Flow Cytometric Method Research Articles

Trait positive affect buffers the association between experimental sleep disruption and inflammation

BackgroundSleep disturbances and insufficient sleep are highly prevalent. Both clinical sleep disorders and multiple forms of experimental sleep loss predict heightened inflammation. As such, it is necessary to investigate potential protective factors. Given that trait positive affect (PA) is associated with reduced inflammation, and buffers the proinflammatory effects of stress, it is possible that high trait positive affect might protect individuals from an inflammatory response to sleep disruption. The present study tested this hypothesis in an experimental sleep disruption paradigm with assessment of cellular inflammation. MethodsData were drawn from good sleeping adults (n = 79) who participated in a randomized, within-subjects crossover experiment comparing the effects of two nights of sleep disruption versus two nights of uninterrupted sleep. Stimulated monocytic production of intracellular proinflammatory cytokines tumor necrosis factor (TNF) and interleukin-6 (IL-6) were assayed using flow cytometric methods and indexed as the percentage of monocytes expressing TNF, IL-6, or co-expressing both. Hypotheses were evaluated using linear mixed effects models. ResultsControlling for negative affect, body mass index, age, and sex, PA significantly moderated the associations between sleep condition and stimulated monocyte production of IL-6 (b = −1.03, t = −2.02, p = .048) and its co-expression with TNF (b = −0.93, t = −2.00, p = .049), such that inflammatory responses were blunted among those high in PA with increases principally among those low in PA. The effect on TNF was similar in terms of effect size, but marginally significant. ConclusionsActivation of cellular inflammation in response to sleep disruption is buffered by PA independent of negative affect. Interventions that promote PA might protect persons from the inflammatory activation following sleep loss, with the potential to mitigate the adverse health consequences of sleep disturbance.

Anticancer, anticholinesterase and antidiabetic activities of tunceli garlic (Allium tuncelianum): determining its phytochemical content by LC–MS/MS analysis

We aimed to investigate the phytochemical content and pharmaceutical activity of methanol extracts of Allium tuncelianum (MAT). In this study, phytochemical contents of MAT were analyzed by liquid chromatography mass-spectrometry (LC–MS/MS) technique. Anticancer activity was determined on six different cell lines. By flow cytometric method, apoptotic activity was examined with annexin V in human prostate carcinoma (PC3) cell lines where the highest cytotoxic activity was observed. Real Time PCR was used to investigate the molecular genetic size of the effect. Anticholinesterase and antidiabetic activities were performed using enzyme inhibition technique. Enzyme inhibitory activity of MAT was determined using AChE, α-glucosidase enzymes. In these extracts, “μg Analyte/g MAT” as its unit; quinic acid (6458.45 ± 2.389), malic acid (4187.25 ± 0.544) and vanilic acid (349.4 ± 0.052) were found to be major components. The highest cytotoxic activity was found in PC3 cells (IC50: 105.72 µg/mL). Therefore, the mechanism of apoptotic effect in this cell line was also investigated. In a statistically meaningful proportion, MAT increased Caspase 3 and Caspase 9 gene expressions and suppressed IL6, IL8, MMP2 and MMP9 gene expressions. On the other hand, MAT demonstrated effective inhibition effects against AChE (IC50: 11.25 μg/mL), and α-glycosidase (IC50: 9.85 μg/mL) enzymes. As a result, it can be argued that Allium tuncelianum can be used as an alternative treatment for prostate cancer, diabetes and some neurodegenerative diseases.

To Ki or Not to Ki: Re-Evaluating the Use and Potentials of Ki-67 for T Cell Analysis.

This study discusses substantive advances in T cell proliferation analysis, with the aim to provoke a re-evaluation of the generally-held view that Ki-67 is a reliable proliferation marker per se, and to offer a more sensitive and effective method for T cell cycle analysis, with informative examples in mouse and human settings. We summarize recent experimental work from our labs showing that, by Ki-67/DNA dual staining and refined flow cytometric methods, we were able to identify T cells in the S-G2/M phases of the cell-cycle in the peripheral blood (collectively termed “T Double S” for T cells in S-phase in Sanguine: in short “TDS” cells). Without our refinement, such cells may be excluded from conventional lymphocyte analyses. Specifically, we analyzed clonal expansion of antigen-specific CD8 T cells in vaccinated mice, and demonstrated the potential of TDS cells to reflect immune dynamics in human blood samples from healthy donors, and patients with type 1 diabetes, infectious mononucleosis, and COVID-19. The Ki-67/DNA dual staining, or TDS assay, provides a reliable approach by which human peripheral blood can be used to reflect the dynamics of human lymphocytes, rather than providing mere steady-state phenotypic snapshots. The method does not require highly sophisticated “-omics” capabilities, so it should be widely-applicable to health care in diverse settings. Furthermore, our results argue that the TDS assay can provide a window on immune dynamics in extra-lymphoid tissues, a long-sought potential of peripheral blood monitoring, for example in relation to organ-specific autoimmune diseases and infections, and cancer immunotherapy.

SATB2-AS1 acts as miR-299-3p sponge to facilitate tumorigenesis in human non-small cell lung cancer

IntroductionNon-small cell lung cancer (NSCLC), the most common pathological type of lung cancer, is partly responsible for an increasing number of tumor-related deaths worldwide. This study aimed to explore the biological role of the anti-sense transcript of special AT-rich sequence binding protein 2 (SATB2-AS1), a novel cancer-related long non-coding RNA (lncRNA), and illustrate the potential molecular mechanisms.Material and methodsThe expression patterns of SATB2-AS1 were determined via qPCR analysis in clinical samples and tumor cell lines. Kaplan-Meier survival analysis was conducted to assess the relationship between SATB2-AS1 expression and survival time of NSCLC patients. NSCLC tumors were transfected with SATB2-AS1 expression vectors or specific short hairpin RNAs (sh-SATB2-AS1). Tumor cell proliferation, cell cycle progression and apoptosis was detected by MTT assays and flow cytometric method, respectively. Nude mouse transplantation models were applied to investigate the effects of SATB2-AS1 on tumor cell growth in vivo. Bio-informatics analysis, luciferase reporter assays and rescue assays were performed to elucidate possible molecular mechanisms.ResultsSATB2-AS1 up-regulation was observed in tumorous tissues and cell lines. Up-regulated SATB2-AS1 expression was associated with shorter overall survival time of patients. SATB2-AS1 over-expression facilitated tumor cell proliferation, cell cycle progression and survival, while its knockdown inhibited tumor cell proliferation, cell cycle progression and survival. SATB2-AS1 depletion suppressed tumor growth in vivo. SATB2-AS1 was revealed to act as a miR-299-3p sponge to exert carcinogenic role.ConclusionsOur data indicate that SATB2-AS1 acts as a miR-299-3p sponge to facilitate NSCLC development, providing a novel candidate therapeutic target for NSCLC.

Peripheral Plasma Cells Associated with Mortality Benefit in Severe COVID-19: A Marker of Disease Resolution

BackgroundCytokines seen in severe coronavirus disease 2019 (COVID-19) are associated with proliferation, differentiation, and survival of plasma cells. Plasma cells are not routinely found in peripheral blood, though may produce virus-neutralizing antibodies in COVID-19 later in the course of an infection. MethodsUsing the Johns Hopkins COVID-19 Precision Medicine Analytics Platform Registry, we identified hospitalized adult patients with confirmed severe acute respiratory coronavirus 2 (SARS-CoV-2) infection and stratified by presence of plasma cells and World Health Organization (WHO) disease severity. To identify plasma cells, we employed a sensitive flow cytometric screening method for highly fluorescent lymphocytes and confirmed these microscopically. Cox regression models were used to evaluate time to death and time to clinical improvement by the presence of plasma cells in patients with severe disease. ResultsOf 2301 hospitalized patients with confirmed infection, 371 had plasma cells identified. Patients with plasma cells were more likely to have severe disease, though 86.6% developed plasma cells after onset of severe disease. In patients with severe disease, after adjusting for age, sex, body mass index, race, and other covariates associated with disease severity, patients with plasma cells had a reduced hazard of death (adjusted hazard ratio: 0.57; 95% confidence interval: 0.38-0.87; P value: .008). There was no significant association with the presence of plasma cells and time to clinical improvement. ConclusionsPatients with severe disease who have detectable plasma cells in the peripheral blood have improved mortality despite adjusting for known covariates associated with disease severity in COVID-19. Further investigation is warranted to understand the role of plasma cells in the immune response to COVID-19.

Chronic Running Exercise Regulates Cytotoxic Cell Functions and Zinc Transporter SLC39A10/ZIP10 Levels in Diabetic Rats.

The aim of this study is to investigate how chronic running exercise affects ZIP10 levels in thymus and spleen tissue as well as immune parameters in diabetic rats. A total of 40 adult male Wistar rats were divided into 4 equal groups: group 1, control; group 2, exercise control; group 3, diabetes; group 4, diabetes + exercise. Diabetes was induced by injecting intraperitoneal streptozotocin (STZ) at a dose of 40 mg/kg twice with 24-h intervals to the animals in groups 3 and 4. The animals in group 2 and group 4 underwent exercise for 45 min on the rat treadmill for 4 weeks at 20 m/min. Twenty-four hours after the last running exercise, the animals were sacrificed under general anesthesia. Immunological parameters were determined by flow cytometric method; tissue ZIP 10 levels were determined by ELISA method. The diabetic group had the lowest natural killer (NK) and natural killer T (NKT) cells percentages. Chronic exercise partially improved NK and NKT cell percentages in diabetic rats. The diabetic group had the lowest ZIP10 levels in spleen and thymus tissue. ZIP10 values in spleen and thymus tissue of diabetes exercise group were significantly higher than diabetes group. The results of our study show that the impaired cytotoxic cell functions in diabetes are partially corrected with 4 weeks of chronic exercise, and that the suppressed ZIP 10 levels in diabetic rats are reversed by 4 weeks of chronic exercise.

A flow cytometric approach to study glucocorticoid receptor expression in immune cell subpopulations of genetically engineered mice

Glucocorticoids (GCs) constitute one of the most powerful classes of anti-inflammatory agents and are used for the treatment of a plethora of diseases related to autoimmunity, allergy, cancer, and infection. In the last two decades, multiple studies using genetically engineered mice with targeted deletions of the GC receptor (GR) in individual cell types have provided insights into the mechanisms of GCs in the control of the immune system. The characterization of GR expression in these mouse models, however, mostly relied on the analysis of mRNA expression or reporter gene activity. In contrast, approaches directly detecting the GR protein on a cellular level are scarce. Thus, we here used a flow cytometric method to analyze mice in which the GR gene locus was disrupted with the help of a Cre recombinase expressed under the control of either the lck or the lysM promoter. Measuring GR protein expression in immune cell subpopulations unveiled an efficient and highly selective depletion in both strains of knock-out mice in accordance with the expected cellular specificity of the employed promoters for T cells or myeloid cells, respectively. The flow cytometric data were well in line with those from the analysis of GR mRNA expression in magnetically sorted immune cell subpopulations but they could be obtained much more quickly. In summary, our data indicate that flow cytometry is a powerful tool with which to define GR protein content at a single cell level when studying the function of GCs in the immune system.

Eupafolin induces autophagy and apoptosis in B-cell non-Hodgkin lymphomas.

Eupafolin, an extract from Artemisia princeps, possesses multiple pharmacological activities. However, the effect of eupafolin on B-cell non-Hodgkin lymphomas is currently unknown. In this study, we report that eupafolin shows anticancer activity against B-cell non-Hodgkin lymphomas cell line, OCI-LY-3. A CCK-8 assay was used to detect the proliferation inhibition of OCI-LY-3 cells treated with additional concentrations of eupafolin. Flow cytometric analysis method of the cell apoptosis was detected after cells stained with Annexin-V-FITC/PI according to the manufacturer's instructions. The proteins in the cell were detected by western blot after treatment with eupafolin. Eupafolin induced apoptosis in this cell line evidenced by the caspases activation, cleavage of PARP and downregulation of Bcl-2 and Bcl-xl. Eupafolin-induced autophagy was verified by accumulation of LC3-II and beclin-1. Eupafolin induced autophagy promoting apoptosis by the treatment of eupafolin combined with autophagy inhibitors 3-methyladenine and bafilomycin A1, respectively. Moreover, we disclose that the expression levels of p-Akt, p-mTOR,p-P70S6K and p-4EBP1 decrease in the Akt/mTOR signalling pathway, and the expression levels of proteins in the NF-ΚB signalling pathway, such as p-p65, p-IκBα, is downregulation. Together, these results provide crucial evidences explaining the antitumour activity of eupafolin in human NHL cell line, OCI-LY-3.

COMPARISON BETWEEN FLOW CYTOMETERIC METHOD AND OTHER METHODS FORRETICULOCYTE COUNT IN DIAGNOSIS OF PHENYLHYDRAZINE-INDUCED HEMOLYTIC ANEMIA

Reticulocyte count is the salient evidence of the effectiveness of bone marrow to produce red blood cells. Currently, the reticulocyte counting is a challenge for clinical laboratoriesmainly for the ordinary ones, which still use the manual method.This study was designed to evaluate the performance of flow cytometer for reticulocytes counting comparing to traditional and optimized manual methods which helpful in diagnosis of phenylhydazine-induced anemia.For that 45 male white Albino rats were divided into 5 groups, control group,phenylhydrazine group (PHZ) which injected by phz(20 mg/kg b.w, I/P),quercetin+phz group (quercetin, 50 mg/kgb.w per os), silymarin+phz group (silymarin, 100 mg/kgb.w per os) and quercetin group. Whole blood samples of these groups were collected at day 3, 5 and 10 after 1st injection of phz which used for reticulocyte counts by flow cytometeric method and other manual methods in addition to measurement ofCBC and osmotic fragility. Analysis of the results showed that phenylhydrazine injection induced hemolytic anemia with significant reticulocytosis and using of flow cytometer in reticulocyte count more precise, easy and fast than traditional and optimized manual methods. Furthermore, degree of hemolysis was significantly increases in phz group comparing to other groups. Therefore, we concluded that flow cytometric method for reticulocyte counts was simple, fast and highly reliable comparable to traditional and optimized manual methods. Also optimized manual showed that more perfect than traditional manual method and nearly to accuracy of flow cytometeric method.

Association of different Candida species with catheter-related candidemia, and the potential antifungal treatments against their adhesion properties and biofilm-forming capabilities.

BackgroundTo compare the adhesion properties and biofilm‐forming capabilities of 27 Candida isolates obtained from catheter‐related candidemia patients and to evaluate the inhibitory effects of antifungal agents on different Candida species.Material and MethodsSeven C. albicans, six C. parapsilosis, five C. guilliermondii, five C. tropicalis, and four C. glabrata clinical isolates were investigated. We quantified the adherence of these Candida species by flow cytometric method and evaluated the formation of biofilms by XTT reduction and crystal violet methods. Actions of micafungin (MF), fluconazole (FZ), and N‐acetylcysteine (NAC) on the adhesion and biofilm formation of different Candida species were determined.ResultsNon‐albicans Candida species were demonstrated to have stronger adhesion abilities compared with C. albicans. The biofilm‐forming capabilities of different Candida species were varied considerably, and the degree of biofilm formation might be affected by different assay approaches. Interestingly, C. parapsilosis displayed the highest biofilm formation abilities, while C. glabrata exhibited the lowest total biomass and metabolic activity. Furthermore, the inhibitory activities of MF, FZ, and NAC on fungal adhesion and biofilm formation were evaluated, and the results indicated that MF could reduce the adhesion ability and biofilm metabolism more significantly (p < 0.05), and its antifungal activity was elevated in a dose‐dependent manner.ConclusionNon‐albicans Candida species, especially C. guilliermondii, C. tropicalis, and C. parapsilosis, exhibited higher adhesion ability in catheter‐related candidemia patients. However, these Candida species had varied biofilm‐forming capabilities. MF tended to have stronger inhibitory effects against both adhesion and biofilm formation of different Candida species.

A Novel Branched DNA-Based Flowcytometric Method for Single-Cell Characterization of Gene Therapy Products and Expression of Therapeutic Genes.

Many preclinical and clinical studies of hematopoietic stem cell-based gene therapy (GT) are based on the use of lentiviruses as the vector of choice. Assessment of the vector titer and transduction efficiency of the cell product is critical for these studies. Efficacy and safety of the modified cell product are commonly determined by assessing the vector copy number (VCN) using qPCR. However, this optimized and well-established method in the GT field is based on bulk population averages, which can lead to misinterpretation of the actual VCN per transduced cell. Therefore, we introduce here a single cell-based method that allows to unmask cellular heterogeneity in the GT product, even when antibodies are not available. We use Invitrogen’s flow cytometry-based PrimeFlow™ RNA Assay with customized probes to determine transduction efficiency of transgenes of interest, promoter strength, and the cellular heterogeneity of murine and human stem cells. The assay has good specificity and sensitivity to detect the transgenes, as shown by the high correlations between PrimeFlow™-positive cells and the VCN. Differences in promoter strengths can readily be detected by differences in percentages and fluorescence intensity. Hence, we show a customizable method that allows to determine the number of transduced cells and the actual VCN per transduced cell in a GT product. The assay is suitable for all therapeutic genes for which antibodies are not available or too cumbersome for routine flow cytometry. The method also allows co-staining of surface markers to analyze differential transduction efficiencies in subpopulations of target cells.

In-Depth Immune-Oncology Studies of the Tumor Microenvironment in a Humanized Melanoma Mouse Model.

The presence and interaction of immune cells in the tumor microenvironment is of significant importance and has a great impact on disease progression and response to therapy. Hence, their identification is of high interest for prognosis and treatment decisions. Besides detailed phenotypic analyses of immune, as well as tumor cells, spatial analyses is an important parameter in the complex interplay of neoplastic and immune cells—especially when moving into focus efforts to develop and validate new therapeutic strategies. Ex vivo analysis of tumor samples by immunohistochemistry staining methods conserves spatial information is restricted to single markers, while flow cytometry (disrupting tissue into single cell suspensions) provides access to markers in larger numbers. Nevertheless, this comes at the cost of scarifying morphological information regarding tissue localization and cell–cell contacts. Further detrimental effects incurred by, for example, tissue digestion include staining artifacts. Consequently, ongoing efforts are directed towards methods that preserve, completely or in part, spatial information, while increasing the number of markers that can potentially be interrogated to the level of conventional flow cytometric methods. Progression in multiplex immunohistochemistry in the last ten years overcame the limitation to 1–2 markers in classical staining methods using DAB with counter stains or even pure chemical staining methods. In this study, we compared the multiplex method Chipcytometry to flow cytometry and classical IHC-P using DAB and hematoxylin. Chipcytometry uses frozen or paraffin-embedded tissue sections stained with readily available commercial fluorophore-labeled antibodies in repetitive cycles of staining and bleaching. The iterative staining approach enables sequential analysis of a virtually unlimited number of markers on the same sample, thereby identifying immune cell subpopulations in the tumor microenvironment in the present study in a humanized mouse melanoma model.

Diverse Effect of Vitamin C and N-Acetylcysteine on Aluminum-Induced Eryptosis.

Purpose The role of oxidative stress in Aluminum (Al)-induced apoptotic effects has been investigated and suicidal death of erythrocytes, eryptosis, is characterized by cell shrinkage and phosphatidylserine externalization (PSE) at the surface of the erythrocyte cell membrane. Eryptosis is stimulated by an increase in cytosolic Ca2+ concentration and reactive oxygen species (ROS). This ex vivo study was conducted to evaluate the effect of well-known antioxidants including vitamin C (vit C) and N-acetylcysteine (NAC), against Al-induced hemolysis and eryptosis. Methods Isolated erythrocytes from the healthy volunteers were partitioned into various groups (6 replicates/group) and treated by various concentrations of Al (3–100 µM) in the presence and absence of vit C (0.6 mM) and NAC (1 mM). After 24 hours of treatment, hemolysis was determined from hemoglobin levels in the supernatant. Flowcytometric methods were applied to measure PSE, cell shrinkage, Ca2+ content, and ROS abundance using annexin V-binding, forward scatter, Fluo3-fluorescence, and DCFDA dependent fluorescence, respectively. Reduced glutathione (GSH) was measured by the ELISA method. Results The results showed that a 24 hours' exposure of the erythrocytes to Al (10–100 µM) significantly increased hemolysis in a dose and Ca2+dependent manner. Al also dramatically decreased forward scatter. The percentage of PSE cells, Fluo3-fluorescence, and DCFDA fluorescence were increased by Al. Furthermore, cotreatment with NAC inhibited the effect of Al on hemolysis, eryptosis, and ROS production. Vit C decreased Al-induced ROS production. However, increased Al-induced eryptosis. There were no significant changes in glutathione after the ALCL3 treatment. Conclusions Al-induced eryptosis and hemolysis through triggering oxidative stress, while NAC could diverse this effect. In contrast, vit C might intensify Al-induced eryptosis at particular doses through a less known mechanism.

Molecular methods for measurable residual disease in acute myeloid leukemia: where are we and where are we going?

Measurable residual disease (MRD) testing has become a standard practice for patients with acute myeloid leukemia (AML) following therapy. However, MRD testing in AML is not straightforward, and both molecular methods and multiparameter flow cytometric (MFC) methods demonstrate clinical utility. While MFC methods are potentially applicable to all AML patients, current molecular MRD methods must be individually tailored to the patient’s AML disease genetics, a strategy that is currently applied for patients with acute promyelocytic leukemia, core binding factor AML, and AML with mutated NPM1. However, there is great interest in next-generation sequencing (NGS) methods for MRD assessment, an approach that could potentially be applied to all patients with AML. Current NGS methods have limited analytic sensitivity when compared with other molecular methods and MFC, but advances in NGS methods and informatic algorithms may improve NGS MRD testing in the near future. In this review, we discuss current recommendations for molecular MRD assessment in AML and discuss opportunities and challenges for MRD assessment by NGS methods.

A DNA/Ki67-Based Flow Cytometry Assay for Cell Cycle Analysis of Antigen-Specific CD8 T Cells in Vaccinated Mice.

The cell cycle of antigen-specific T cells in vivo has been examined by using a few methods, all of which possess some limitations. Bromodeoxyuridine (BrdU) marks cells that are in or recently completed S-phase, and carboxyfluorescein succinimidyl ester (CFSE) detects daughter cells after division. However, these dyes do not allow identification of the cell cycle phase at the time of analysis. An alternative approach is to exploit Ki67, a marker that is highly expressed by cells in all phases of the cell cycle except the quiescent phase G0. Unfortunately, Ki67 does not allow further differentiation as it does not separate cells in S-phase that are committed to mitosis from those in G1 that can remain in this phase, proceed into cycling, or move into G0. Here, we describe a flow cytometric method for capturing a "snapshot" of T cells in different cell cycle phases in mouse secondary lymphoid organs. The method combines Ki67 and DNA staining with major histocompatibility complex (MHC)-peptide-multimer staining and an innovative gating strategy, allowing us to successfully differentiate between antigen-specific CD8 T cells in G0, in G1 and in S-G2/M phases of the cell cycle in the spleenand draining lymph nodes of mice after vaccination with viral vectors carrying the model antigen gag of human immunodeficiency virus (HIV)-1. Critical steps of the method were the choice of the DNA dye and the gating strategy to increase the assay sensitivity and to include highly activated/proliferating antigen-specific T cells that would have been missed by current criteria of analysis. The DNA dye, Hoechst 33342, enabled us to obtain a high-quality discriminationof the G0/G1 and G2/M DNA peaks, while preserving membrane and intracellular staining. The method has great potential to increase knowledge about T cell response in vivo and to improve immuno-monitoring analysis.

A DNA/Ki67-Based Flow Cytometry Assay for Cell Cycle Analysis of Antigen-Specific CD8 T Cells in Vaccinated Mice

The cell cycle of antigen-specific T cells in vivo has been examined by using a few methods, all of which possess some limitations. Bromodeoxyuridine (BrdU) marks cells that are in or recently completed S-phase, and carboxyfluorescein succinimidyl ester (CFSE) detects daughter cells after division. However, these dyes do not allow identification of the cell cycle phase at the time of analysis. An alternative approach is to exploit Ki67, a marker that is highly expressed by cells in all phases of the cell cycle except the quiescent phase G0. Unfortunately, Ki67 does not allow further differentiation as it does not separate cells in S-phase that are committed to mitosis from those in G1 that can remain in this phase, proceed into cycling, or move into G0. Here, we describe a flow cytometric method for capturing a "snapshot" of T cells in different cell cycle phases in mouse secondary lymphoid organs. The method combines Ki67 and DNA staining with major histocompatibility complex (MHC)-peptide-multimer staining and an innovative gating strategy, allowing us to successfully differentiate between antigen-specific CD8 T cells in G0, in G1 and in S-G2/M phases of the cell cycle in the spleen and draining lymph nodes of mice after vaccination with viral vectors carrying the model antigen gag of human immunodeficiency virus (HIV)-1. Critical steps of the method were the choice of the DNA dye and the gating strategy to increase the assay sensitivity and to include highly activated/proliferating antigen-specific T cells that would have been missed by current criteria of analysis. The DNA dye, Hoechst 33342, enabled us to obtain a high-quality discrimination of the G0/G1 and G2/M DNA peaks, while preserving membrane and intracellular staining. The method has great potential to increase knowledge about T cell response in vivo and to improve immuno-monitoring analysis.

Elucidation of underlying molecular mechanism of 5-Fluorouracil chemoresistance and its restoration using fish oil in experimental colon carcinoma.

Latest strategies for cancer treatment primarily focus on the use of chemosensitizers to enhance therapeutic outcome. N-3 PUFAs have emerged as the strongest candidate for the prevention of colorectal cancer (CRC). Our previous studies have demonstrated that fish oil (FO) rich in n-3 PUFAs not only increased therapeutic potential of 5-Fluorouracil(5-FU) in colon cancer but also ameliorated its toxicity. Henceforth, the present study is designed to elucidate mechanistic insights of FO as a chemosensitizer to circumvent drug resistance in experimental colon carcinoma. The colon cancer was induced by 1,2-dimethylhydrazine(DMH)/dextran sulfate sodium(DSS) in male Balb/c mice and these animals were treated with 5-FU(12.5mg/kg b.w.), FO(0.2ml), or 5-FU + FO(12.5mg/kg b.w + 0.2ml) orally for 14days. The molecular mechanism of overcoming 5-FU resistance using FO in colon cancer was delineated by estimating expression of cancer stem cell markers using flowcytometric method and drug transporters by immunohistochemistry and immunoblotting. Additionally, distribution profile of 5-FU and its cytotoxic metabolite, 5-FdUMP at target(colon), and non-target sites (serum, kidney, liver, spleen) was assessed using high-performance liquid chromatography(HPLC) method. The observations revealed that expression of CSCs markers was remarkably reduced after using fish oil along with 5-FU in carcinogen-treated animals. Interestingly, the use of FO alongwith 5-FU also significantly declined the expression of drug transporters (ABCB1,ABCC5) and consequently resulted in an increased cellular uptake of 5-FU and its metabolite, 5-FdUMP at target site (colon). It could be possibly associated with change in permeability of cell membrane owing to the alteration in membrane fluidity. The present study revealed the mechanistic insights of FO as a MDR revertant which successfully restored 5-FU-mediated chemoresistance in experimental colon carcinoma.

Protective role of melatonin against testicular damage caused by polymicrobial sepsis in adult rats.

This study aimed to investigate the possible protective effects of melatonin (MEL) against the damage to testicular tissue in rats caused by polymicrobial sepsis as a result of cecal ligation and perforation (CLP). In this study, 21 male Wistar albino rats were used. The rats were randomly divided into three groups (n=7): Sham Control (Group 1), CLP (Group 2), and CLP + MEL (Group 3). Sepsis was created using the CLP method. MEL was administered intraperitoneally in two equal doses of 10 mg/kg at 30 min before and 6 h after perforation. Tissue sections taken from paraffin blocks were stained with hematoxylin and eosin (H and E) and examined histopathologically under a light microscope. Intracellular H2O2 and apoptosis evaluations were carried out using the flow cytometric method. Sepsis caused a significant reduction in all sperm parameters. There was a significant decrease in sperm density, motility and cell numbers with normal morphology (p<0.05). Intracellular H2O2 level and apoptotic cell percentages increased in sperm cells in the CLP group. MEL treatment was found to significantly reduce sperm abnormalities, testicular damage, intracellular H2O2 levels, and apoptosis. This study showed that melatonin administration could be a potential treatment option to reduce acute testicular tissue damage due to sepsis.

Interleukin 8 Elicits Rapid Physiological Changes in Neutrophils That Are Altered by Inflammatory Conditions

A sufficient response of neutrophil granulocytes stimulated by interleukin (IL)-8 is vital during systemic inflammation, for example, in sepsis or severe trauma. Moreover, IL-8 is clinically used as biomarker of inflammatory processes. However, the effects of IL-8 on cellular key regulators of neutrophil properties such as the intracellular pH (pH_i) in dependence of ion transport proteins and during inflammation remain to be elucidated. Therefore, we investigated in detail the fundamental changes in pH_i, cellular shape, and chemotactic activity elicited by IL-8. Using flow cytometric methods, we determined that the IL-8-induced cellular activity was largely dependent on specific ion channels and transporters, such as the sodium-proton exchanger 1 (NHE1) and non-NHE1-dependent sodium flux. Exposing neutrophils in vitro to a proinflammatory micromilieu with N-formyl-Met-Leu-Phe, LPS, or IL-8 resulted in a diminished response regarding the increase in cellular size and pH. The detailed kinetics of the reduced reactivity of the neutrophil granulocytes could be illustrated in a near-real-time flow cytometric measurement. Last, the LPS-mediated impairment of the IL-8-induced response in neutrophils was confirmed in a translational, animal-free human whole blood model. Overall, we provide novel mechanistic insights for the interaction of IL-8 with neutrophil granulocytes and report in detail about its alteration during systemic inflammation.

Sex selection techniques and their implications

Abstract Sex selection techniques provide economic benefits to dairy and beef herd management. Therefore, the development of such techniques has attracted the attention of reproductive biologists. There have been numerous studies concerning the development of sex selection techniques. As the sex of the offspring is determined by certain chromosomes, namely the X and Y chromosomes in mammals, most studies have focused on sperm sexing, attempting to separate the X and Y chromosome-bearing sperm based on differences in deoxyribonucleic acid (DNA) content, head size/volume, motility, and immunological specificity. However, most of these methods have failed to show reproducibility. Only the flow cytometric method has been confirmed to be accurate and reliable thus far. More than three decades have passed since this technique was first developed. The sexed semen produced with the method is currently commercialized and widely used for artificial insemination (AI) in cattle around the world. Recently, however, another technique based on differences in DNA contents using a fluidics device was developed by a commercial company. Studies focused on immunological approaches and the modification of sperm motility have also described the successful separation of the two types of sperm. Therefore, the present review evaluated the available sex selection techniques and their implications.