Identify Target Cells Research Articles

Mechanisms of inducing cancer extracellular vesicles secretion and transfer for cancer malignancy

Assistant Professor Nao Nishida-Aoki, Waseda Institute for Advanced Study (WIAS), is investigating the role of extracellular vesicles (EVs) in physiological tumour tissue environments in order to improve cancer treatments. Communication tools like EVs are used by cancer cells to educate the surrounding cells and make them supportive to cancer cell growth and metastasis. Nishida-Aoki’s research focuses on understanding EV-mediated communication within the complex tissue environment of patient tumours, where they are secreted in densely packed cells and non-cellular components forming a 3D structure. She has developed ex vivo brain and lung metastatic models using organotypic tissue slice culture (TSC). This enables her to observe EV transfer within tissue architecture in real time, identify target cells affected by cancer EVs and determine how they are distributed. Nishida-Aoki is also exploring the mechanisms of inducible secretion of cancer extracellular vesicles upon cell recognition and contribution to cancer malignancy. Her research is investigating the molecular mechanics by which cancer cells regulate and the extent of interactions with neighbouring cells using EVs at metastatic initiation. She has hypothesised that cancer cells should increase communication frequency using EVs when they meet other types of cells in a new environment to benefit themselves. By elucidating the molecular basis of cancer-normal cell interactions mediated by EVs, Nishida-Aoki aims to develop novel cancer therapeutic strategies.

Retrieving single-cells and cell clusters from cytology direct smear slides for genomic evaluation

Abstract Introduction/Objective The key role of cytopathologists is to generate diagnoses based on cytologically stained samples. However, diagnoses increasingly need genomic data in addition to morphologic information. By current methods, morphologically diagnostic samples may be inadequate to perform comprehensive ancillary testing. Therefore, there is a great need for a workflow that can reliably identify and retrieve target cells based on their morphological features. This workflow should also enable highly sensitive molecular profiling from individual cells or small clusters of cells, allowing accurate detection of genomic alterations like copy number variations (CNVs) and single nucleotide variations (SNVs). Methods/Case Report Previously, we established a workflow that combines the RareCyte (Seattle, WA) cell-retrieval platform with primary template-directed amplification (PTA) to generate whole genome libraries from Propidium iodide (PI) stained effusion tumor single-cells or cell clusters and detected copy number variation. In this study, we modified and expanded the assay to identify and retrieve malignant or suspicious cells from Diff-Quik direct smear slides from patient pleural effusion specimens. The samples were further subjected to PTA amplification and library preparation for future copy number variation (CNV) evaluation. In parallel, patient cells from PI-stained smear slides were also obtained and processed to serve as controls. Results (if a Case Study enter NA) Direct smear slides were made from patient pleural fluid (N=5). In contrast to the previous PI staining, which only highlighted nuclei under fluorescence microscopy, Diff-Quik staining allowed nuclear and cytoplasmic morphology evaluation to identify target cells via light microscope. The XYZ coordinates of each target cell or cluster were further documented in the RareCyte Imager software to facilitate cell retrieval. We have successfully collected 39 single-cell and 18 bulk-cell samples from 5 different cases. PTA amplification has been completed and DNA yields of amplified libraries are promising. Conclusion We demonstrated that our modified cell retrieval platform can reliably obtain both single-cell or cluster samples based on morphology from cytologic smear slides made from patient effusion specimens. These collected samples were shown to be compatible with downstream amplification and library preparation enabling CNV evaluation and future deep sequencing for diagnostic, prognostic, and predictive pathogenic mutation identification.

Targeting Stem Cells and Dysplastic Features With Dual MEK/ERK and STAT3 Suppression in Gastric Carcinogenesis

Backgrounds & AimsPre-cancerous metaplasia progression to dysplasia can increase the risk of gastric cancers. However, effective strategies to specifically target these pre-cancerous lesions are currently lacking. To address this, we aimed to identify key signaling pathways which are upregulated during metaplasia progression and critical for stem cell survival and function in dysplasia. MethodsTo assess the response to chemotherapeutic drugs, we utilized metaplastic and dysplastic organoids derived from Mist1-Kras mice and twenty human pre-cancerous organoid lines established from gastric cancer patients. Phospho-antibody array analysis and scRNA-seq were performed to identify target cell populations and signaling pathways affected by Pyrvinium, a putative anti-cancer drug. Pyrvinium was administered to Mist1-Kras mice to evaluate drug effectiveness in vivo. ResultsWhile pyrvinium treatment resulted in growth arrest in metaplastic organoids, it induced cell death in dysplastic organoids. Pyrvinium treatment significantly downregulated phosphorylation of ERK and STAT3 as well as STAT3-target genes. scRNA-seq data analyses revealed that Pyrvinium specifically targeted CD133+/CD166+ stem cell populations, as well as proliferating cells in dysplastic organoids. Pyrvinium inhibited metaplasia progression and facilitated the restoration of normal oxyntic glands in Mist1-Kras mice. Furthermore, Pyrvinium exhibited suppressive effects on the growth and survival of human organoids with dysplastic features, through simultaneous blocking the MEK/ERK and STAT3 signaling pathways. ConclusionsThrough its dual blockade of MEK/ERK and STAT3 signaling pathways, Pyrvinium can effectively induce growth arrest in metaplasia and cell death in dysplasia. Therefore, our findings suggest that Pyrvinium is a promising chemotherapeutic agent for reprogramming the pre-cancerous milieu to prevent of gastric cancer development.

FINDER: A Fluidly Confined CRISPR‐Based DNA Reporter on Living Cell Membranes for Rapid and Sensitive Cancer Cell Identification

AbstractThe accurate, rapid, and sensitive identification of cancer cells in complex physiological environments is significant in biological studies, personalized medicine, and biomedical engineering. Inspired by the naturally confined enzymes on fluid cell membranes, a fluidly confined CRISPR‐based DNA reporter (FINDER) was developed on living cell membranes, which was successfully applied for rapid and sensitive cancer cell identification in clinical blood samples. Benefiting from the spatial confinement effect for improved local concentration, and membrane fluidity for higher collision efficiency, the activity of CRISPR‐Cas12a was, for the first time, found to be significantly enhanced on living cell membranes. This new phenomenon was then combined with multiple aptamer‐based DNA logic gate for cell recognition, thus a FINDER system capable of accurate, rapid and sensitive cancer cell identification was constructed. The FINDER rapidly identified target cells in only 20 min, and achieved over 80 % recognition efficiency with only 0.1 % of target cells presented in clinical blood samples, indicating its potential application in biological studies, personalized medicine, and biomedical engineering.

FINDER: A Fluidically Confined CRISPR-Based DNA Reporter on Living Cell Membranes for Rapid and Sensitive Cancer Cell Identification.

The accurate, rapid, and sensitive identification of cancer cells in complex physiological environments is significant in biological studies, personalized medicine, and biomedical engineering. Inspired by the natural confined enzyme on fluidic cell membrane, afluidically confined CRISPR-based DNA reporter (FINDER) was developed on living cell membranes, which was successfully applied for rapid and sensitive cancer cell identification in clinical blood samples. Benefiting from the spatial confinement effect for improved local concentration, and membrane fluidity for higher collision efficiency, the activity of CRISPR-Cas12a was, for the first time, found to be significantly enhanced on living cell membranes. This new phenomenon was then combined with multiple aptamers-based DNA logic gate for cell recognition, thus a FINDER system with accurate, rapid and sensitive cancer cell identification was constructed. The FINDER can rapidly identified target cells in only 20 min, and achieved over 80% recognition efficiency with only 0.1% of target cells presented in clinical blood samples, indicating its potential application in biological studies, personalized medicine, and biomedical engineering.

Extracellular ATP-activated hybridization chain reaction for accurate and sensitive detection of cancer cells

Accurate and sensitive detection of cancer cells is of significant importance for early diagnosis and treatment of cancer. Here, we developed an extracellular ATP-activated hybridization chain reaction (HCR) amplification strategy to meet this purpose. This strategy relies on three DNA probes, Apt-trigger, H1-ATP aptamer duplex and hairpin H2. The Apt-trigger probe consists of two components: an aptamer sequence for specific recognition of the target cells, and a trigger sequence for the HCR assembly. The duplex structure of H1-ATP aptamer causes the toehold in hairpin H1 to be hidden, preventing the strand-displacement reaction between hairpin H1 and Apt-trigger. Upon activation with ATP, the ATP aptamer will bind to ATP to dissociate from hairpin H1, thus leading to an Apt-trigger-induced strand-displacement reaction and subsequent HCR with hairpin H2 on the target cell surface. Benefiting from aptamer recognition and ATP-activated HCR amplification, this strategy can not only perform sensitive quantitative analysis with a detection limit of 25 cells in 200 µL of binding buffer, but also show desirable specificity and accuracy for identifying target cells from control cells and mixed cell samples. Importantly, this method retains stable and good performance for target cell detection in 10% fetal bovine serum, demonstrating great potential for clinical diagnosis in complex biological matrices. Furthermore, this strategy can be adapted to detect various types of cancer cells by changing the corresponding aptamer sequence.

Study on the effect of γδ T cells expanded in vitro to kill hepatocellular carcinoma cells.

γδ T cells for tumor cell immunotherapy has recently become a hot topic. To investigate the stimulation of expanded γδ T cells in vitro to kill liver cancer cells and its mechanism, and in vivo validation. Peripheral blood mononuclear cells (PBMCs) were isolated and amplified. The proportion of γδ T cells in T cells was determined using flow cytometry. γδ T cells were selected as effector cells, and HepG2 cells as target cells in the cytotoxicity experiment. NKG2D blocker was used to block effector cells from identifying target cells, and PD98059 was used to block intracellular signaling pathways. The nude mice tumor model was established in two batches, the tumor growth curve was drawn, and the tumor formation effect was tested using small animal imager to verify the killing effect of γδ T cells. The γδ T cells in the three experimental groups exhibited a large amount of amplification (P < 0.01). In the killing experiment, the killing rate of γδ T cells stimulated by zoledronate (ZOL) in the experimental group was significantly higher than that in the HDMAPP group and the Mycobacterium tuberculosis H37Ra strain (Mtb-Hag) group (P < 0.05). The blocking effect of PD98059 is stronger than that of the NKG2D blocker (P < 0.05). Among them, in the HDMAPP group, when the target ratio was 40:1, the NKG2D blocker exhibited a significant blocking effect (P < 0.05). Alternatively, in the ZOL group, when the effect ratio was 10:1, the effector cells were blocked significantly after treatment using PD98059 (P < 0.05). In vivo experiments verified the killing effect of γδ T cells. According to the tumor growth curve, there was a difference between the experimental and control groups after cell treatment (P < 0.05). ZOL has high amplification efficiency and a positive effect on killing tumor cells.

Characterization of experimental Shuni virus infection in the mouse.

Shuni virus (SHUV), an orthobunyavirus of the Simbu serogroup, was initially isolated in Nigeria in the 1960s, further detected in other African countries and in the Middle East, and is now endemic in Israel. Transmitted by blood-sucking insects, SHUV infection is associated with neurological disease in cattle and horses, and with abortion, stillbirth, or the birth of malformed offspring in ruminants. Surveillance studies also indicated a zoonotic potential. This study aimed to test the susceptibility of the well-characterized interferon (IFN)-α/β receptor knock-out mouse model (Ifnar-/-), to identify target cells, and to describe the neuropathological features. Ifnar-/-mice were subcutaneously infected with two different SHUV strains, including a strain isolated from the brain of a heifer showing neurological signs. The second strain represented a natural deletion mutant exhibiting a loss of function of the S-segment-encoded nonstructural protein NSs, which counteracts the host's IFN response. Here it is shown that Ifnar-/-mice are susceptible to both SHUV strains and can develop fatal disease. Histological examination confirmed meningoencephalomyelitis in mice as described in cattle with natural and experimental infections. RNA in situ hybridization was applied using RNA Scope™ for SHUV detection. Target cells identified included neurons and astrocytes, as well as macrophages in the spleen and gut-associated lymphoid tissue. Thus, this mouse model is particularly beneficial for the evaluation of virulence determinants in the pathogenesis of SHUV infection in animals.

Дозозависимый эффект снижения слуха на фоне приема цисплатина

Cisplatin is a cystostatic agent with a ototoxic side effect. Modern chemotherapy protocols aim to maintain high efficiency against malignant tumors with minimal complications. It is known that one of the side effects of the drug is cisplatin-induced sensoneural hearing loss. Our study research hearing loss with the use of cisplatin depending on the total dose of the drug in clinical practice and identifying target cells in the structure of the inner ear in experiment. A total of 117 patients using cisplatin to treat tumors of various locations were studied, among whom hearing loss was revealed in 28% of cases. Hearing loss predominantly affected the high-frequency region above 8000 Hz and depended on the total dose of cisplatin. We carried out the experimental part of the work on BALB/C mice. To model toxic hearing loss, we used 2 cisplatin administration protocols: a single dose of 12 mg per kg intraperitoneally and a dose of 3 mg/kg sequentially for 7 days. After the administration of cisplatin, a decrease in hearing was recorded according to ABR and DPOAE in all animals, which was more prominent among animals with a single dose of the drug. Histological examination of preparations of the cochlea of animals exposed to the toxic effects of cisplatin showed damage to the structures of the spiral ganglion, stria vascularis and outer hair cells. Variety of damage depended on the dose of the drug.

627 Temporal-spatial CRISPR base editing to identify target cells for genetic-based therapies

627 Temporal-spatial CRISPR base editing to identify target cells for genetic-based therapies

Abstracts from the NIH Office of Research on Women's Health 2022 Annual BIRCWH Meeting:Building Interdisciplinary Research Careers in Women's Health November 2, 2022

Abstracts from the NIH Office of Research on Women's Health 2022 Annual BIRCWH Meeting:Building Interdisciplinary Research Careers in Women's Health November 2, 2022

Imaging flow cytometry challenges the usefulness of classically used extracellular vesicle labeling dyes and qualifies the novel dye Exoria for the labeling of mesenchymal stromal cell–extracellular vesicle preparations

Background aimsExtracellular vesicles (EVs) are involved in mediating intercellular communication processes. An important goal within the EV field is the study of the biodistribution of EVs and the identification of their target cells. Considering that EV uptake is assumed to be important for EVs in mediating intercellular communication processes, labeling with fluorescent dyes has emerged as a broadly distributed strategy for the identification of EV target cells and tissues. However, the accuracy and specificity of commonly utilized labeling dyes have not been sufficiently analyzed. MethodsBy combining recent advances in imaging flow cytometry for the phenotypic analysis of single EVs and aiming to identify target cells for EVs within therapeutically relevant mesenchymal stromal cell (MSC)-EV preparations, the authors explored the EV labeling efficacy of various fluorescent dyes, specifically carboxyfluorescein diacetate succinimidyl ester, calcein AM, PKH67, BODIPY TR ceramide (Thermo Fisher Scientific, Darmstadt, Germany) and a novel lipid dye called Exoria (Exopharm Limited, Melbourne, Australia). ResultsThe authors’ analyses qualified Exoria as the only dye that specifically labeled EVs within the MSC-EV preparations. Furthermore, the authors demonstrated that Exoria labeling did not interfere with the immunomodulatory properties of the MSC-EV preparations as tested in a multi-donor mixed lymphocyte reaction assay. Within this assay, labeled EVs were differentially taken up by different immune cell types. ConclusionsOverall, the results qualify Exoria as an appropriate dye for the labeling of EVs derived from the authors’ MSC-EV preparations. This study also demonstrates the need for the development of next-generation EV characterization tools that are able to localize and confirm the specificity of EV labeling.

V-CARMA: A tool for the detection and modification of antigen-specific T cells

T cells promote our body's ability to battle cancers and infectious diseases but can act pathologically in autoimmunity. The recognition of peptides presented by major histocompatibility complex (pMHC) molecules by T cell receptors (TCRs) enables T cell-mediated responses. To modify disease-relevant T cells, new tools to genetically modify T cells and decode their antigen recognition are needed. Here, we present an approach using viruses pseudotyped with peptides loaded on MHC called V-CARMA (Viral ChimAeric Receptor MHC-Antigen) to specifically target T cells expressing cognate TCRs for antigen discovery and T cell engineering. We show that lentiviruses displaying antigens on human leukocyte antigen (HLA) class I and class II molecules can robustly infect CD8+ and CD4+ T cells expressing cognate TCRs, respectively. The infection rates of the pseudotyped lentiviruses (PLVs) are correlated with the binding affinity of the TCR to its cognate antigen. Furthermore, peptide-HLA pseudotyped lentivirus V-CARMA constructs can identify target cells from a mixed T cell population, suppress PD-1 expression on CD8+ T cells via PDCD1 shRNA delivery, and induce apoptosis in autoreactive CD4+ T cells. Thus, V-CARMA is a versatile tool for TCR ligand identification and selective T cell manipulation.

Interleukin-22 by altering type O-glycan profile of mucin plays a protective role in animal model of experimental colitis

Abstract IL-22 is known to play both protective and inflammatory role in the intestine. Thus, the effect of IL-22 in intestine is complex, and cell type-specific function of this cytokine requires further investigation. To identify target cell type of IL-22, we have used entire intestinal epithelial cell (IL-22Ra1ΔIEC), Paneth (IL-22Ra1ΔDefa6) and Lgr5+ intestinal stem cell (IL-22Ra1ΔLgr5)-specific IL-22Ra1 knockout mice models. Experimental colitis was developed by providing dextran sulphate sodium (DSS) in drinking water for first 8 days, and change to normal water for next two days. On examining DSS induced colitis development, significant increase in disease severity as revealed by reduced colon length, greater weight loss and histopathological changes in tissues were observed in IL-22ra1ΔIEC as compared to IL-22ra1fl/fl mice. No difference in disease severity was observed in IL-22ra1ΔDefa6 and IL-22ra1ΔLgr5 mice compared to their respective littermate control IL-22Ra1fl/fl mice. Quantitative PCR data show increased expression of inflammatory cytokines in distal colon of IL-22ra1ΔIEC compared to IL-22ra1fl/fl mice. Alcian blue staining revealed no difference in goblet cell number, however, expression of selective glycosyltransferase, required for mucin type-O glycosylation core extension, was significantly reduced in distal colon of IL-22Ra1ΔIEC compared to IL-22Ra1fl/fl mice on day 10 DSS/water treatment. Mucin type-O glycan analysis by MALDI-TOF revealed an increase in core 1 and decrease in core 4 structure in IL-22Ra1ΔIEC mice compared to IL-22Ra1fl/fl mice at steady state. Thus, IL-22 through glycosyltransferase activity may maintain intestinal barrier integrity and alleviate severity of experimental colitis.

Electrochemical carbon dioxide reduction to isopropanol using novel carbonized copper metal organic framework derived electrodes

It is well known in sustainable energy research that metallic copper functions as an electrocatalyst for the reduction of CO2 to multicarbon products such as alcohols and hydrocarbons. However, it remains a great challenge to develop a cost-effective, selective, and stable catalyst/electrode material for this reaction. This work furthers previous studies concerning the potential of carbonized copper MOF-derived electrocatalysts as catalyst materials for the electrochemical reduction of CO2. Two commercial copper-decorated metal organic frameworks (MOFs), HKUST-1 and PCN-62, pyrolyzed at variable temperatures, 400−800 °C, were coated on both metallic nickel and copper supports as inks. The electrocatalysts’ potential to reduce CO2 was gauged using an electrochemical cell with both GC-TCD and GC-FID analyses. Many of the previously reported products of this reaction were formed, however, most notably, GC-FID analysis confirmed the formation of isopropanol, a product not previously reported to the best of our knowledge. It was observed that MOF-derived coatings can produce electrodes with both better current density and selectivity towards isopropanol compared to that of uncoated copper electrodes. Amongst all the carbon products observed, the best performing electrocatalyst demonstrated isopropanol faradaic efficiency (FE) of over 72 %. Preliminary techno-economic analysis was conducted to identify target cell operating voltage in order to make the proposed electrochemical CO2 reduction process economically feasible. The desired cell operating voltage is less than 1.9 V, preferably less than 1.6 V. Future work will focus on electrochemical cell design and electrocatalyst morphology to further improve isopropanol selectivity and minimize hydrogen FE.

Identifying target cells for a tick-borne virus that causes fatal hemorrhagic fever.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging disease in China, South Korea, and Japan caused by the tick-borne SFTS virus (SFTSV). Severe and fatal SFTS presents as a hemorrhagic fever characterized by high viral load, uncontrolled inflammatory response, dysregulated adaptive immunity, coagulation abnormalities, hemorrhage, and multiorgan failure with up to 33% case fatality rates (CFRs). Despite its public health significance in Asia, vaccines and specific therapeutics against SFTS are still unavailable. A better understanding of the pathogenesis of SFTS is crucial to improving medical countermeasures against this devastating disease. In this issue of the JCI, Suzuki and colleagues analyzed histopathological samples from 22 individuals who succumbed to SFTS, and identified antibody-producing B cell-lineage plasmablasts and macrophages as principal target cells for SFTSV infection in fatal SFTS. Their results suggest that SFTSV-infected post-germinal center B cells, plasmablasts, and macrophages affect systemic immunopathology and dysregulation, which likely leads to fatal outcomes.

Indoleamine-2,3-Dioxygenase in Thyroid Cancer Cells Suppresses Natural Killer Cell Function by Inhibiting NKG2D and NKp46 Expression via STAT Signaling Pathways.

Natural killer (NK) cells are key players in the immune system. They use receptors on their cell surface to identify target cells. However, to escape being killed by the immune system, cancer cells such as thyroid cancer cells, use various methods to suppress the function of NK cells. Thus, this study aims to elucidate how thyroid cancer cells downregulate NK cell function in a co-culture system. We found that thyroid cancer cells suppress NK cell cytotoxicity and inhibit the expression of activating receptors, such as NKG2D and NKp46, by regulating indoleamine 2,3-dioxygenase (IDO). Also, thyroid cancer cells produce kynurenine using IDO, which causes NK cell dysfunction. Kynurenine enters NK cells via the aryl hydrocarbon receptor (AhR) on the surfaces of the NK cells, which decreases NK cell function and NK receptor expression via the signal transducer and activator of transcription (STAT) 1 and STAT3 pathways. In addition, STAT1 and STAT3 directly regulated the expression of NKG2D and NKp46 receptors by binding to the promoter region. Conclusively, NK cell function may be impaired in thyroid cancer patients by IDO-induced kynurenine production. This implies that IDO can be used as a target for thyroid cancer therapeutics aiming at improving NK cell function.

Feasibility of single-cell analysis of model cancer and foetal cells in blood after isolation by cell picking.

The objective of the present feasibility study was to transfer single cell line cells to either microscopy slides for downstream immune characterization or to polymerase chain reaction tubes for downstream DNA quantitation. Tumour cell lines, SKBR3 and MCF7 and trophoblast cell line JEG-3 were spiked in healthy donor blood. The CytoTrack system was used to scan the spiked blood samples to identify target cells. Individual target cells were identified, picked by use of a CytoPicker and deposited to either a microscopic slide or a polymerase chain reaction tube (PCR). Single tumour cells on microscopic slides were further immunostained with human epidermal growth factor receptor 2 (Her2) and epithelial cell adhesion molecule (EpCAM). From the picked cells in polymerase chain reaction tubes, DNA was amplified, quantified and used for Short Tandem Repeat genotyping. Depositing rare cells to microscopy slides was laborious with only five cells per hour. In this study with a trained operator, the picked cells had an 80.5% recovery rate. Depositing single trophoblast cells in PCR tubes was a faster process with 10 cells in 5 min. Immunostaining of isolated cells by both Her2 and EpCAM was possible but showed varying staining intensity. Presence of trophoblasts and contaminating white blood cells in PCR tubes after cell picking was confirmed based on DNA yield and mixed Short Tandem Repeat profiles in five out of eight samples. Using the CytoPicker tool, single tumour and trophoblast cells were successfully isolated and moved from blood samples, allowing subsequent immunostaining or Short Tandem Repeat genotyping.

PO-129 Effect of endurance training on liver NK cells in mice

Objective NK cell (natural killer cell) is a large granular lymphocyte distinct from a group of T and B lymphocytes. At present, the research shows that NK cells can specifically identify target cells and release killing media and then play a killing effect. It is confirmed that the expression of IL-15 is closely related to the differentiation and maturation of NK cells. Furthermore, skeletal muscle is an endocrine tissue and plays a key role in regulating the whole-body metabolic health by synthesizing and releasing humoral factors called myokines, such as IL-15. Whether the IL-15 induced by exercise training can promote the maturation of NK cells remain unsolved. This study aimed to explore the effects of moderate endurance training on NK cells and relative mechanism.&#x0D; Methods Twenty male C57BL/6J mice were randomly divided into 2 groups: control group (YC) and exercise group (YE). YC animals were fed normally for 12 weeks, YE animals were trained for 12 weeks on moderate intensity treadmill (12 m/min).Then the samples were isolated and RT-PCR was used to detect IL-15 and Nkg2d genes in the liver, Western blotting was used to detect the killer factor IFN-γ released by NK cells. Flow cytometry was used to detect NK1.1 cell markers in primary liver cells .&#x0D; Results 1)Compared with the YC group, the expression level of IL-15 and Nkg2d gene in the liver tissue of YE mice increased significantly (P &lt; 0.05,P &lt; 0.01); 2) Compared with the YC group, the expression of IFN-γ protein in the liver tissue of the YE mice increased significantly (P &lt; 0.05); 3) Compared with two group. The proportion of NK cells in liver cells of group YE increased significantly (P &lt; 0.05).&#x0D; Conclusions Moderate intensity endurance training can enhance the content and killing ability of NK cells through induced IL-15 in the liver.

Novel and simple method of double-detection using fluorescence in situ hybridization and fluorescence immunostaining of formalin-fixed paraffin-embedded tissue sections.

The importance of fluorescence in situ hybridization (FISH) for pathological diagnosis has been increasing. However, the procedures utilized for a conventional FISH method with formalin-fixed paraffin-embedded tissue sections are complicated and it is difficult to perform as a routine laboratory test. In addition, there are difficulties with differentiation of targeted cells in observations with a fluorescence microscope. The present study reported a novel method that utilizes FISH in combination with fluorescence immunostaining as a simple double-detection technique that addresses these problems. Using this novel method, various genetic aberrations, as well as protein overexpression were easily visualized in isologous sections. In particular, FISH signals with our method clearly identify target cells in samples with poor differentiation between tumor cells coexisting with normal cells. It is proposed that this simple technique is widely applicable as a routine laboratory test and future developments are expected.