Cultures Of Bovine Cells Research Articles

Grifola frondosa extract as a fetal bovine serum supplement for the culture of bovine muscle satellite cells under low serum conditions

Expensive fetal bovine serum (FBS) is a major obstacle to the production of cultivated meat. However, because FBS substitutes do not sufficiently induce cell proliferation, a good alternative is to reduce the amount of FBS and use ingestible additives to promote cell proliferation. In this study, Grifola frondosa extract (GFE) was used to investigate its potential as an additive to promote myogenesis of bovine muscle satellite cells from Hanwoo cattle under low serum conditions (10 % FBS). GFE treated with 10 % FBS only during the proliferation period not only increased cell proliferation and related biomarkers in a concentration-dependent manner (0.78–12.5 μg/mL), but also increased cell differentiation. Additionally, differentiation was promoted when cells were with GFE treated only during the differentiation period. Especially GFE at 12.5 µg/mL induced significantly higher proliferation and differentiation rates than 20 % FBS medium. In particular, compared to treatment alone in the proliferation or differentiation periods, GFE treatment in both periods contributed to an increase in the differentiation rate and significantly enhanced total protein production. The integration of GFE into cultivated meat production presents a promising approach to reducing FBS dependence, lowering costs, and enhancing scalability, aligning with sustainability and consumer acceptance goals.

352 Supplementing bovine satellite cells with anabolic hormones and/or zinc at differentiation impacts abundance of mRNA involved in growth, mineral transport, and oxidative stress

Abstract Zinc (Zn) is the most utilized metal in biological processes and supports growth through critical roles in protein turnover. The Zn requirements of skeletal muscle may be augmented when growth promoting technologies, such as anabolic hormones are being utilized. Both anabolic hormones and Zn affect protein synthesis and degradation in skeletal muscle. However, the molecular mechanisms through which Zn may interact with anabolic hormones to support skeletal muscle growth remains unknown. The objective of this study was to determine whether trenbolone acetate (TBA), estradiol (E2) and Zn interact to promote differentiation of bovine satellite cell (BSC) cultures by assessing abundance of mRNA involved in growth, mineral transport, differentiation, protein turnover and oxidative stress. To do this, primary BSC cultures were isolated from three different steers and grown to 80% confluency and induced to differentiate in 3% horse serum, at which time a factorial design was employed to assess the impact of Zn (0, 10, 20 or 40 μM) as ZnCl2 and/or the impact of hormone (10 nM TBA,10 nM E2, 10 nM TBA and 10 nM E2). Zinc concentrations represent physiologically relevant plasma Zn concentrations. Total mRNA was isolated from the treated cultures at 4 h, 12 h and 24 h post-treatment. Assays utilizing cultures from each steer were run in duplicate (n = 6). The mRNA abundance of 45 genes were examined and analyzed using the PROC MIXED procedure of SAS with time as a repeated measure to evaluate the fixed effects of hormone, mineral, time, and all interactions. No effects (P ≥ 0.19) of hormone x mineral x time, hormone x time, hormone x mineral or hormone were noted. A mineral x time effect (P ≤ 0.04) was noted for the igf1r, slc39a7, mapk1, mmp2, and G6pd. A mineral effect was found for hormone receptors (ir, ar, esr1, esr2, and gper; P ≤ 0.03), mRNA involved in mineral transport (slc30a10, slc30a7, sod1, sod2, slc2a4, and slc39a8; P ≤ 0.009), and genes related to skeletal muscle differentiation (pax7, myf5, myf6, spry1, myog, mef2a, and myod; P ≤ 0.02) and protein turnover (akt1, fox01, fox04, mstn,fbox32, fox03, mtor, trim63, and CKM; P ≤ 0.03). These differences are generally driven by abundance being increased (P ≤ 0.05) in cultures treated with 40 or 20 μM Zn compared with cultures receiving 0 or 10 μM Zn. These results indicate that while Zn and hormone do not appear to interact to influence gene expression of differentiating primary BSC cultures at the time points measured, increased concentrations of Zn appear to increase mRNA abundance of hormone receptors, mineral transporters, and genes involved in skeletal muscle differentiation and protein turnover.

Identification of infectious viruses for risk-based virus testing of CHO unprocessed bulk using next-generation sequencing.

It is important to increase manufacturing speed to make medicines more widely available. One bottleneck for CHO-based drug substance release is the in vitro viral (IVV) cell-based assay on unprocessed bulk. To increase process speed, we evaluate the suitability of replacing the IVV cell-based assay with next-generation sequencing (NGS). First, we outline how NGS is currently used in the pharmaceutical industry, and how it may apply to CHO virus testing. Second, we examine CHO virus contamination history. Since prior virus contaminants can replicate in the production bioreactor, we perform a literature search and classify 159 viruses as high, medium, low, or unknown risk based on their ability to infect CHO cells. Overall, the risk of virus contamination during the CHO manufacturing process is low. Only six viruses were reported to have contaminated CHO bioprocesses over the past several decades, and were primarily caused by fetal bovine serum or cell culture components. These virus contamination events can be mitigated through limitation and control of raw materials, combined with virus testing and virus clearance technologies. The list of CHO infectious viruses provides a starting framework for virus safety risk assessment and NGS development. Furthermore, ICH Q5A (R2) includes NGS as a molecular method for adventitious agent testing, paving a path forward for modernizing CHO virus testing.

Efficient expansion culture of bovine myogenic cells with differentiation capacity using muscle extract-supplemented medium

The production of tissue-engineered skeletal muscles for cell-based therapy and cultivated meat production requires a large number of myogenic cells capable of differentiating into myotubes. A suspension culture with a stirred bioreactor is a system that is feasibly scalable, but scaling up the production of myogenic cells while maintaining differentiation capacity requires an efficient culture technique. This remains challenging in suspension culture. On the other hand, conventional meat processing generates substantial by-products, including meat trimmings containing effective substances for myogenic cell cultures. Efficiently utilizing these by-products can reduce environmental impact and disposal costs. Here, we describe an expansion culture method for bovine myogenic cells (BMCs) using Dulbecco's Modified Eagle's medium (DMEM) supplemented with muscle extracts. During the 6-day planar culture, the proliferation rate using the culture medium supplemented with muscle extracts (CMME) and 10% fetal bovine serum (FBS) was 18 ± 3.3, three times higher than that achieved using DMEM with 10% FBS. Furthermore, a combination of CMME supplemented with 10% FBS and an iMatrix-511-coated dish could maintain differentiation capacity through passaging culture. Finally, we demonstrated a suspension culture with microcarriers using CMME with 10% FBS. Through 3 passages, the cumulative proliferation rate was 191.7 ± 35.1, four times higher than that of produced DMEM with 10% FBS. Using CMME, the cells maintained differentiation capacity in comparison to that using DMEM. This approach will contribute to both the reduction of wasted by-products from conventional meat production and the development of a suspension culture system for maintaining high-quality myogenic cells.

Extracellular vesicles in cattle infected with bovine leukaemia virus: isolation and molecular analysis.

Exosomes are nanosized lipid bilayer membranous microvesicles, extracellularly released from a variety of mammalian cells. They mediate intercellular signalling by transporting several types of RNA, lipids and proteins and participate in the intercellular exchange of DNA, RNA, micro RNA, proteins and other components. These microvesicles are present in all body fluids in physiological and pathological conditions and reflect the state of the host organism. The aim of the study was the isolation and molecular determination of exosomes in blood and supernatant fluids of bovine dendritic cell cultures infected with bovine leukaemia virus (BLV). Exosomes were isolated by ultracentrifugation from the blood sera, plasma and supernatant of bovine BLV-infected and uninfected control dendritic cell cultures and their presence was confirmed with scanning electron and transmission electron microscopy. Western blot analysis of the structural BLV glycoprotein 51 (Env) and protein 24 (Gag) and of the tetraspanin exosomal markers CD9, CD63 and flotillin-1 was undertaken in BLV+ and control BLV- cattle. In exosomes of leukaemic cattle both BLV proteins and exosomal markers were detected. In healthy control animals only exosomal markers were determined. Proteins of BLV were released with exosomes and could be transferred into recipient cells as an alternative propagation route not requiring virus infection.

Role of steroid hormones in the maintenance of focal adhesions in bovine oviductal epithelial cells

The oviduct, the organ of the female reproductive system where fertilization and early embryonic development occur, provides an optimal environment for the final maturation of oocytes, storage, and sperm capacitation and transport of gametes and embryos. During the estrous cycle, the oviduct is affected by ovarian sex hormones, resulting in changes aimed at maintaining an appropriate microenvironment. Normal cell migration is tightly regulated, its role being essential for the development and maintenance of organ and tissue functions as well as for regeneration following injury. Due to their involvement in focal contact formations, focal adhesion kinase (PTK2) and paxillin (PXN) are key proteins in the study of cell migration and adhesion. The objective of this work was to compare the expression of PTK2 and PXN in oviductal cells along the estrous cycle and to determine if their expression is regulated by the presence of 17-β estradiol (E2) and/or progesterone (P4). No transcripts of PTK2 or of PXN were detected in cells corresponding to the luteal phase. Additionally, hormonal stimulation experiments on bovine oviductal cell cultures (BOECs) were carried out, where P4 inhibited the expression of both genes. Migration assays demonstrated that P4 reduced BOECs migration capacity. P4 treatment also reduced cell adhesion, while E2 increased the number of adhered cells. In conclusion, the presence of E2 and P4 regulates the expression of genes involved in the formation of focal contacts and modifies the migration and adhesion of BOECs. Understanding the effect of steroid hormones on BOECs is critical to grasp the impact of steroid control on oviductal function and its contribution to establishing successful pregnancies.

Animal-free scaffold from brown algae provides a three-dimensional cell growth and differentiation environment for steak-like cultivated meat

Scaffolds for the production of cultivated meat, a promising sustainable meat alternative, should exhibit physical and chemical properties that enable three-dimensional animal cell culture, along with biological characteristics that support cell attachment, proliferation, and differentiation. Additionally, the scaffold should be crafted from edible materials and offer textural similarities to meat and have minimal influence on flavor and taste. Herein, an edible alginate-based alginate-cellulose hydrogel (ACe-gel) scaffold derived from the brown alga Undaria pinnatifida is developed. In terms of physical characteristics, the scaffold had porosity (119.5 ± 37.2 μm) and moisture-holding capacity (73.03 ± 3.82, 68.66 ± 9.54, and 84.17 ± 9.94 at 25 °C, 37 °C, and 60 °C, respectively) suitable for three-dimensional culture and differentiation of bovine muscle stem cells (bMuSCs). Accordingly, the scaffold was superior to a commercial alginate scaffold in terms of the attachment and proliferation of bMuSCs (5.5-fold over 72 h), and its performance was comparable with that of a lyophilized collagen scaffold (7.8-fold over 72 h, compared with the pure alginate). The bMuSCs cultured on the ACe-gel scaffold were capable of differentiating into muscle fibers, as verified by gene expression profile analysis. Furthermore, the scaffold exhibited minimal heavy metal contents and distinct seaweed odorants, while the stress-strain characteristics of the scaffold cultured with bMuSC (Young's modulus of raw ACe-gel: 285.19 ± 83.37 kPa, cooked ACe-gel meat: 880.60 ± 485.60 kPa) closely resembled that of meat (raw beef: 267.76 ± 156.42 kPa, cooked beef: 1331.94 ± 762.43 kPa). These findings highlight that the seaweed-derived and animal-free ACe-gel scaffold has strong potential for utilization as a food technology for cultured meat production in the future.

Genes and pathways revealed by whole transcriptome analysis of milk derived bovine mammary epithelial cells after Escherichia coli challenge

Mastitis, inflammation of the mammary gland, is the costliest disease in dairy cattle and a major animal welfare concern. Mastitis is usually caused by bacteria, of which staphylococci, streptococci and Escherichia coli are most frequently isolated from bovine mastitis. Bacteria activate the mammary immune system in variable ways, thereby influencing the severity of the disease. Escherichia coli is a common cause of mastitis in cattle causing both subclinical and clinical mastitis. Understanding of the molecular mechanisms that activate and regulate the host response would be central to effective prevention of mastitis and breeding of cows more resistant to mastitis. We used primary bovine mammary epithelial cell cultures extracted noninvasively from bovine milk samples to monitor the cellular responses to Escherichia coli challenge. Differences in gene expression between control and challenged cells were studied by total RNA-sequencing at two time points post-challenge. In total, 150 and 440 (Padj < 0.05) differentially expressed genes were identified at 3 h and 24 h post-challenge, respectively. The differentially expressed genes were mostly upregulated at 3 h (141/150) and 24 h (424/440) post-challenge. Our results are in line with known effects of E. coli infection, with a strong early inflammatory response mediated by pathogen receptor families. Among the most significantly enriched early KEGG pathways were the TNF signalling pathway, the cytokine-cytokine receptor interaction, and the NF-kappa B signalling pathway. At 24 h post-challenge, most significantly enriched were the Influenza A, the NOD-like receptor signalling, and the IL-17 signaling pathway.

Bovine umbilical vein endothelial cells conditioned medium prevents TMT-induced neurotoxicity mediated by the upregulation of brain-derived neurotropic factors on the human neuroblastoma SH-SY5Y cells

Background: Neurodegenerative diseases are characterized by damage to the central nervous system through the progressive decrease in the structure and function of neurons, followed by neuronal death. Current therapy for these diseases is still limited. Bovine umbilical vein endothelial cells conditioned medium (BUVEC-CM) is a medium harvested from culture of bovine umbilical vein endothelial cells (BUVEC). This study aimed to evaluate the capability of BUVEC-CM to prevent neurotoxicity induced by trimethyltin (TMT) on human neuroblastoma SH-SY5Y cells. Methods: The tests carried out were a viability test with MTT and CCK-8 assay, scratch wound assay to determine cell migration, Hoechst 33342 and AO/PI staining to detect apoptosis, ELISA test to measure the concentration of BDNF, and RT-qPCR to determine the expression of CD68, caspase-7 and caspase-9, and DCFH-DA staining to detect reactive oxygen species (ROS). Results: BUVEC-CM maintained the viability of cells, increase cell migration, prevent cell apoptosis, increase BDNF levels, decrease the expression of CD68, as well as the caspase-7 and -9 genes, and suppress ROS generation. Conclusions: BUVEC-CM can protect from neurodegeneration in SH-SY5Y cells through upregulation of BDNF and downregulation of CD68, caspase-7 and caspase-9.

Função do fator de crescimento fibroblástico 18 na regulação da cascata de eventos pré-ovulatórios em bovinos

ABSTRACT: Suboptimal LH surges can lead to delayed or absent ovulation during reproductive management programs in cattle. Several signaling pathways may enhance the LH signal, potentially including growth factors. In the present study, we determined whether Fibroblast growth factor 18 (FGF18), which mimics some actions of the LH surge on granulosa cells, may potentiate the action of a suboptimal dose of LH using an established bovine granulosa cell (GC) culture system. Addition of recombinant FGF18 increased the concentration of progesterone (P4) in the culture medium. A combination of LH and FGF18 in the culture medium increased the abundance of mRNA encoding EREG, EGR1 and EGR3, proteins that are early modulators of the LH-induced pre-ovulatory cascade. This study demonstrated that FGF18 may be a co-factor in the regulation of the pre-ovulatory cascade in bovine ovarian granulosa cells.

Bioactivity of cerium dioxide nanoparticles as a function of size and surface features.

Nano-dispersed cerium dioxide is promising for use in medicine due to its unique physicochemical properties, including low toxicity, the safety of in vivo usage, active participation in different redox processes occurring in living cells, and its regenerative potential, manifested in the ability of CeO2 to participate repeatedly in redox reactions. In this work, we examined the biological activity of cerium dioxide nanoparticles (CeO2 NPs) synthesized by precipitation in mixed water/alcohol solutions at a constant pH of 9. This synthesis method allowed controlling the size and Ce3+/Ce4+ proportion on the surface of NPs, changing the synthesis conditions and obtaining highly stable suspensions of "naked" CeO2 NPs. Changes in the surface properties upon contact of CeO2 NPs with protein-rich media, e.g., bovine serum albumin and DMEM cell culture medium supplemented with 10% fetal bovine serum, the characteristics of nanoparticle uptake by mouse aortic endothelial cells and the antioxidant activity of the nanoparticles of different sizes were investigated by various state-of-the-art analytical methods.

Characterization of an iPSC-based barrier model for blood-brain barrier investigations using the SBAD0201 stem cell line

BackgroundBlood-brain barrier (BBB) models based on primary murine, bovine, and porcine brain capillary endothelial cell cultures have long been regarded as robust models with appropriate properties to examine the functional transport of small molecules. However, species differences sometimes complicate translating results from these models to human settings. During the last decade, brain capillary endothelial-like cells (BCECs) have been generated from stem cell sources to model the human BBB in vitro. The aim of the present study was to establish and characterize a human BBB model using human induced pluripotent stem cell (hiPSC)-derived BCECs from the hIPSC line SBAD0201.MethodsThe model was evaluated using transcriptomics, proteomics, immunocytochemistry, transendothelial electrical resistance (TEER) measurements, and, finally, transport assays to assess the functionality of selected transporters and receptor (GLUT-1, LAT-1, P-gp and LRP-1).ResultsThe resulting BBB model displayed an average TEER of 5474 ± 167 Ω·cm2 and cell monolayer formation with claudin-5, ZO-1, and occludin expression in the tight junction zones. The cell monolayers expressed the typical BBB markers VE-cadherin, VWF, and PECAM-1. Transcriptomics and quantitative targeted absolute proteomics analyses revealed that solute carrier (SLC) transporters were found in high abundance, while the expression of efflux transporters was relatively low. Transport assays using GLUT-1, LAT-1, and LRP-1 substrates and inhibitors confirmed the functional activities of these transporters and receptors in the model. A transport assay suggested that P-gp was not functionally expressed in the model, albeit antibody staining revealed that P-gp was localized at the luminal membrane.ConclusionsIn conclusion, the novel SBAD0201-derived BBB model formed tight monolayers and was proven useful for studies investigating GLUT-1, LAT-1, and LRP-1 mediated transport across the BBB. However, the model did not express functional P-gp and thus is not suitable for the performance of drug efflux P-gp reletated studies.

Uncovering the Role of CD59 As a Novel Therapeutic Target Against High-Risk Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a hematologic malignancy driven by acquired genetic mutations in hematopoietic stem and progenitor cells. Continued disease progression leads to the unregulated growth of immature cells of the myeloid lineage, resulting in impaired hematopoiesis, organ failure, and death. High-risk AML is a disease subtype, classified by cytogenetic abnormalities, TP53 mutations, chemotherapy resistance, and dismal overall survival. To uncover the driving factors of poor outcomes in high-risk AML, we performed a multivariable analysis of the BEAT-AML-2.0 patient cohort. We identified that overexpression of the membrane regulatory protein, CD59, serves as a prognostic marker for an increased risk of death. Furthermore, we uncovered that CD59 is overexpressed in high-risk AML patients, including samples with TP53 mutations and refractory disease. CD59 prevents the formation of the membrane attack complex of the complement system and its overexpression is linked with resistance to cancer therapies that activate the complement cascade. However, the intracellular role of CD59 is poorly understood and it is unexplored in AML. Given that the standard of care for AML treatment, which consists of a combination therapy of cytarabine and an anthracycline, does not activate the complement system, we hypothesize that CD59 confers a survival advantage to AML cells in a complement-independent manner. To study the role of CD59 in AML, we constructed doxycycline-inducible short-hairpin RNA (shRNA) expression vectors against CD59 (shCD59) and a non-targeting control (shNT) and transduced them into SKM-1, HL-60, EOL-1, and OCI-M1 AML cell lines. First, we examined the impact of CD59 knockdown on cell growth through a cell competition assay between shCD59 or shNT cells and untransduced cells by culturing them at a 1:1 ratio. We observed that upon the addition of doxycycline, shCD59 cells are outcompeted by untransduced cells whereas the percentage of shNT cells remains unchanged. Cell viability analysis revealed an increased level of cell death in shCD59 cells in comparison to shNT cells. Moreover, we quantified the level of cellular proliferation in shCD59 and shNT cells through 5-ethyl-2'-deoxyuridine (EdU) staining. We observed that shCD59 cells have a signification reduction in cellular proliferation in comparison to shNT cells, highlighting changes in both cellular division and survival pathways upon CD59 knockdown. Importantly, to rule out the impact of alternative complement activation on the observed phenotype, we repeated the previously described experiments in heat-inactivated fetal bovine serum supplement cell culture medium and observed an identical response, confirming a complement-independent role of CD59. Based on our findings, we investigated the mechanism through which CD59 exerts pro-survival signals by performing RNA-sequencing on shCD59 and shNT cells. Through differential expression and pathway analyses, we discovered shCD59 cells exhibit increased inflammatory signalling and apoptotic pathway activation. Conversely, mTOR and MYC signaling pathways are significantly downregulated upon CD59 knockdown, including a reduction in mitotic spindle activity, DNA repair machinery, and DNA replication checkpoints. These results indicate that upon CD59 knockdown, AML cells undergo mitotic catastrophe, driven by deficient cell-cycle checkpoints, DNA damage, and ultimately, cell death. We further examined changes in the signaling landscape of shCD59 and shNT cells by analyzing the cellular phospho-proteome through mass spectrometry. In agreement with our RNA-sequencing analysis, shCD59 cells have increased activation of DNA damage and inflammation markers, such as BCLAF1 and IKBKG, respectively. In contrast, shNT cells display elevated activation of mTOR pathway markers, including EIF4B, and cell cycle drivers, such as CDC20. In summary, we present CD59 overexpression as a driver of AML progression and poor clinical outcomes. Through functional and computational studies, we have shown CD59 expression is critical for cell growth and cell survival, and as such, identifying it as a novel therapeutic target. The survival outlook for high-risk AML patients has remained unchanged for the past 25 years. Our findings offer valuable insights that can significantly alter the current treatment landscape of AML.

Valine and nonessential amino acids affect bidirectional transport rates of leucine and isoleucine in bovine mammary epithelial cells

A more complete understanding of the mechanisms controlling AA transport in mammary glands of dairy cattle will help identify solutions to increase nitrogen feeding efficiency on farms. It was hypothesized that Ala, Gln, and Gly (NEAAG), which are actively transported into cells and exchanged for all branched-chain AA (BCAA), may stimulate transport of BCAA, and that Val may antagonize transport of the other BCAA due to transporter competition. Thus, we evaluated the effects of varying concentrations of NEAAG and Val on transport and metabolism of the BCAA Ala, Met, Phe, and Thr by bovine mammary epithelial cells. Primary cultures of bovine mammary epithelial cells were assigned to treatments of low (70% of mean in vivo plasma concentrations of lactating dairy cows) and high (200%) concentrations of Val and NEAAG (LVal and LNEAAG, HVal and HNEAAG, respectively) in a 2 × 2 factorial design. Cells were preloaded with treatment media containing [15N]-labeled AA for 24 h. The [15N]-labeled media were replaced with treatment media containing [13C]-labeled AA. Media and cells were harvested from plates at 0, 0.5, 1, 5, 15, 30, 60, and 240 min after application of the [13C]-labeled AA and assessed for [15N]- and [13C]-AA label concentrations. The data were used to derive transport, transamination, irreversible loss, and protein-synthesis fluxes. All Val fluxes, except synthesis of rapidly exchanging tissue protein, increased with the HVal treatment. Interestingly, the rapidly exchanging tissue protein, transamination, and irreversible-loss rate constants decreased with HVal, indicating that the significant flux increases were primarily driven by mass action with the cells resisting the flux increases by downregulating activity. However, the decreases could also reflect saturation of processes that would drive down the mass-action rate constants. This is supported by decreases in the same rate constants for Ile and Leu with HVal. This could be due to either competition for shared transamination and oxidation reactions or a reduction in enzymatic activity. Also, NEAAG did not affect Val fluxes, but influx and efflux rate constants increased for both Val and Leu with HNEAAG, indicating an activating substrate effect. Overall, AA transport rates generally responded concordantly with extracellular concentrations, indicating the transporters are not substrate-saturated within the in vivo range. However, BCAA transamination and oxidation enzymes may be approaching saturation within in vivo ranges. In addition, System L transport activity appeared to be stimulated by as much as 75% with high intracellular concentrations of Ala, Gln, and Gly. High concentrations of Val antagonized transport activity of Ile and Leu by 68% and 15%, respectively, indicating competitive inhibition, but this was only observable at HNEAAG concentrations. The exchange transporters of System L transport 8 of the essential AA that make up approximately 40% of milk protein, so better understanding this transporter is an important step for increased efficiency.

Divergent proinflammatory immune responses associated with the differential susceptibility of cattle breeds to tuberculosis.

Tuberculosis (TB) in the bovine is one of the most predominant chronic debilitating infectious diseases primarily caused by Mycobacterium bovis. Besides, the incidence of TB in humans due to M. bovis, and that in bovines (bovine TB, bTB) due to M. tuberculosis- indicates cattle as a major reservoir of zoonotic TB. While India accounts for the highest global burden of both TB and multidrug-resistant TB in humans, systematic evaluation of bTB prevalence in India is largely lacking. Recent reports emphasized markedly greater bTB prevalence in exotic and crossbred cattle compared to indigenous cattle breeds that represent more than one-third of the total cattle population in India, which is the largest globally. This study aimed at elucidating the immune responses underlying the differential bTB incidence in prominent indigenous (Sahiwal), and crossbred (Sahiwal x Holstein Friesian) cattle reared in India. Employing the standard Single Intradermal Tuberculin Test (SITT), and mycobacterial gene-targeting single as well as multiplex-PCR-based screening revealed higher incidences of bovine tuberculin reactors as well as Mycobacterium tuberculosis Complex specific PCR positivity amongst the crossbred cattle. Further, ex vivo mycobacterial infection in cultures of bovine peripheral blood mononuclear cells (PBMC) from SITT, and myco-PCR negative healthy cattle exhibited significantly higher intracellular growth of M. bovis BCG, and M. tuberculosis H37Ra in the crossbred cattle PBMCs compared to native cattle. In addition, native cattle PBMCs induced higher pro-inflammatory cytokines and signaling pathways, such as interferon-gamma (IFN-γ), interleukin-17 (IL-17), tank binding kinase-1 (TBK-1), and nitric oxide (NO) upon exposure to live mycobacterial infection in comparison to PBMCs from crossbred cattle that exhibited higher expression of IL-1β transcripts. Together, these findings highlight that differences in the innate immune responses of these cattle breeds might be contributing to the differential susceptibility to bTB infection, and the resultant disparity in bTB incidence amongst indigenous, and crossbred cattle.

Adipocyte-Derived Paracrine Factors Regulate the In Vitro Development of Bovine Mammary Epithelial Cells.

The mammary gland is composed of epithelial tissue forming ducts and lobules, and the stroma, composed of adipocytes, connective tissue, and other cell types. The stromal microenvironment regulates mammary gland development by paracrine and cell-cell interactions. In the present study, primary cultures of bovine mammary epithelial cells (bMEC) and bovine adipose-derived stem cells (bASC) subjected to adipogenic differentiation were used to investigate the influence of paracrine factors secreted by preadipocytes and adipocytes on bMEC development. Four types of conditioned media (CM) were collected from undifferentiated preadipocytes (preA) and adipocytes on days: 8, 12, 14 of differentiation. Next, bMEC were cultured for 24 h in CM and cell viability, apoptosis, migratory activity, ability to form spheroids on Matrigel, and secretory activity (alpha S1-casein concentration) were evaluated. CM derived from fully differentiated adipocytes (12 d and 14 d) significantly decreased the number of apoptotic cells in bMEC population and increased the size of spheroids formed by bMEC on Matrigel. CM collected from preadipocytes significantly enhanced bMEC's migration, and stimulated bMEC to produce alpha S1-casein, but only in the presence of prolactin. These results confirm that preadipocytes and adipocytes are important components of the stroma, providing paracrine factors that actively regulate the development of bovine mammary epithelium.

POLYCAPROLACTONE FIBER-BASED BOVINE DENDRITIC CELL DEVELOPMENT AND THREE- DIMENSIONAL CULTURE IN VITRO

Two-dimensional (2D) cell culture systems are normally used for in vitro experiments. These systems have some drawbacks which affect results of in vitro experiments because cultured cells have different morphology and physiological properties comparing to in vivo models. Therefore, there are current technique to develop three-dimensional (3D) cell culture systems which are closer to the environment in real tissues. In our study, we used polycaprolactone fiber scaffold for culture of bovine monocytes to develop dendritic cells (DCs) from those cells. Cultured monocytes in the polycaprolactone nanofiber scaffold differentiated to DCs. The differentiated DCs showed a 3D structure in the scaffold. These results suggest that this method is suitable for the development of DCs in a manner of three-dimension. These method is perspective for bovine cell culture and has advantages compare to conventional 2D culture systems.

The effect of 1,25-dihydroxyvitamin D3 on the Wnt signaling pathway in bovine intestinal epithelial cells is mediated by the DKK2 (dickkopf2) Wnt antagonist

The Wnt/β-catenin signaling pathway is aberrantly activated in most colorectal cancers. High-dose 1,25(OH)2D3 has anticancer effect by regulating Wnt signal pathway. However, it is not clear whether high-dose of 1,25(OH)2D3 have an effect on normal cells. The aim of the present study was to investigate the mechanism of high-dose 1,25(OH)2D3 on the Wnt signaling pathway in bovine intestinal epithelial cells. The potential mechanism of action was investigated after knockdown and overexpression of the Wnt pathway inhibitor, DKK2, in intestinal epithelial cells by observing the effects of 1,25(OH)2D3 on proliferation, apoptosis, pluripotency and the expression of genes related to the Wnt/β-catenin signaling pathway. In the present study, we introduced the method of isolation and culture of primary bovine intestinal epithelial cells. After cells were treated with 50 ng/mL 1,25(OH)2D3 or DMSO for 48 h, total RNA was extracted, and six differentially expressed genes, including SERPINF1, SFRP2, SFRP4, FZD2, WISP1 and DKK2 were identified by transcriptome sequencing, which were related to Wnt signaling pathway. To further explore the mechanism of 1,25(OH)2D3 on the Wnt/β-catenin signaling pathway, we constructed knockdown and overexpression plasmids of DKK2. After transfecting these plasmids into bovine intestinal epithelial cells, we measured the expression of DKK2 mRNA and protein through GFP expression, qRT-PCR and western blot analyses to verify the transfection efficiency. In addition, the CCK-8 assay was used to detect the cell proliferation rate after transfection. Subsequently, the transfected cells were treated with 1,25(OH)2D3 for 48 h, and the proliferation- (Ki67 and PCNA), apoptosis- (Bcl-2, p53, casp3 and casp8), pluripotency- (Bmi-1, Lrig1, KRT19 and TUFT1) and Wnt/ β-catenin signaling pathway- related genes (LGR5, DKK2, VDR, β- Catenin, SFRP2, WISP1 and FZD2) were detected by qRT-PCR and western blot analyses. Our results showed that the expression trend of some genes in bovine intestinal epithelial cells under high-dose 1,25(OH)2D3 was consistent with the sequencing results, including SFRP2 (P < 0.001), SFRP4 (P < 0.05), FZD2 (P < 0.01), WISP1 (P < 0.001) and DKK2 (P < 0.001). In addition, knockdown of DKK2 inhibited cell proliferation (P < 0.01), but DKK2 overexpression promoted cell proliferation (P < 0.01). Compared to the control group, 1,25(OH)2D3 promoted the expression of Wnt/β-catenin signaling pathway-related proteins in bovine intestinal epithelium, thus maintaining intestinal homeostasis in normal intestinal epithelium. In addition, knockdown and overexpression of DKK2 indicated that 1,25(OH)2D3 weakened the inhibitory effect of DKK2 on the Wnt/β-catenin signaling pathway. Together, these results suggest that high-dose 1,25(OH)2D3 has no killing effect on normal intestinal epithelial cells and regulates Wnt/β-catenin signaling pathway through DKK2.

Establishment of bovine trophoblast stem cells

Here, we report that a chemical cocktail (LCDM: leukemia inhibitory factor [LIF], CHIR99021, dimethinedene maleate [DiM], minocycline hydrochloride), previously developed for extended pluripotent stem cells (EPSCs) in mice and humans, enables de novo derivation and long-term culture of bovine trophoblast stem cells (TSCs). Bovine TSCs retain developmental potency to differentiate into mature trophoblast cells and exhibit transcriptomic and epigenetic (chromatin accessibility and DNA methylome) features characteristic of trophectoderm cells from early bovine embryos. The bovine TSCs established in this study will provide a model to study bovine placentation and early pregnancy failure.

Celecoxib alleviates nociceptor sensitization mediated by interleukin-1beta-primed annulus fibrosus cells

PurposeThis study aims to analyze the effect of pro-inflammatory cytokine-stimulated human annulus fibrosus cells (hAFCs) on the sensitization of dorsal root ganglion (DRG) cells. We further hypothesized that celecoxib (cxb) could inhibit hAFCs-induced DRG sensitization.MethodshAFCs from spinal trauma patients were stimulated with TNF-α or IL-1β. Cxb was added on day 2. On day 4, the expression of pro-inflammatory and neurotrophic genes was evaluated using RT-qPCR. Levels of prostaglandin E2 (PGE-2), IL-8, and IL-6 were measured in the conditioned medium (CM) using ELISA. hAFCs CM was then applied to stimulate the DRG cell line (ND7/23) for 6 days. Then, calcium imaging (Fluo4) was performed to evaluate DRG cell sensitization. Both spontaneous and bradykinin-stimulated (0.5 μM) calcium responses were analyzed. The effects on primary bovine DRG cell culture were performed in parallel to the DRG cell line model.ResultsIL-1ß stimulation significantly enhanced the release of PGE-2 in hAFCs CM, while this increase was completely suppressed by 10 µM cxb. hAFCs revealed elevated IL-6 and IL-8 release following TNF-α and IL-1β treatment, though cxb did not alter this. The effect of hAFCs CM on DRG cell sensitization was influenced by adding cxb to hAFCs; both the DRG cell line and primary bovine DRG nociceptors showed a lower sensitivity to bradykinin stimulation.ConclusionCxb can inhibit PGE-2 production in hAFCs in an IL-1β-induced pro-inflammatory in vitro environment. The cxb applied to the hAFCs also reduces the sensitization of DRG nociceptors that are stimulated by the hAFCs CM.