Magnetic Cell Separation Research Articles

Optimization of pre-enrichment strategies for mouse hematopoietic stem cell isolation and metabolomic analysis

Blood cell production arises from the activity of hematopoietic stem cells (HSCs), defined by their self-renewal capacity and ability to give rise to all mature blood cell types. The mouse remains one of the most studied species in hematological research, and markers to define and isolate mouse HSCs are well-established. Given the very low frequency of HSCs in the bone marrow, stem cell pre-enrichment by red blood cell lysis and magnetic cell separation is often performed as part of the isolation process to reduce sorting times. Several pre-enrichment strategies are available, differing in their speed, degree of enrichment, final cell yield, and cost. In the current study, we performed a side-by-side comparison and provide a decision tree to help researchers select a pre-enrichment strategy for mouse HSC isolation depending on their downstream application. We then compared different pre-enrichment techniques in combination with metabolomics analysis of HSCs, where speed, yield and temperature during pre-enrichment are crucial factors, and found that the choice of pre-enrichment strategy significantly impacts the number of metabolites detected and levels of individual metabolites in HSCs.

CD19-chimeric antigen receptor-invariant natural killer T cells transactivate NK cells and reduce alloreactivity

Invariant natural killer T (iNKT) cells are a small fraction of T lymphocytes with strong cytotoxic and immunoregulatory properties. We previously showed that human culture-expanded iNKT cells prevent alloreactivity and lyse primary leukemia blasts. Here, iNKT cells have several advantages over T cells based on their immunoregulatory capabilities. Since chimeric antigen receptors (CARs) increase the benefit of immune effector cells, they play a crucial role in improvement of cytotoxic abilities of novel cellular therapeutics such as iNKT cells. In the present study, we investigated transactivation of NK cells and prevention of alloreactivity through iNKT cells transduced with a CD19-directed CAR. iNKT cells were isolated by magnetic cell separation from peripheral blood mononuclear cells and transduced with a CD19-CAR retrovirus. Transduction efficiency, purity and cell subsets were measured by flow cytometry. Transactivation and cytotoxicity assays have been established to investigate the ability of CD19-CAR-iNKT cells to transactivate primary NK cells. A mixed lymphocyte reaction (MLR) was performed to explore the inhibition of alloreactive CD3+ T cells by CD19-CAR-iNKT cells. CD19-CAR-iNKT cells are able to transactivate NK cells independent of cell contact: The expression of activation marker CD69 was significantly increased and also production of the proinflammatory cytokine interferon-gamma was higher in NK cells pretreated with CD19-CAR-iNKT cells. Consequently, the cytotoxic activity of such NK cells was significantly increased being able to lyse leukemia cells more effectively than without prior transactivation. Adding CD19-CAR-iNKT cells to an MLR resulted in a decreased expression of the T cell activation marker CD25 on alloreactive CD3+ T lymphocytes stimulated with HLA mismatched dendritic cells. Also, the proliferation of alloreactive CD3+ T lymphocytes was significantly reduced in this setting. We demonstrate that CD19-CAR-iNKT cells keep their immunoregulatory properties despite transduction with a CAR making them an attractive effector cell population for application after allogeneic hematopoietic cell transplantation. By transactivating NK cells, increasing their cytotoxic activity and suppressing alloreactive T cells, they might further improve outcomes through prevention of both relapse and graft-versus-host disease.

Effect of Annealing Temperature on the Characterizations of Cobalt Ferrite Nanoparticles Synthesized via Co-precipitation

This study investigates the effect of annealing temperature on the structural and magnetic properties of cobalt ferrite (CoFe2O4) nanoparticles synthesized via the co-precipitation method. The results indicate that the annealing temperature significantly enhances the crystal size and quality, yielding well-defined, impurity-free CoFe2O4 crystals. Magnetic measurements reveal that both saturation magnetization (Ms) and coercivity (Hc) are improved with annealing. The nanoparticles exhibit a soft-magnetic, single-domain state up to an annealing temperature of 1200°C. They reach a critical size of 39.7 nm and begin transitioning to a multi-domain state. These findings highlight the crucial role of annealing temperature in optimizing the properties of CoFe2O4 nanoparticles for potential applications in magnetic recording media, magnetic resonance imaging, magnetic fluid hyperthermia, biosensors, drug delivery, and cell separation.

Increasing the Particle Size and Magnetic Property of Iron Oxide Nanoparticles through a Segregated Nucleation and Growth Process.

Iron oxide nanoparticles (IONs) with good water dispersibility were prepared by the thermal decomposition of iron acetylacetonate (Fe(acac)3) in the high-boiling organic solvent polyethylene glycol (PEG) using polyethyleneimine (PEI) as a modifier. The nucleation and growth processes of the crystals were separated during the reaction process by batch additions of the reaction material, which could inhibit the nucleation but maintain the crystal growth, and products with larger particle sizes and high saturation magnetization were obtained. The method of batch addition of the reactant prepared IONs with the largest particle size and the highest saturation magnetization compared with IONs reported using PEG as the reaction solvent. The IONs prepared by this method also retained good water dispersibility. Therefore, these IONs are potentially suitable for the magnetic separation of cells, proteins, or nucleic acids when large magnetic responses are needed.

Abstract 2022: E7386, a Wnt/β-catenin signaling modulator, suppresses the differentiation of regulatory T cells in combination with lenvatinib plus anti-PD-1 antibody

Abstract Background: E7386, a selective inhibitor of the interaction between β-catenin and CREB binding protein (CBP), showed antitumor activity in preclinical tumor models with aberrant activation of the Wnt/β-catenin signaling pathway. Previously, we reported the enhanced antitumor activity of a triple combination (E7386 in combination with Lenvatinib [LEN, a multiple receptor tyrosine kinase inhibitor mainly targeting VEGFRs and FGFRs] plus anti-PD-1 antibody) versus a 2-drug combination (LEN plus anti-PD-1 antibody). In this study, we further investigated the immunomodulatory functions of E7386 as a triple combination by single-cell RNA-sequencing (scRNA-Seq) analysis and subsequent functional assays. Methods: Mice bearing subcutaneous RAG (mouse renal cell carcinoma) tumors were randomized into 4 groups (n=6 in each group), including ⑴ Nontreatment, ⑵ E7386 at 25 mg/kg (PO, QD), ⑶ LEN at 10 mg/kg (PO, QD) plus anti-PD-1 antibody at 10 mg/kg (IP, BIW), and ⑷ triple combination. Tumor tissues were collected after a 2-week treatment period. CD45+ immune cells were isolated from the tumors by magnetic cell separation (MACS) and subjected to scRNA-Seq using 10x Genomics pipelines. Bioinformatics data analysis was conducted with Clarivate Analytics. To evaluate the effects of E7386 on Treg differentiation, an in vitro-induced regulatory T cells (Tregs) differentiation assay was conducted. Naïve CD4+ T cells were isolated by MACS and plated under iTreg differentiation conditions (stimulation of TGF-β and IL-2) with or without treatment of E7386. Induced Tregs and conditional culture medium were collected for evaluation of Treg differentiation, including gene expression and protein analysis. Results: CD45+ immune cells were clustered into 14 cell-type groups. Increased proportion of monocytes, NK cells, Tregs, CD4+ T cells, CD8+ T cells, eosinophils, and neutrophils were observed after LEN plus anti-PD-1 treatment. Among these subpopulations, Treg were most significantly suppressed by E7386 plus LEN plus anti-PD-1. Gene expression analysis revealed that the triple combination suppressed the activated gene expression of the Stat5/Foxp3 axis in CD4+ T cells, particularly Tregs, induced by LEN plus anti-PD-1 treatment. Consistently, the iTreg differentiation assay confirmed the suppressive function of E7386 on the Treg differentiation rate. Multiple subsequent in vitro assays also demonstrated the inhibitory effects of E7386 on TGF-β secretion and the expression levels of TGF-β downstream target genes. Conclusion: In summary, these data suggest a suppressive role of E7386 on Treg differentiation, which leads to antitumor activity of E7386 in combination with lenvatinib and anti-PD-1 antibody in a preclinical tumor model. Citation Format: Jialing Shen, Ryoga Ishida, Masahiko Kume, Takayuki Kimura, Yusuke Adachi, Yu Kato. E7386, a Wnt/β-catenin signaling modulator, suppresses the differentiation of regulatory T cells in combination with lenvatinib plus anti-PD-1 antibody [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2022.

Abstract 2026: Single-cell RNA-sequencing analysis of tumor endothelial cells reveals suppression of specific endothelial subtypes by the combination treatment of Wnt/β-catenin signaling modulator E7386 plus lenvatinib

Abstract Background: Aberrant activation of Wnt/β-catenin signaling plays an important role in both carcinogenesis and modulation of the tumor microenvironment. E7386, a novel orally active antitumor agent, inhibits the interaction between β-catenin and CREB-binding protein (CBP) and thereby modulates Wnt/β-catenin signaling. We previously reported that E7386 in combination with lenvatinib (LEN), a multiple receptor tyrosine kinase inhibitor mainly targeting VEGFRs and FGFRs, showed a greater antitumor activity and reduction of tumor microvessels than each treatment alone in preclinical tumor models. Multiple clinical studies are in progress including the combination of E7386 plus LEN (NCT04008797). Here, we further tried to elucidate the mechanism underlying the antitumor and antiangiogenic effect of the combination of E7386 plus LEN by using single-cell RNA sequencing (scRNA-seq) of tumor endothelial cells (ECs). Methods: Mice bearing subcutaneous RAG (mouse renal cell carcinoma cell line) tumors were treated with E7386 at 25 mg/kg and/or with LEN at 10 mg/kg by daily oral gavage for 8 days. Tumor ECs (CD31+/CD45−) were isolated by magnetic cell separation and flow cytometry and were subjected to scRNA-seq using 10x Genomics pipeline. Bioinformatics data analysis was conducted by Clarivate Analytics. After quality control and filtering, ECs were gated by their expression of pan-EC markers and were further classified into 9 subtypes (tip, capillary, arterial, venous, proliferating, immature, interferon, activated artery, and lymphatic) according to previously reported markers for each subtype. Results: Among classified subtypes, proliferating and immature subtypes showed clear reduction in the percentage of total ECs with combination of E7386 plus LEN versus LEN monotherapy. In both proliferating and immature subtypes, a subset of Wnt/β-catenin downstream genes showed increased expression with LEN monotreatment. Conversely, these Wnt/β-catenin downstream genes were downregulated with the combination of E7386 plus LEN, suggesting that Wnt/β-catenin signaling may be associated with a drug resistance mechanism against LEN in these subtypes. Pathway analysis indicated that genes involved in cell proliferation and survival were downregulated by the E7386 plus LEN combination versus LEN monotreatment in both proliferating and immature subtypes. Conclusion: Taken together, these results suggest a role for E7386 in suppressing the activation of Wnt/β-catenin signaling induced by LEN monotreatment in specific EC subtypes; this activity leads to enhanced antiangiogenic and antitumor activity with E7386 plus LEN combination therapy in a preclinical tumor model. Citation Format: Masahiko Kume, Ryoga Ishida, Yoichi Ozawa, Jialing Shen, Hiroto Konishi, Megumi Kuronishi, Takayuki Kimura, Yusuke Adachi, Yu Kato. Single-cell RNA-sequencing analysis of tumor endothelial cells reveals suppression of specific endothelial subtypes by the combination treatment of Wnt/β-catenin signaling modulator E7386 plus lenvatinib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2026.

Adoptive transfer of donor B lymphocytes: a phase 1/2a study for patients after allogeneic stem cell transplantation

AbstractImmune reconstitution after allogeneic hematopoietic stem cell transplantation (allo-HSCT) is slow and patients carry a high and prolonged risk of opportunistic infections. We hypothesized that the adoptive transfer of donor B cells can foster after HSCT immuno-reconstitution. Here, we report, to our knowledge, the results of a first-in-human phase 1/2a study aimed to evaluate the feasibility and safety of adoptively transferred donor B cells and to test their activity upon recall vaccination. Good manufactoring practice (GMP) B-cell products were generated from donor apheresis products using 2-step magnetic cell separation. Fifteen patients who had undergone allo-HSCT were enrolled and treated after taper of immunosuppression (median, day +148; range, 130-160). Patients received 4 different doses of B cells (0.5 × 106 to 4.0 × 106 B cells per kg body weight). To test the activity of infused donor memory B cells in vivo, patients were vaccinated with a pentavalent vaccine 7 days after B-cell transfer. We observed the mobilization of plasmablasts and an increase in serum titers against vaccine antigens, with a stronger response in patients receiving higher B-cell numbers. Analysis of immunoglobulin VH-sequences by next-generation sequencing revealed that plasmablasts responding to vaccination originated from memory B-cell clones from the donor. Donor B-cell transfer was safe, as no Epstein-Barr virus (EBV) reactivation was observed, and only low-grade graft-versus-host disease (GVHD) occurred in 4 out of 15 patients. This pilot trial may pave the way for further studies exploring the adoptive transfer of memory B cells to reduce the frequency of infections after allo-HSCT. This trial was registered at ClinicalTrial.gov as #NCT02007811.

A comprehensive review on the applications of ferrite nanoparticles in the diagnosis and treatment of breast cancer.

Various conventional treatments including endocrine therapy, radiotherapy, surgery, and chemotherapy have been used for several decades to treat breast cancer; however, these therapies exhibit various life-threatening and debilitating adverse effects in patients. Additionally, combination therapies are required for prompt action as well as to prevent drug resistance toward standard breast cancer medications. Ferrite nanoparticles (NPs) are increasingly gaining momentum for their application in the diagnosis and treatment of breast cancer. Spinel ferrites are particularly used against breast cancer and have shown in vitro and in vivo better efficacy as compared to conventional cancer therapies. Magnetic resonance imaging contrast agents, magnetic particle imaging tracers, cell separation, and immune assays are some aspects related to the diagnosis of breast cancer against which different ferrite NPs have been successfully evaluated. Moreover, citrate-coated nickel ferrite, Mg/Zn ferrites, poly amidoamine dendrimers, cobalt ferrites, graphene oxide cobalt ferrites, doxorubicin functionalized cobalt ferrites, chitosan-coated zinc ferrites, PEG-coated cobalt ferrite, and copper ferrite NPs have demonstrated antiproliferative action against different breast cancer cells. Oxaliplatin-loaded polydopamine/BSA-copper ferrites, functionalized cobalt and zinc ferrites of curcumin, oxaliplatin-copper ferrite NPs, tamoxifen/diosgenin encapsulated ZnO/Mn ferrites, and fabricated core-shell fibers of doxorubicin have been developed to increase the bioavailability and anti-proliferative effect and decrease the toxicity of anticancer drugs. These ferrite NPs showed an anticancer effect at different doses in the presence or absence of an external magnetic field. The present review covers the in-depth investigations of ferrite NPs for the diagnosis and management of breast cancer.

Magnetic Separation of Micro Beads and Cells on a Paper-Based Lateral Flow System

Paper based lateral flow systems are widely used biosensor platforms to detect biomolecules in a liquid sample. Proteins, bacteria, oligonucleotides, and nanoparticles were investigated in the literature. In this work we designed a magnetic platform including dual magnets and tested the flow of micron size immunomagnetic particles alone and when loaded with cells on two different types of papers. The prewetting conditions of the paper and the applied external magnetic field are the two dominant factors affecting the particle and cell transport in paper. The images recorded with a cell phone, or with a bright field optical microscope were analyzed to measure the flow of particles and cells. The effect of prewetting conditions and magnetic force were measured, and it was shown that in the worst case, minimum 90% of the introduced cells reached to the edge of the paper. The paper based magnetophoretic lateral flow systems can be used for cell assays.

Functional integration of natural killer cells in a microfluidically perfused liver on-a-chip model

ObjectiveThe liver acts as an innate immunity-dominant organ and natural killer (NK) cells, are the main lymphocyte population in the human liver. NK cells are in close interaction with other immune cells, acting as the first line of defense against pathogens, infections, and injury.A previously developed, three-dimensional, perfused liver-on-a-chip comprised of human cells was used to integrate NK cells, representing pivotal immune cells during liver injury and regeneration. The objective of this study was to integrate functional NK cells in an in vitro model of the human liver and assess utilization of the model for NK cell-dependent studies of liver inflammation.ResultsNK cells from human blood and liver specimen were isolated by Percoll separation with subsequent magnetic cell separation (MACS), yielding highly purified blood and liver derived NK cells. After stimulation with toll-like-receptor (TLR) agonists (lipopolysaccharides, Pam3CSK4), isolated NK cells showed increased interferon (IFN)-gamma secretion. To study the role of NK cells in a complex hepatic environment, these cells were integrated in the vascular compartment of a microfluidically supported liver-on-a-chip model in close interaction with endothelial and resident macrophages. Successful, functional integration of NK cells was verified by immunofluorescence staining (NKp46), flow cytometry analysis and TLR agonist-dependent secretion of interleukin (IL)-6 and tumor necrosis factor (TNF)-alpha. Lastly, we observed that inflammatory activation of NK cells in the liver-on-a-chip led to a loss of vascular barrier integrity. Overall, our data shows the first successful, functional integration of NK cells in a liver-on-a-chip model that can be utilized to investigate NK cell-dependent effects on liver inflammation in vitro.

The CD44std and CD44v9 subpopulations in non-tumorigenic invasive SNU-423 cells present different features of cancer stem cells

Hepatocellular carcinoma (HCC) is a type of liver cancer, in which CD44 isoforms have been proposed as markers to identify cancer stem cells (CSCs). However, it is unclear what characteristics are associated with CSCs that exclusively express CD44 isoforms. The objective of the present study was to determine the expression of CD44 isoforms and their properties in CSCs. Analysis of transcriptomic data from HCC patient samples identified CD44v8-10 as a potential marker in HCC. In SNU-423 cells, CD44 expression was detected in over 99% of cells, and two CD44 isoforms, namely, CD44std and CD44v9, were identified in this cell line. CD44 subpopulations, including both CD44v9+ (CD44v9) and CD44v9- (CD44std) cells, were obtained by purification using a magnetic cell separation kit for human CD44v9+ cancer stem cells. CD44v9 cells showed greater potential for colony and spheroid formation, whereas CD44std cells demonstrated significant migration and invasion capabilities. These findings suggested that CD44std and CD44v9 may be used to identify features in CSC populations and provide insights into their roles in HCC.

All-in-One Microfluidic Chip for Online Labeling, Separating, and Focusing Rare Circulating Tumor Cells from Blood Samples Followed by Inductively Coupled Plasma Mass Spectrometry Detection.

Circulating tumor cell (CTC) detection is essential for early cancer diagnosis and evaluating treatment efficacy. Despite the growing interest in isolating CTCs and further quantifying surface biomarkers at the single-cell level, highly efficient separation of rare CTCs from massive blood cells is still a big challenge. Here, we developed an all-in-one microfluidic chip system for the immunolabeling, magnetic separation, and focusing of HepG2 cells (as a CTC model) and online combined it with single cell-inductively coupled plasma mass spectrometry (SC-ICP-MS) for quantitative analysis of the asialoglycoprotein receptor (ASGPR) on single HepG2 cells. Lanthanide-labeled anti-ASGPR monoclonal antibody and antiepithelial cell adhesion molecule-modified magnetic beads were prepared as signal and magnetic probes, respectively. Target cells were highly efficiently labeled with signal and magnetic probes in the mixing zone of the microfluidic chip and then focused and sorted in the separation zone by specific magnetic separation techniques to avoid matrix contamination. The average cell recovery of HepG2 cells was derived to be 94.1 ± 5.7% with high separation efficiency and purity. The sorted cells with signal probes were detected for enumeration and quantification of ASGPR on their surface by SC-ICP-MS. The developed method showed good specificity and high sensitivity, detecting an average of (1.0 ± 0.2) × 105 ASGPR molecules per cell surface. This method can be used for absolute quantitative analysis of ASGPR on the surface of single hepatocellular carcinoma cells in real-world samples, providing a highly efficient analytical platform for studying targeted drug delivery in cancer therapy.

Magnetohydrodynamic conjugate heat transfer analysis on a viscous fluid past a vertical permeable plate

The influence of conjugate heat transfer based on radiation and magnetohydrodynamic utilization is an attractive research area, with progressive features; it has many applications in thermal engineering, heat exchangers, cooling phenomenon, magnetic cell separation, energy production, hyperthermia, etc. Following the motivating significances of the current research topic, this paper explores the influences of an electrically conducting and radiating fluid with internal friction, heat generation and thermal radiation embedded in a porous medium past a flat permeable plate. The boundary layer equations are dimensionally transformed and solved numerically using implicit finite difference technique called the Keller-box method. The effect of various fluid obeying parameters such as magnetic field, viscous dissipation and heat generation on the flow factors such as velocity and temperature are graphed and discussed in this paper. Growing heat source, energy inside the fluid boosts, thereby increasing the energy of the flow. Increase in generation of energy reduces the viscosity of the flow reduces thereby increasing the velocity of the flow particles.

CAR NK-92 cell–mediated depletion of residual TCR+ cells for ultrapure allogeneic TCR-deleted CAR T-cell products

AbstractGraft-versus-host disease (GVHD) is a major risk of the administration of allogeneic chimeric antigen receptor (CAR)-redirected T cells to patients who are HLA unmatched. Gene editing can be used to disrupt potentially alloreactive T-cell receptors (TCRs) in CAR T cells and reduce the risk of GVHD. Despite the high knockout rates achieved with the optimized methods, a subsequent purification step is necessary to obtain a safe allogeneic product. To date, magnetic cell separation (MACS) has been the gold standard for purifying TCRα/β– CAR T cells, but product purity can still be insufficient to prevent GVHD. We developed a novel and highly efficient approach to eliminate residual TCR/CD3+ T cells after TCRα constant (TRAC) gene editing by adding a genetically modified CD3-specific CAR NK-92 cell line during ex vivo expansion. Two consecutive cocultures with irradiated, short-lived, CAR NK-92 cells allowed for the production of TCR– CAR T cells with <0.01% TCR+ T cells, marking a 45-fold reduction of TCR+ cells compared with MACS purification. Through an NK-92 cell–mediated feeder effect and circumventing MACS-associated cell loss, our approach increased the total TCR– CAR T-cell yield approximately threefold while retaining cytotoxic activity and a favorable T-cell phenotype. Scaling in a semiclosed G-Rex bioreactor device provides a proof-of-principle for large-batch manufacturing, allowing for an improved cost-per-dose ratio. Overall, this cell-mediated purification method has the potential to advance the production process of safe off-the-shelf CAR T cells for clinical applications.

Inhibition of the Sonic Hedgehog Pathway using Small Molecule Inhibitors: Targeting Colon Cancer Stem Cells

&lt;p&gt;Background: The Hedgehog (HH) pathway is a key regulator of many important processes in vertebrate embryonic development, including stem cell maintenance, cell differentiation, tissue polarity and cell proliferation. During pathway activation, Ptch no longer inhibits Smo and the full length Gli translocates to the nucleus resulting in the transcription of oncogenes. When constitutively activated, this leads to tumorigenesis in several human cancers. Cyclopamine acts as an antagonist of the HH signalling pathway by directly binding to the Smo heptahelical domain. The involvement of this pathway in metastasis, and its presence in cancer stem cells (CSCs), makes it a valid option for developing a targeted therapeutic against it. &lt;p&gt; Methods: CSC were isolated from DLD1 and HT29 cell lines using magnetic cell separation labelling the CD133 receptor. The growth patterns of isolated CSCs (CD133 positive) in comparison to non-stem cells (CD133 negative) were analysed using real-time cell impedance assays (RTCA). Thereafter, adhesion, invasion and migration assays were performed with the application of small molecule inhibitors. The expression levels of CD133 and SHH were evaluated using confocal microscopy following treatment with cyclopamine. &lt;p&gt; Results and Discussion: Growth of CSCs appeared to be slower than non-CSCs. Adhesion, invasion and cell migration were inhibited when CSCs were pharmacologically treated either with cyclopamine or SANT-2 (a synthetic analogue of cyclopamine), small molecule inhibitors of the HH pathway. Using confocal microscopy the cell surface expression of Sonic Hedgehog (SHH) was significantly decreased following treatment with cyclopamine, while the expression of CD133 remained unaffected. &lt;p&gt; Conclusion: Considering these in vitro results, small molecule inhibitors targeting the SHH pathway appear to be promising therapeutic tools for the treatment of metastatic colon CSCs.&lt;/p&gt;

The magnetic cell separation method reveals protective effect of melatonin on human spermatozoa from peroxide-induced apoptosis

BackgroundThe selection of non-apoptotic sperm is related to successful fertilization. This study investigated the protective effects of melatonin and the role of the MACS (magnetically activated cell sorting) method to prevent oxidative damage in vitro and improve sperm quality parameters such as viability and DNA integrity.Materials and methodsSemen analysis was performed on 20 different eligible donors participating in the study. Sperm viability and concentration were checked at time of 0 (0 h). In order to conduct more studies after 24 h (24 h), the remaining sperm suspension was divided into a control group and six experimental groups. DNA fragmentation was assessed using the TUNEL assay.ResultsThe treatment of human spermatozoa with 100 µM hydrogen peroxide for 24 h induced a significant increase in phosphatidylserine externalization and significantly increases apoptotic sperm (p ≤ 0.001). TUNEL analysis of human sperm pretreated with 100 µM hydrogen peroxide for 24 h showed that the percentage of sperm with fragmented DNA was significantly reduced after sorting by MACS (P ≤ 0.001). However, pretreated human sperm with 1 μM melatonin for 24 h could effectively maintain sperm motility and progressive motility.ConclusionsPretreated human spermatozoa with 1 µM melatonin for 24 h could be effective for maintenance of sperm motility and progressive motility. Although 100 µM hydrogen peroxide-treated sperm were used, MACS was used to retain the appropriate sperm and select high-quality sperm.

Differentiated Mouse Adipocytes in Primary Culture: A Model of Insulin Resistance.

Insulin resistance is a reduced effect of insulin on its target cells, usually derived from decreased insulin receptor signaling. Insulin resistance contributes to the development of type 2 diabetes (T2D) and other obesity-derived diseases of high prevalence worldwide. Therefore, understanding the mechanisms underlying insulin resistance is of great relevance. Several models have been used to study insulin resistance both in vivo and in vitro; primary adipocytes represent an attractive option to study the mechanisms of insulin resistance and identify molecules that counteract this condition and the molecular targets of insulin-sensitizing drugs. Here, we have established an insulin resistance model using primary adipocytes in culture treated with tumor necrosis factor-α (TNF-α). Adipocyte precursor cells (APCs), isolated from collagenase-digested mouse subcutaneous adipose tissue by magnetic cell separation technology, are differentiated into primary adipocytes. Insulin resistance is then induced by treatment with TNF-α, a proinflammatory cytokine that reduces the tyrosine phosphorylation/activation of members of the insulin signaling cascade. Decreased phosphorylation of insulin receptor (IR), insulin receptor substrate (IRS-1), and protein kinase B (AKT) are quantified by western blot. This method provides an excellent tool to study the mechanisms mediating insulin resistance in adipose tissue.

Vitamin D induced microbicidal activity against Mycobacterium bovis BCG is dependent on the synergistic activity of bovine peripheral blood cell populations

A growing appreciation is emerging of the beneficial role of vitamin D for health and resistance against infectious diseases, including tuberculosis. However, research has predominantly focused on murine and human species and functional data in bovines is limited. Therefore, the objective of this study was to assess the microbicidal activity and immunoregulatory effect of the vitamin D metabolite 1,25(OH)2D3 on bovine peripheral blood leukocytes (PBL) in response to Mycobacterium bovis BCG (BCG) infection using a combination of functional assays and gene expression profiling. Blood from Holstein-Friesian bull calves with low circulating levels of 25(OH)D was stimulated with 1,25(OH)2D3 for 2 h, and then infected with M. bovis BCG. Results showed that 1,25(OH)2D3 supplementation significantly increased BCG killing by on average 16 %, although responses varied between 1 % and 38 % killing. Serial cell subset depletion was then performed on PBL prior to 1,25(OH)2D3 incubation and BCG infected as before to analyse the contribution of major cell types to mycobacterial growth control. Specific antibodies and either magnetic cell separation or density gradient centrifugation of monocytes, granulocytes, CD3+, CD4+, and CD8+ T lymphocytes were used to capture each cell subset. Results showed that depletion of granulocytes had the greatest impact on BCG growth, leading to a significant enhancement of bacterial colonies. In contrast, depletion of CD4+ or CD8+ T cells individually, or in combination (CD3+), had no impact on mycobacterial growth control. In agreement with our previous data, 1,25(OH)2D3 significantly increased bacterial killing in PBL, in monocyte depleted samples, and a similar trend was observed in the granulocyte depleted subset. In addition, specific analysis of sorted neutrophils treated with 1,25(OH)2D3 showed an enhanced microbicidal activity against both BCG and a virulent strain of M. bovis. Lastly, data showed that 1,25(OH)2D3 stimulation increased reactive oxygen species (ROS) production and the expression of genes encoding host defence peptides (HDP) and pathogen recognition receptors (PRRs), factors that play an important role in the microbicidal activity against mycobacteria. In conclusion, the vitamin D metabolite 1,25(OH)2D3 improves antimycobacterial killing in bovine PBLs via the synergistic activity of monocytes and granulocytes and enhanced activation of innate immunity.

Features of hybridized AA7072 and AA7075 alloys nanomaterials with melting heat transfer past a movable cylinder with Thompson and Troian slip effect

A number of scientists have indicated interest in the promising area of research that involves improving heat transmission by utilizing nanoparticles. The dynamic properties of nanofluids reflect several applications in the cooling process, thermal engineering, heat exchangers, magnetic cell separation, energy production, hyperthermia, etc. In light of the appealing implications of nanofluids, the current research aims to explore the melting process of MHD hybrid AA7072 and AA7075 alloy nanoparticles from a movable cylinder. It also incorporates the irregular Thompson and Troian slip effect to simulate the intricate features of the nanofluid flow. The primary nonlinear partial differential equations (NPDEs) are converted into nonlinear ordinary differential equations (NODEs) with the use of the similarity technique. These converted equations are unraveled numerically via applying the built-in program bvp4c in MATLAB. It is visually examined that physical parameters affect the temperature and skin friction. The outcomes suggest that double solutions are found in a certain range of movable cylinder parameter. In addition, the skin friction is decelerated due to the critical shear rate and velocity slip factors while it is enhanced with the influence of suction and nanoparticles volume fraction. Moreover, the temperature is uplifted due to nanoparticles volume fraction and is declined due to Stefan number. In case of quanitative descriptions, the skin friction boosted up by almost 3.64% and 1.18% for the particular upper and lower branch outcomes due to the superior influences of nanoparticle volume fraction. Also, the skin friction decelerated for the branch of upper and lower solutions at about 5.26% and 22.55%, respectively, due to the larger velocity slip parameter.

Magnetic bead-based separation of sperm cells from semen-vaginal fluid mixed stains using an anti-ACRBP antibody.

Forensic DNA analysis of semen-vaginal fluid mixed stains is essential and necessary in sexual assault cases. Here, we used a magnetic bead conjugated acrosin binding protein (ACRBP) antibody to separate and enrich sperm cells from mixed stains. Previously, western blotting indicated that ACRBP was specifically expressed in sperm cells, but not in female blood and epithelial cells, while immunofluorescence data showed ACRBP was localized to the acrosome in sperm cells. In our study, sperm were separated from mixed samples at three sperm cell/female buccal epithelial cell ratios (103:103; 103:104; and 103:105) using a magnetic bead conjugated ACRBP antibody. Subsequently, 23 autosomal short tandem repeat (STR) loci were amplified using the Huaxia™ Platinum PCR Amplification System and genotyped using capillary electrophoresis. The genotyping success rate for STR loci was 90% when the sperm to female buccal epithelial cell ratio was > 1:100 in mixed samples. Our results suggest that the magnetic bead conjugated ACRBP antibody is effective for isolating sperm cells in sexual assault cases.