeloid cultures led to minimal proliferation of FA MNCs, we observed a 6-fold increase in total cell numbers in the presence of EPO. Erythroid-expanded FA cells were transduced with SeV2 harboring four standard reprogramming transcription factors (SOX2, OCT4, KLF4 and c-MYC). Cells were subsequently cultured under hypoxia for at least 30 days in a reprogramming cocktail supplemented or not with 0.3uM, 0.6µM or 1.2µM cyclic pifthrin-α (cPFT), a reversible small molecule inhibitor of p53-dependent gene transcription. In the absence of cPFT, no FA-iPSC clones were obtained, consistent with reported reprogramming barriers in FA cells. In contrast, several iPSC colonies were generated with cPFT-treated FA cells, albeit at much slower kinetics (~30 days) compared to MNCs without FA mutations (~20 days). The overall efficiency of reprogramming was ~0.01%, with a trend toward enhanced efficiencies in cultures containing the highest concentration of cPFT (1.2µM). To investigate the need for p53 inhibition beyond the reprogramming period, half of the FA-iPSC clones were passaged and further cultured in 5% O2 with cPFT 1.2µM, whereas the remaining clones were similarly treated but without p53 inhibitor. None of the FA-iPSC clones that were maintained in cPFT-containing cultures following reprogramming survived further passaging. In contrast, when cPFT was removed after reprogramming, all iPSC clones could stably undergo repeated passages provided that hypoxia (5% O2) was maintained. The FA-iPSC clones expressed high levels alkaline phosphatase and were TRA 1-60- and NANOG-positive, preliminarily validating their pluripotent nature. Karyotyping, trilineage differentiation, and in vivo teratoma assays are ongoing. Overall, this study provides a practical approach for successful reprogramming of FA peripheral blood cells based on commercially available non-integrating SeV2 reprogramming strategies supplemented with p53 inhibition. Regulated addition of variable concentrations of a small molecule inhibitor of p53 at various steps of the reprogramming process will allow further enhancement of reprogramming efficiencies in FA. DisclosuresNo relevant conflicts of interest to declare.

Antioxidants improve IVF outcome and subsequent embryo development in the mouse

What is the effect of a combination of three antioxidants (Acetyl-L-Carnitine, N-Acetyl-L-Cysteine and α-Lipoic Acid), present in IVF medium during mouse oocyte and sperm collection, on fertilization and subsequent IVF embryo development? A combination of antioxidants resulted in faster developmental times from the 2-cell stage through to expanded blastocyst stage, accompanied by a significant increase in blastocyst cell number and a reduction of intracellular hydrogen peroxide (H2O2) levels. The antioxidant combination Acetyl-L-Carnitine, N-Acetyl-L-Cysteine and α-Lipoic Acid, when present in embryo culture media, has a significant beneficial effect on in vitro fertilized mouse pronucleate oocyte development, especially under oxidative stress. IVF was conducted with combined antioxidants supplemented in IVF medium that was used for mouse oocyte collection and fertilization (oocyte IVF medium, 4 h exposure) and sperm collection and preparation (sperm IVF medium, 1 h exposure). IVF was conducted under 20% oxygen, in the presence or absence of a combination of antioxidants (10 μM Acetyl-L-Carnitine, 10 μM N-Acetyl-L-Cysteine, 5 μM α-Lipoic Acid) and resultant embryos cultured with and without antioxidants under 20% oxygen. Subsequently, the effects of antioxidants on either oocytes or sperm was evaluated. Embryo development was analysed through time-lapse microscopy followed by differential nuclear staining to determine cell allocation in the blastocyst. Intracellular levels of H2O2 were assessed using an aryl boronate probe after 4 h of incubation with antioxidants. Controls were gametes and embryos that had no antioxidants in the medium. In a separate series of experiments, pronucleate oocytes were collected in handling medium with and without antioxidants for 20 min and subsequent cell numbers analysed. Antioxidant treatment during both IVF and culture resulted in significantly faster development times to two cell cleavage (P < 0.01), which continued through to the expanded blastocyst stage (P < 0.05). Resultant blastocysts had a significant increase in both trophectoderm (TE) cell numbers, inner cell mass (ICM) and total cell numbers (P < 0.001). The addition of antioxidants to IVF medium or embryo culture media exclusively also resulted in a significant increase in both blastocyst TE and ICM numbers leading to an increase in total cell numbers (P < 0.001). Antioxidant supplementation of either oocyte IVF medium alone, or in both oocyte and sperm IVF medium, lead to significantly faster times to two cell cleavage, which continued through to the expanded blastocyst stage. Blastocyst cell number in both these groups had significantly higher TE cell numbers resulting in an increase in total cell numbers. In contrast, there were no differences in embryo developmental rates and blastocyst cell number when antioxidants were present only in the sperm IVF medium. Levels of H2O2 were significantly reduced in pronucleate oocytes that were cultured in the presence of antioxidants (P < 0.001) compared to control, untreated embryos. Similarly, pronucleate oocytes treated with the combined antioxidants during pronucleate oocyte collection resulted in significantly increased blastocyst ICM numbers compared with controls (P < 0.05). Embryo development was only examined in the mouse. These findings suggest that supplementation of antioxidants to the IVF medium, as well as to embryo culture media, may further assist in maintaining the viability of human embryos in ART, conceivably through the reduction of oxidative stress. This work was funded by a research grant from Vitrolife AB (Sweden). The authors have no conflict of interest to declare.

Mitochondrial biogenesis and neural differentiation of human iPSC is modulated by idebenone in a developmental stage-dependent manner

Idebenone, the synthetic analog of coenzyme Q10 can improve electron transport in mitochondria. Therefore, it is used in the treatment of Alzheimer’s disease and other cognitive impairments. However, the mechanism of its action on neurodevelopment is still to be elucidated. Here we demonstrate that the cellular response of human induced pluripotent stem cells (hiPSC) to idebenone depends on the stage of neural differentiation. When: neural stem cells (NSC), early neural progenitors (eNP) and advanced neural progenitors (NP) have been studied a significant stimulation of mitochondrial biogenesis was observed only at the eNP stage of development. This coexists with the enhancement of cell viability and increase in total cell number. In addition, we report novel idebenone properties in a possible regulation of neural stem cells fate decision: only eNP stage responded with up-regulation of both neuronal (MAP2), astrocytic (GFAP) markers, while at NSC and NP stages significant down-regulation of MAP2 expression was observed, promoting astrocyte differentiation. Thus, idebenone targets specific stages of hiPSC differentiation and may influence the neural stem cell fate decision.

Hydroalcoholic Extract from Copaifera Langsdorffii Has Skin Wound Healing Activity in Rats

The loss of skin integrity causes wounds, and in response, the organism promotes the healing process, which is divided into three phases: inflammatory, proliferative and remodeling. Several plant species have been tested in healing experimental wounds due to popular application. Among the species used in skin wound healing related in popular knowledge is Copaifera langsdorffii. By this way, we aim to analyze wound healing changes in rats with skin lesions after treatment with hydroalcoholic extract from Copaifera langsdorffii leaves. Male Wistar rats were submitted to excisional injury with a 2 cm diameter in the dorsal region. They were divided into groups (n=6) with daily treatments of hydroalcoholic extract at 1; 5 and 10% concentrations or a negative control (saline) during 3, 7 and 21 days. The lesions were analyzed and measured daily. Blood was collected for toxicity analysis, and dorsal lesions were collected for histological analysis and measurements of cellularity in normal skin, border and center of the lesion. The toxicity data did not show any significant differences between the groups. Microscopic analysis showed an increase in total cell number at the edges of the extract treated groups after 3 days. Macroscopically, the groups treated with 5% and 10% extract demonstrated better wound retraction compared with saline treatment. In summary, the groups treated with hydroalcoholic extract of Copaifera langsdorffii showed the efficacy of the plant as treatment of skin wound healing.

Human Cord Blood and Bone Marrow CD34+ Cells Generate Macrophages That Support Erythroid Islands.

Recently, we developed a small molecule responsive hyperactive Mpl-based Cell Growth Switch (CGS) that drives erythropoiesis associated with macrophages in the absence of exogenous cytokines. Here, we compare the physical, cellular and molecular interaction between the macrophages and erythroid cells in CGS expanded CD34+ cells harvested from cord blood, marrow or G-CSF-mobilized peripheral blood. Results indicated that macrophage based erythroid islands could be generated from cord blood and marrow CD34+ cells but not from G-CSF-mobilized CD34+ cells. Additional studies suggest that the deficiency resides with the G-CSF-mobilized CD34+ derived monocytes. Gene expression and proteomics studies of the in vitro generated erythroid islands detected the expression of erythroblast macrophage protein (EMP), intercellular adhesion molecule 4 (ICAM-4), CD163 and DNASE2. 78% of the erythroblasts in contact with macrophages reached the pre reticulocyte orthochromatic stage of differentiation within 14 days of culture. The addition of conditioned medium from cultures of CD146+ marrow fibroblasts resulted in a 700-fold increase in total cell number and a 90-fold increase in erythroid cell number. This novel CD34+ cell derived erythroid island may serve as a platform to explore the molecular basis of red cell maturation and production under normal, stress and pathological conditions.

Targeting the transforming growth factor-β signaling during pre-implantation development in embryos of cattle, sheep and goats

Recently, application of chemical inhibitors against differentiation signaling pathways has improved establishment of mESCs. In this study, we applied inhibitors of TGF-β (SB431542) and BMP4 (Noggin) from cleavage to blastocyst stage in cattle, goat and sheep embryos. SB significantly decreases blastocyst rate and total cell number (TCN) in sheep blastocysts, whereas only TCN was significantly decreased in cattle blastocysts. In contrast to SB, Noggin significantly improved cattle blastocyst development but decreased TCN. However, Noggin treatment led to a significant increase in TCN in sheep blastocysts. Regarding pluripotency triad (OCT4, NANOG, SOX2) and cell lineage commitment (REX1, CDX2, GATA4), SB led to a significant reduction in SOX2 expression in goat and cattle, while Noggin increased at least one or two of pluripotent markers in these species. Taken together, this data suggests that inhibition of TGF-β by Noggin may be more favorable for derivation of stem cells in farm animals.

2.3 THz radiation: Absence of genotoxicity/mutagenicity in Escherichia coli and Salmonella typhimurium

The mutagenicity and genotoxicity in bacteria of 2.3THz radiation (THz) produced by a free-electron laser (NovoFEL) were evaluated; exposures were 5, 10, or 15min at average power 1.4W/cm2. Two Ames mutagenicity test strains of Salmonella typhimurium, TA98 and TA102, were used. For the genotoxicity test, we measured SOS induction in Escherichia coli PQ37. No significant differences were found between exposed and control cells, indicating that THz radiation is neither mutagenic nor genotoxic under these conditions. Nevertheless, a small increase in total cell number of S. typhimurium after 15min exposure, and an increase in β-galactosidase and alkaline phosphatase activities in E.coli PQ37, were observed, indicating some effect of THz radiation on cell metabolism. We also examined the combined effect of 4-NQO (8μM; positive control) and THz exposure (5min) on genotoxicity in E.coli PQ37. Unexpectedly, THz radiation decreased 4-NQO genotoxicity.

A New Approach to Expand Cord Blood Derived Hematopoietic Stem Cells, Using Bioengineered Human Fetal Liver Tissue 3D-Constructs

Despite advances in ex-vivo expansion of cord blood-derived hematopoietic stem/progenitor cells (CB-HSPC), challenges still remain regarding the ability to obtain, from a single unit, sufficient numbers of both long- and short-term repopulating cells, capable of functional engraftment, to enable the treatment of an adolescent or adult patient. We have previously shown that CB-HSPC can be effectively expanded in a 2D serum-free culture system, using a feeder layer of adult human bone marrow-derived stromal cells; still, the percentage of the most primitive stem cells decreased with time. Because, during development, the fetal liver is the main site of HSC expansion and differentiation, we hypothesized that efficient expansion of functional HSPC could be achieved in vitro under more physiologic conditions provided by surrogate fetal liver microenvironments. Therefore, we compared bioengineered liver constructs made from a natural 3D liver extracellular matrix (3DExM) seeded with hepatoblasts (HB), fetal liver-derived stromal cells (FLSC), or bone marrow-derived stromal cells (BMSC), with a 2D culture system using FLSC or BMSC. Overall, 2D culture systems generated a higher yield of mature blood cells by day 14, mostly within the myelomonocytic lineages, with fold increases in total cell number in 2D-HB, 2D-FLSC, and 2D-BMSC cultures of 1145, 16151, and 229, respectively, while 3D cultures generated fold increases of 94.3, 492, and 110, respectively. Nevertheless, the output and expansion of more primitive HSPC was significantly higher in 3D cultures, as determined by flow cytometry and colony-forming assays. Specifically, in 3D-HB cultures, the percentage of CD34+CD38- cells increased by 100% at day 2, while 3D-FLSC and 3D-BMSC cultures each supported a 90% increase in the percentage of CD34+CD38- cells during this period. By contrast, all of the 2D cultures experienced a 30% decrease in the percentage of CD34+CD38- cells during this same time period. In addition, only 3D conditions maintained CD34+CD38- cells until day 12 of culture. Colony-forming assays demonstrated that the CFU-GEMM output was higher in 3D cultures when compared with their 2D counterpart. Among the 3D cultures, the 3D-HB cultures had the highest number of CFU-GEMM at day 2 and day 4. In conclusion, we demonstrate that by integrating biological components in vitro to obtain structures that contain all the necessary elements to mimic the fetal liver microenvironmental niches, which are known to promote rapid expansion of HSC during development, we were able to achieve significant expansion and maintenance of CD34+CD38- cells. In addition, since little is known about fetal liver niches that support HSPC expansion, the 3D constructs will provide, for the first time, a model system in which to dissect the role of the individual cellular and matrix niche components in supporting CB HSPC maintenance, expansion, and differentiation. DisclosuresNo relevant conflicts of interest to declare.

Caspase-9 inhibitor Z-LEHD-FMK enhances the yield of in vitro produced buffalo (Bubalus bubalis) pre-implantation embryos and alters cellular stress response

The present investigation was done to study the effect of caspase-9 inhibitor Z-LEHD-FMK, on in vitro produced buffalo embryos. Z-LEHD-FMK is a cell-permeable, competitive and irreversible inhibitor of enzyme caspase-9, which helps in cell survival. Buffalo ovaries were collected from slaughterhouse and the oocytes were subjected to in vitro maturation (IVM), in vitro fertilization (IVF) and in vitro culture (IVC). The culture medium was supplemented with Z-LEHD-FMK at different concentrations i.e. 0μM (control), 10μM, 20μM, 30μM and 50μM during IVM and IVC respectively. After day-2 post-insemination, the cleavage rate was significantly higher (74.20±5.87% at P<0.05) in the group treated with 20μM of Z-LEHD-FMK than at any other concentration. Same trend was observed in the blastocyst production rate which was higher at 20μM (27.42±2.94% at P<0.05). The blastocysts obtained at day-8 of the culture at different concentrations were subjected to TUNEL assay, to determine the level of apoptosis during the culture medium supplied with 20μM Z-LEHD-FMK which showed apoptotic index significantly lower (1.88±0.87 at P<0.05). There was a non-significant increase in total cell number in all Z-LEHD-FMK treated blastocysts. The quantitative gene expression of CHOP and HSP10 genes showed significant increase (P<0.05) in the group treated with 50μM Z-LEHD-FMK, while, HSP40 showed significant increase (P<0.05) at 30μM and 50μM Z-LEHD-FMK concentrations. From the afore mentioned results we conclude that, Z-LEHD-FMK at 20μM increased the cleavage and blastocyst rate of buffalo pre-implantation embryos also affecting the rate of apoptosis and cellular stress at various concentrations.

Characterization of the draining lymph node response in the mouse drug allergy model: A model for drug hypersensitivity reactions

The mouse drug allergy model (MDAM) was developed as a tool to predict the potential of systemically administered drugs to produce hypersensitivity reactions (HR). Drugs associated with HR in the clinic produce a marked increase in the cellularity of the draining lymph nodes (DLN) in the MDAM. The objective of this study was to characterize the changes in the DLN following exposure to drugs associated with HR and to investigate whether lymphocyte migration and/or proliferation play a role in the response. These endpoints were also investigated in the local lymph node assay (LLNA) to determine whether responses between the two assays occur via similar mechanisms. Results demonstrated that total numbers of T- and B-cells were proportionally increased in the DLN of mice treated with positive control drugs (i.e. abacavir, amoxicillin, ofloxacin, and sulfamethoxazole) compared to animals administered the vehicle or negative control drugs (metformin and cimetidine). In contrast, a significant increase in the B-cell population of the DLN was observed for 2,4-dinitrofluorobenzene (DNFB) following the LLNA protocol. Down-regulation of CD62L and up-regulation of CCR7 were observed for T-cells from the DLN of the positive control treated mice in the MDAM, but not with DNFB in the LLNA. A mild increase in T-cell proliferation was observed in the MDAM with positive control drugs, while DNFB in the LLNA induced proliferation within the B-cell population only. Anti-CD40L antibody administration inhibited MDAM responses to positive control drugs, but did not affect DNFB-induced increases in total cell number in the LLNA. These results suggest that the increased cellularity of the DLN in the MDAM may be the result of drug-induced alterations in lymphocyte migration and/or effects on lymphocyte proliferation. Moreover, it appears that different mechanisms may be involved in driving the MDAM and LLNA responses.

Abstract 4225: Estrogen regulated oncogene SHON mediates the efficacy of anti-estrogen treatment in breast cancer

Abstract Background: Estrogen receptors (ER) play a pivotal role in the etiology and progression of breast cancer. Hormone receptor positive breast cancers usually respond to endocrine therapy at the beginning, but a substantial proportion will eventually develop anti-estrogen resistance. However, the mechanism remains largely unclear. Cross-talk between ER and growth factor signaling pathways is known to contribute to the development of anti-estrogen resistance. In addition, activation of JNK signaling pathway has been implicated in tamoxifen resistance of mammary carcinoma MCF-7 cells. We have demonstrated that SHON is a novel human oncogene and its expression is inducible by estradiol (E2). Moreover, SHON expression is highly positively correlated with ER, progesterone receptor (PR) and androgen receptor (AR) expression in breast cancer, and may also serve as a prognostic biomarker for predicting patient response to anti-estrogen therapy in ER+ breast cancer. Purpose: This study aimed to investigate how SHON modulated estrogen signaling and its role in anti-estrogen treatment and resistance in ER+ breast cancer. Methods: Estrogen responsive luciferase reporter assays were used to measure the activation of ER signaling by SHON. Total cell number and soft agar colony formation assays were used to determine the effects of SHON expression on the efficacy of anti-estrogens. Protein expression was measured by Western blotting. Results: Forced expression of SHON in MCF-7 cells increased ER transcriptional activity while depletion of endogenous SHON decreased this activity. Upon tamoxifen or fulvestrant treatment, MCF-7 cells stably transfected with SHON expression plasmid displayed an increase in total cell number and cell migration compared with the control vector transfected MCF-7 cells. Anti-estrogen treatment inhibited the colony formation of MCF-7 cells in soft agar, and this inhibitory effect was significantly abrogated by forced expression of SHON. SHON also increased the protein level of both ERα and JNK in MCF-7 cells under estrogen deprivation or stimulation, or under estrogen stimulation in combination with tamoxifen or fulvestrant treatment. Conclusion: There was a positive feedback of SHON and ERα expression. SHON may mediate the efficacy of anti-estrogen drugs via modulation of ERα and JNK expression. These results improve our understanding of the role of SHON in anti-estrogen treatment and provide evidence for the use of SHON antagonism in enhancing the efficacy of anti-estrogen treatment. Citation Format: Bing Xu, Yewon Jung, Jo Perry, Tao Zhu, Peter E. Lobie, Baiqu Huang, Jun Lu, Dong-Xu Liu. Estrogen regulated oncogene SHON mediates the efficacy of anti-estrogen treatment in breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4225. doi:10.1158/1538-7445.AM2014-4225

Differential effects of lipopolysaccharide and cigarette smoke exposure on genes associated with contractile agonist responsiveness in mouse lungs (660.2)

Introduction: Inflammation is a key driver of many lung diseases, including asthma and chronic obstructive pulmonary disease. Exposure to lipopolysaccharide (LPS) and cigarette smoke (CS) are two mouse models that induce inflammation, however studies assessing changes in expression of contractile receptors in these models are limited.Methods: Female Balb/C mice were exposed to inhaled LPS once a day for 4 days or PBS. Male Balb/C mice were exposed to 9 cigarettes 3 times a day for 4 days and sham mice were exposed to air. On day 5, mice were euthanized, bronchoalveolar lavage fluid (BALF) was taken and lungs were harvested for PCR.Results: Inflammation was confirmed in both LPS and CS models by an increase in total cell number in the BALF. In both groups, there were no changes in gene expression levels of serotonin 2A, bradykinin, or muscarinic M3 receptors. There were no changes in G‐protein expression or downstream inositol trisphosphate receptor expression. In the LPS‐treated mice, there was an upregulation of muscarinic M2 receptors and ryanodine isoform 2 receptors (RyR2). In contrast, these receptors were down regulated in CS mice.Discussion: Despite both LPS and CS inducing inflammation, these stimuli have opposite effects on expression of M2 and RyR2 receptors. The influence of these changes on responsiveness to muscarinic and other contractile agonists remain to be determined.Grant Funding Source: Supported by NHMRC