Flow Cytometric Results Research Articles

Urinary CD8+HLA-DR+ T Cell Abundance Non-invasively Predicts Kidney Transplant Rejection.

Early detection of kidney transplant (KT) rejection remains a challenge in patient care. Non-invasive biomarkers hold high potential to detect rejection, adjust immunosuppression, and monitor KT patients. So far, no approach has fully satisfied requirements to innovate routine monitoring of KT patients. In this two-center study we analyzed a total of 380 urine samples. T cells and tubular epithelial cells were quantified in KT patients with graft deterioration using flow cytometry. Epigenetic urine cell quantification was used to confirm flow cytometric results. Moreover, a cohort of KT patients was followed up during the first year after transplantation, tracking cell subsets over time. Abundance of urinary cell counts differed in patients with and without rejection. Most strikingly, various T cell subsets were enriched in patients with T cell-mediated rejection (TCMR) compared to patients without TCMR. Among T cell subsets, CD8+HLA-DR+ T cells were most distinctive (AUC = 0.91, Spec.: 95.9%, Sens.: 76.5%). Epigenetic analysis confirmed T cell and tubular epithelial cell quantities as determined by flow cytometry. Urinary T cell abundance in new KT patients decreased during their first year after transplantation. In conclusion urinary T cells reflect intrarenal inflammation in TCMR. T cell subsets yield high potential to monitor KT patients and detect rejection. Hereby we present a promising biomarker to non-invasively diagnose TCMR.

Enhanced morphologic traits and medicinal constituents of octaploids in Satureja mutica, a high-yielding medicinal savory

Satureja mutica is a tetraploid and perennial semi-bushy plant cultivated for different medicinal purposes. To induce polyploidy, two-leafed seedlings were exposed to different concentrations (0.00, 0.05, 0.1, and 0.2 % w/v) and durations (6, 12, and 24 h) of colchicine. The seedlings were then transferred to a culture medium for recovery and propagation. After clones were prepared from each seedling, octaploid clones were identified using flow cytometry. Chromosome counting was also used to confirm flow cytometric results in tetraploid (2n = 4x = 60; 2C DNA= 1.90 ± 0.01 pg) and octaploid (2n = 8x = 120; 2C DNA= 3.82 ± 02 pg) plants. The highest polyploidy induction efficiency with 32% was related to 0.05 % colchicine and 12 h duration. The results showed that the phenotypic traits of anatomical (stomata size, leaf guard cell size), morphological (stem diameter, length, width, and leaf area), physiological (soluble sugars, phenols, and flavonoids), and phytochemical (essential oil yield, P-cymene, γ-Terpinene, α-Thujene, and α-Pinene) properties significantly increased in octaploid plants, while the density of leaf stomata decreased compared to tetraploid plants. Our results verified that octaploid induction in Satureja mutica is an effective breeding method, remarkably increasing the quantitative and qualitative characteristics, which could be used as a new genetic resource in future breeding programs.

Gold nanoparticles induce apoptosis in HCT-116 colon cancer cell line.

This study aimed to investigate the apoptotic and anti-cancer effect of gold nanoparticles (AuNPs) on apoptosis in HCT-116 colon cancer cells. The level of ROS and apoptosis were determined by fluorimetric method and flow cytometry and Hoechst 33,258 staining, respectively. Furthermore, the mRNA expression of Bax, Bcl-2, CCNB1, P53 genes was evaluated by qRT-PCR method in HCT116 cells. The experimental results of this study showed that treatment with nanoparticles led to a significant increase in expression of Bax, P53 genes and a significant decrease in the expression of Bcl-2, CCNB1 genes at concentrations of 25 and 50µg/ml during 48h of incubation, compared to control cells (p < 0.05). The flow cytometric results (Annexin-pI) and Hoechst 33,258 staining also showed a significant increase in the level of apoptosis in the treated cells, depending on the concentration and time. The results of this study showed that AuNPs cause apoptosis at the half-maximal inhibitory concentration in the HCT-116 tumor cells during 48h of incubation.

Combined therapy with dapagliflozin and entresto offers an additional benefit on improving the heart function in rat after ischemia-reperfusion injury

BackgroundThis study tested whether combined dapagliflozin and entresto treatment would be superior to either one alone for preserving the left-ventricular ejection-fraction (LVEF) in rat after ischemia-reperfusion (IR) injury. MethodsCell culture using H9C2 cells and IR injury in rat with dapagliflozin-entresto treatment were conducted in the present study. ResultsIn vitro flow-cytometric result showed that the intracellular and mitochondrial reactive oxygen species and mitochondrial permeability transition pore, and protein levels of oxidative-stress/DNA-damaged markers [NADPH-oxidase-1 (NOX-1)/NOX-2/oxidized-protein/γ-H2A-histone-family member X (γ-H2AX)] were significantly higher in hydrogen peroxide (H2O2) (300μM)-treated H9C2 cells as compared with the controls that were significantly reversed in sacubitril/valsartan and dapagliflozin therapy in the same H2O2-treated condition, whereas the protein expressions of antioxidants [Sirtuin-1 (SIRT1)/SIRT3/superoxide dismutase/catalase/glutathione peroxidase) exhibited an opposite pattern among the groups (all p<0.001). Adult-male-Sprague-Dawley rat (n=40) were equally categorized into group 1 (sham-operated control), group 2 (IR), group 3 (IR+dapagliflozin/20mg/kg/orally at 3h and post-days 1/2/3 after IR), group 4 (IR+entresto/100mg/kg/orally at 3h and post-days 1/2/3 after IR) and group 5 (IR+dapagliflozin+entresto) and the hearts were harvested by day 3 after IR. The 3rd day’s LVEF was highest in group 1, lowest in group 2 and significantly higher in group 5 than in groups 3/4, but it was similar between the latter two groups (p<0.001). The protein expressions of oxidative-stress (NOX-1/NOX-2/oxidized protein), fibrotic (transforming-growth factor-ß/phosphorylated-Smad3), apoptotic [mitochondrial-Bax/cleaved-caspase-3/cleaved-poly (ADP-ribose) polymerase], mitochondria/DNA damaged (cytosolic-cytochrome-c/γ-H2AX), pressure-overload/heart-failure [brain natriuretic peptide (BNP)/ß-myosin heavy chain] and autophagic (ratio of meiotic cyclins CLB3-II/CLB3-I) biomarkers, and the upstream (high-mobility group box 1/Toll-like receptor-4/MyD88/phosphorylated-nuclear factor-κB and downstream [interleukin (IL)-1ß/IL-6/tumor necrosis factor-α] inflammatory signalings revealed an antithetical features of LVEF among the groups (all p<0.0001). The cellular levels of inflammatory (myeloperoxidase+/CD68+), pressure-overload/heart-failure (BNP+) and DNA-damage (γ-H2AX+) biomarkers as well as infarct area demonstrated an opposite pattern of LVEF among the groups (all p<0.0001). ConclusionIncorporated entresto-dapagliflozin treatment was superior to either one alone on protecting the heart against IR injury.

Design, synthesis and anticancer activity studies of novel 4-butylaminophenyl hydrazide-hydrazones as apoptotic inducers

In this study, a series of the novel Tetracaine derivatives bearing hydrazide-hydrazone moiety were designed, synthesized, and evaluated for anticancer activity. The structures of these compounds were characterized by spectral (1H–13C NMR, FT-IR, and HR-MS analyses) methods. All synthesized compounds (2a-l) were screened for anticancer activity against human hepatocellular carcinoma (HepG2) and lung carcinoma (A549) cell lines. Against HepG2 and A549 cell lines, among the synthesized compounds, 4-(Butylamino)-N'-[(2,4-dichlorophenyl)methylidene]benzohydrazide (2i) demonstrated the most potent anticancer activity with IC50 values 28 and 7 μM, respectively. Possible cytotoxic effects of compounds (2a-l) on both normal human lung fibroblast (MCR-5) and normal human dermal fibroblast (HDF) cell lines were assessed. Inhibition of anti-apoptotic protein Bax and Bcl-2 was investigated in HepG2 and A549 cells treated with compound 2i using qRT-PCR. Apoptosis was also detected by Annexin V studies. The flow cytometric analysis results showed that compound 2i treatment of HepG2 and A-549 cells significantly increased apoptotic cell populations while decreasing viabilities in these carcinomas in a dose-dependent manner after 72 h of incubation.

A Single-Domain TCR-like Antibody Selective for the Qa-1b/Qdm Peptide Complex Enhances Tumoricidal Activity of NK Cells via Blocking the NKG2A Immune Checkpoint

The NKG2A/HLA-E axis is an immune checkpoint that suppresses immune effector activity in the tumor microenvironment. In mice, the ligand for the NKG2A/CD94 inhibitory receptor is the nonclassical MHC molecule Qa-1b, the HLA-E ortholog, which presents the peptide AMAPRTLLL, referred to as Qdm (for Qa-1 determinant modifier). This dominant peptide is derived from the leader sequences of murine classical MHC class I encoded by the H-2D and -L loci. To broaden our understanding of Qa-1b/Qdm peptide complex biology and its tumor protective role, we identified a TCR-like Ab from a single domain VHH library using yeast surface display. The TCR-like Ab (EXX-1) binds only to the Qa-1b/Qdm peptide complex and not to Qa-1b alone or Qa-1b loaded with control peptides. Conversely, currently available Abs to Qa-1b bind independent of peptide loaded. Flow cytometric results revealed that EXX-1 selectively bound to Qa-1b/Qdm-positive B16F10, RMA, and TC-1 mouse tumor cells but only after pretreatment with IFN-γ; no binding was observed following genetic knockdown of Qa-1b or Qdm peptide. Furthermore, EXX-1 Ab blockade promoted NK cell-mediated tumor cell lysis in vitro. Our findings show that EXX-1 has exquisite binding specificity for the Qa-1b/Qdm peptide complex, making it a valuable research tool for further investigation of the Qa-1b/Qdm peptide complex expression and regulation in healthy and diseased cells and for evaluation as an immune checkpoint blocking Ab in syngeneic mouse tumor models.

In vitro biological and in silico molecular docking and ADME studies of a substituted triazine-coordinated cadmium(II) ion: efficient cytotoxicity, apoptosis, genotoxicity, and nuclease-like activity plus binding affinity towards apoptosis-related proteins.

A cadmium(II) complex containing dppt ligand with the formula [CdCl2(dppt)2], where dppt is 5,6-diphenyl-3-(2-pyridyl)-1,2,4-triazine was synthesized, elucidated and submitted to in vitro cytotoxicity studies against human breast (MCF-7), glioblastoma (U-87), and lung (A549) cancer cell lines as well as mouse embryo normal cell line (NIH/3T3), in comparison with cisplatin employing MTT assay over 24 and 48h. The complex exhibited the highest cytotoxic effect against MCF-7 cells among the other three cell lines with IC50 values of 8.7 ± 0.5 (24h) and 1.2 ± 0.7µM (48h). Significantly, flow cytometric assessment of the complex-treated MCF-7 and U-87 cells demonstrated a dose-dependent induced apoptotic cell death. The cellular morphological changes were in concord with cytotoxicity and flow cytometric results. The results of comet assay showed that the complex is able to induce DNA damage in MCF-7 cells. These observations are of importance, as sustained damage to cellular DNA could lead to apoptotic cell death. The results of DNA-binding studies indicated that the complex fits into the DNA minor groove and interacts with DNA via a partial intercalation. Moreover, the complex was able to efficiently cleave pUC19 DNA through a hydrolytic mechanism. The binding affinity between the complex and apoptosis-relevant protein targets including APAF1, Bax, Bcl-2, Cas3, Cas7, and Cas9 was evaluated through molecular docking studies. In silico virtual studies revealed the complex's strong affinity towards apoptosis-related proteins; therefore the complex can act as a potential apoptosis inducer. Physicochemical, pharmacokinetics, lipophilicity, drug-likeness, and medicinal chemistry properties of the complex were also predicted through in silico absorption, distribution, metabolism and excretion studies.

Methyl gallate isolated from Mangifera pajang kernel induces proliferation inhibition and apoptosis in MCF-7 breast cancer cells via oxidative stress

Objective: To determine the lead bioactive compound in kernel extract of Mangifera pajang and its anti-cancer activity against human breast cancer cell lines with positive estrogen receptor (MCF-7). Methods: The methanolic extract of dried powder kernel of Mangifera pajang was exposed to column chromatography for isolation. The structural elucidation of the isolated compound was characterized using infrared, nuclear magnetic resonance, mass spectrometry. Furthermore, cytotoxicity, morphological changes, flow cytometry and cell cycle arrest analyses were performed to examine the mechanism of anti-proliferation and apoptosis induced by methyl gallate against MCF-7. Results: One compound was isolated from the methanolic extract of Mangifera pajang kernel and identified as methyl gallate. The flow cytometric results demonstrated induction of apoptosis in MCF-7 cells by three concentrations of methyl gallate. The cell cycle arrest showed a significant (P&lt;0.05) decrease in cell progression at G2/M phase of MCF-7 after treatment with 100 μM of methyl gallate. The cell percentage of early and late apoptosis was significant at 10 and 100 μM of methyl gallate. Also, methyl gallate treatment induced up-regulation of reactive oxygen species levels in MCF-7 cells with a reduction in superoxide dismutase levels. Conclusions: These findings indicate that isolated methyl gallate from Mangifera pajang kernel extracts induces growth inhibition and apoptosis in MCF-7 cells via up-regulating oxidative stress pathway.

The Establishment and Application of Flow Cytometry on the Detection of Jka Antigen in Kidd Blood Group

To establish a based method flow cytometry to identify the antigen Jka in human red blood cells (RBCs) and verify its accuracy. A total of 96 blood samples were enrolled in the study randomly from the voluntary blood donors in Shenzhen Blood Center. The RBCs were incubated with IgG anti-Jka primary antibody, and then labeled with the secondary antibody anti-IgG-Alexa Fluor 647. The fluorescence histograms of each sample were obtained by flow cytometry. Serological agglutination test was used to compare the accuracy of flow cytometry in the detecting of antigen Jka, while PCR-SSP and gene sequencing genotyping were used to verify the accuracy of flow cytometry in the detecting of the antigen in human RBCs. The results of flow cytometry for antigen Jka in human RBCs were consistent with those from serological tests. Samples that demonstrated higher serological agglutination intensity also showed higher fluorescence activity, which indicate more stronger of Jka antigen. The sensitivity of flow cytometry was higher than that of serological test; especially in distinguish Jka weak and negative samples. Flow cytometric results of all samples were consistent with the genotyping results, which confirmed the accuracy of flow cytometry. The study established a new flow cytometry-based method successfully for the identification of Jka antigen of Kidd blood group in human RBCs. The Kidd blood group antigen Jka of different intensities can be accurately distinguished by the technique.

A Diagnostic Pitfall in the Evaluation of CD138-Positive Plasmacytoid Cells in a Fine Needle Aspiration Biopsy

Abstract Introduction/Objective The identification of plasmacytoid cells on rapid assessment leads to a wide range of differential diagnoses. Methods/Case Report We present a 51-year-old female with a parotid mass and cervical lymphadenopathy. The fine needle aspiration biopsy of the lymph node showed clusters of plasmacytoid cells. Flow cytometric studies showed cells positive for CD19, CD45, CD138 and kappa, but negative for CD38 and lambda. The kappa to lambda ratio was greater than 100. Histological sections showed plasmacytoid cells that were positive for AE1/AE3, S100, and SOX10, weakly positive for CK7 and GATA3, but negative for CD79a, CD45, EMA, p40, p63, and pan-melanoma markers. Kappa and lambda had a 1:1 ratio. Due to the discrepancy between the flow cytometric results and the immunohistochemistry, excision was recommended. The subsequent excision specimen showed classic morphology for myoepithelial carcinoma, plasmacytoid variant. Results (if a Case Study enter NA) NA Conclusion Myoepithelial carcinomas of the salivary glands are rare neoplasms, and they have various morphologies. The plasmacytoid variant can pose as a diagnostic pitfall. In fine needle aspiration biopsies, carcinoma cells with plasmacytoid morphology can be confused with a plasma cell neoplasm. CD138 is nonspecific marker, and it can be expressed in plasma cells as well as carcinomas. To further complicate matters, pancytokeratin and SOX10 can aberrantly expressed in some plasma cell myelomas. Flow cytometry studies is a valuable tool to define various cell types. However, it can be misleading if it is used as a sole source for interpreting plasmacytoid cells. Epithelial and myoepithelial neoplasms should be considered as differential diagnoses when CD138-positive plasmacytoid cells are encountered. Extended immunohistochemistry panel including multiple epithelial and myoepithelial markers play pivotal role in settling the diagnosis.

The aggregation of Aspergillus spores and the impact on their inactivation by chlorine-based disinfectants

The formation of fungal biofilm goes through some different states, including monodisperse state, aggregated state, germinated state, hyphal and biofilm. The aggregation of spores is a primary step of fungal biofilm development in aquatic systems. Previous studies on the inactivation of fungi were mostly performed in the monodisperse state of fungal spores and biofilm state, however, the inactivation of aggregated fungal spores is still unclear. In this study, the aggregated characteristics of fungal spores (Aspergillus fumigatus and Aspergillus flavus) at different pH values were firstly studied, and the inactivation efficiency of fungal spores at different aggregation degree by chlorine-based disinfectants was also clarified. The results showed that the aggregation degree of Aspergillus fumigatus was the highest at pH 9.0 while it was the lowest at pH 5.0. Aggregation between fungal spores was mainly mediated by occasional adhesin–adhesin interactions and electrostatic interactions. Compared with monodisperse spores, fungal spores were more resistant to chlorine-based disinfectants with the increase of spore aggregation degree. The inactivation rate constants of Aspergillus fumigatus at 30% and 63% aggregation degree were 1.5- and 4-folds lower than that of monodisperse spores, respectively. The lower proportion of membrane damage and higher intracellular reactive oxygen species level for aggregated spores than monodisperse spores was observed according to the flow cytometric results after chlorine-based disinfectants treatment. The reasons for the lower inactivation efficiency of aggregated spores are as following: the protection of outer layer spores and signals between aggregates lead to the increase of resistance for aggregated spores. This study is meaningful for the control of the fungal spores at different states in water.

Anticancer effects of bifidobacteria on colon cancer cell lines

BackgroundColorectal cancer (CRC), with a growing incidence trend worldwide, is resistant to apoptosis and has uncontrolled proliferation. It is recently reported that probiotic microorganisms exert anticancer effects. The genus Bifidobacterium, one of the dominant bacterial populations in the gastrointestinal tract, has received increasing attention because of widespread interest in using it as health-promoting microorganisms. Therefore, the present study aimed to assess the apoptotic effects of some bifidobacteria species on colon cancer cell lines.Methods The cytotoxicity evaluations performed using MTT assay and FACS-flow cytometry tests. Also, the effects of five species of bifidobacteria secretion metabolites on the expression level of anti- or pro-apoptotic genes including BAD, Bcl-2, Caspase-3, Caspase-8, Caspase-9, and Fas-R studied by real-time polymerase chain reaction (RT-PCR) method.ResultsThe cell-free supernatant of all studied bifidobacteria significantly decreased the survival rates of colon cancer cells compared with control groups. Flow cytometric and RT-PCR results indicated that apoptosis is induced by bifidobacteria secretion metabolites and the mechanism for the action of bifidobacteria species in CRC prevention could be down-regulation and up-regulation of anti-apoptotic and, pro-apoptotic genes.ConclusionsIn the present study, different bifidobacteria species showed anticancer activity on colorectal cancer cells through down-regulation and up-regulation of anti-apoptotic and pro-apoptotic genes. However, further studies are required to clarify the exact mechanism of apoptosis induction by bifidobacteria species.

A recombinant fungal defensin-like peptide-P2 combats Streptococcus dysgalactiae and biofilms.

Streptococcus dysgalactiae, considered one of the main pathogens that causes bovine mastitis, is a serious threat to humans and animals. However, the excessive use of antibiotics and the characteristic of S. dysgalactiae forming biofilms in mastitic teat canal have serious clinical implications. In this study, in vivo and in vitro multiple mechanisms of action of P2, a mutant of fungal defensin plectasin, against S. dysgalactiae were systematically and comprehensively investigated for the first time. P2 showed potent antibacterial activity against S. dysgalactiae (minimum inhibitory concentration, MIC = 0.23-0.46 μM) and rapid bactericidal action by 3.0 lg units reduction in 2-4 h. No resistant mutants appeared after 30-d serial passage of S. dysgalactiae in the presence of P2. The results of electron microscopy and flow cytometer showed that P2 induced membrane damage of S. dysgalactiae, causing the leakage of cellular content and eventually cell death. Besides, P2 effectively inhibited early biofilm formation, eradicated mature biofilms, and killed 99.9% persisters which were resistant to 100 × MIC vancomycin; and confocal laser scanning microscopy (CLSM) also revealed the potent antibacterial and antibiofilm activity of P2 (the thickness of biofilm reduced from 18.82 to 7.94 μm). The in vivo therapeutic effect of P2 in mouse mastitis model showed that it decreased the number of mammary bacteria and alleviated breast inflammation by regulating cytokines and inhibiting bacterial proliferation, which were superior to vancomycin. These data indicated that P2 maybe a potential candidate peptide for mastitis treatment of S. dysgalactiae infections. KEY POINTS: •P2 showed potential in vitro antibacterial characteristics towards S. dysgalactiae. •P2 eradicated biofilms, killed persisters, and induced cell death of S. dysgalactiae. •P2 could effectively protect mice from S. dysgalactiae infection in gland.

Triorganotin (IV) carboxylates as potential anticancer agents: Their synthesis, physiochemical characterization, and cytotoxic activity against HeLa and MCF‐7 cancer cells

Three triorganotin (IV) cyclopentane carboxylates were synthesized and structurally characterized by in solid state by Fourier‐transform infrared spectroscopy and single crystal diffraction, and in solution by NMR (1H, 13C, and 119Sn) spectroscopy. The complexes were tested for their anticancer activity against MCF‐7 and HeLa cells along with normal BHK‐21 cells. As revealed by MTT assay, complex 2 was identified as the most potent derivative with an IC50 value of 2.59 and 0.051 μM against HeLa and MCF‐7 cells, respectively. The results were compared with cisplatin as reference drug. Fluorescent microscopic studies using 4′,6‐diamidino‐2‐phenylindole (DAPI) and propidium iodide (PI) staining confirmed the occurrence of apoptosis in HeLa cells treated with the most active complex 2. The complex 2 also triggered the release of lactate dehydrogenase (LDH) in treated HeLa and MCF‐7 cells whereas a luminescence assay displayed a remarkable increase in the activity of caspase‐9 and ‐3. Moreover, flow cytometric results revealed that complex 2 caused G0/G1 arrest in the treated HeLa cells. The complexes were further screened for DNA binding studies through UV‐vis spectroscopy and cyclic voltammetry. The high activity of complex 2 was attributed to its higher Lewis acidity as indicated by natural bond orbital (NBO) analysis. Theoretical modelling and molecular docking studies were also conducted to study the reactivity of complexes against VEGFR 2 Kinase.

Antiproliferative and Pro-Apoptotic Effects of Thiazolo[3,2-b][1,2,4]triazoles in Breast and Cervical Cancer Cells.

Cancer is one of the leading causes of death in the world affecting millions of people. The commercially available anticancer drugs lack the selectivity and show several undue side effects during the biologically targeted therapy, thus calling for the exploration of wider chemical space to furnish new structural leads with promising anticancer potential. In this endeavor, we synthesized a series of coumarinyl thiazolotriazoles with diverse functional group tolerance and will be tested for their anticancer properties against cancer cell lines (HeLa and MCF-7) and a normal cell line (BHK-21). To overcome such complications, in the current study, we evaluated the cytotoxic effects of coumarinyl thiazolotriazole hybrids on human breast adenocarcinoma (MCF-7), cervical adenocarcinoma (HeLa) cells and normal cells i.e., Baby Hamster Kidney cells (BHK-21) using MTT (dimethyl-2-thiazolyl- 2,5-diphenyl-2H-tetrazolium bromide) assay. DNA binding studies of compound 6c was performed on Herring- Sperm DNA (HS-DNA) and docking studies were also carried out. The mechanistic studies were performed on potent compounds by fluorescent microscopic studies, release of Lactate Dehydrogenase (LDH) and mitochondrial membrane potential, activation of caspase-9 and -3 and flow cytometric analysis. As revealed by MTT assay, compounds 6m and 6c were identified as the most potent derivatives among the tested series with IC50 values of 5.64 and 29.1 μM against HeLa and MCF cells, respectively as compared to cisplatin which gave IC50 values of 11.3 and 6.20 μM, respectively. DNA binding studies of compound 6c showed the binding of compound in DNA with Gibbs free energy of ‒17 KJ/mol and docking studies validated the DNA binding studies. Fluorescent microscopic studies using 4',6-diamidino-2-phenylindole (DAPI) and Propidium Iodide (PI) staining confirmed the occurrence of apoptosis in HeLa cells treated with the most active compound 6m. Moreover, compounds 6m and 6c also triggered the release of Lactate Dehydrogenase (LDH) in treated HeLa and MCF-7 cells while a luminescence assay displayed a remarkable increase in the activity of caspase-9 and -3. Moreover, flow cytometric results revealed that compound 6m caused G0 /G1 arrest in the treated HeLa cells. Our results suggested that the compound possesses chemotherapeutic properties against breast cancer and cervical adenocarcinoma cells, thus warranting further research to test the anticancer efficacy of this compound at clinical level.

Synergic effect of combined cyclosporin and melatonin protects the brain against acute ischemic reperfusion injury

BackgroundThis study tested whether combined cyclosporin-A (CsA) and melatonin (Mel) was superior to either one on protecting the brain against ischemia (occluded left-middle-cerebral-artery for 90-min)-reperfusion (for 14 days) injury. Methods and resultsNeuro-2a cells (N2a) were categorized into groups 1 (N2a), 2 (N2a-IR), 3 (N2a-IR-Mel), 4 (N2a-IR-CsA) and 5 (N2a-IR-CsA-Mel). in vitro results showed the protein expressions of cytosolic-cytochrome-C/mitochondrial-Bax/cleaved-capase-3/NOX-1/NOX-2 and flow-cytometric results of ROS (DCFDA/Mito-SOX) were highest in group 2, lowest in group 1, significantly lower in group 5 than in groups 3/4, but they showed no difference in groups 3/4 (all p < 0.001). Male-adult-SD rats (50) were equally categorized into groups 1 (sham-operated-control), 2 (IR), 3 (IR-CsA/20.0 mg/kg at 0.5/24/48 h intraperitoneally after IR), 4 (IR-Mel/50.0 mg/kg intraperitoneally at 30 min and 30 mg/kg at 6/24/48 h after IR) and 5 (IR-CsA-Mel). The brain-infarct-area (BIA) (at day-3 by TTC-stain) was lowest in group 1, highest in group 2, significantly lower in group 5 than groups 3/4, but it showed no difference between groups 3/4 whereas the brain-infarct-volume (at day 14 by MRI) was similar as BIA except for significantly lower in group 4 than in group 3 (all p < 0.0001). By day 14, microscopic finding showed the numbers of glial+/GFAP+/AQP + cells expressed an identical trend whereas the number of NeuN + cells exhibited an opposite pattern of BIA among the groups (all p < 0.0001). The protein expressions of oxidative-stress (NOX-1/NOX-2/p22phox/oxidized-protein), inflammatory (TNF-α/p-NF-κB/MMP-9), apoptotic (mitochondrial-Bax/caspase-3/PARP) and mitochondrial-damaged (Cyclophilin-D/DRP1/cytosolic-cytochrome-C) biomarkers displayed an identical pattern of BIA among the five groups (all p < 0.0001). ConclusionCombined CsA-Mel was superior to either CsA or Mel on protecting the brain against IR injury.

Therapeutic Effect of SPK1 Gene Transfected Adipose Derived Mesenchymal Stem Cells on Experimental Autoimmune Encephalomyelitis Mice and Its Effect on T Helper Cell 17/Regulatory T Cells Balance

Objective To investigate the therapeutic effect of SPK1 gene transfected adipose derived mesenchymal stem cells(ADMSC)on experimental autoimmune encephalomyelitis mice and the effect on T helper cell 17(Th17)/regulatory T(Treg) cells balance. Methods EAE was induced by myelin oligodendrocyte glycoprotein 35-55 in mice.Totally 44 mice were randomly divided into four groups:normal control group(NC group),model group(EAE group),ADMSC group,and ADMSC-SPK1 group.Forty days after injection,the pathological changes of brain and spinal cord,Th17/Treg-related inflammatory markers in brain tissue,expressions of interleukin-17A(IL-17A)and forkhead box protein p3(Foxp3)in brain and spinal cord tissue,and flow cytometric results of spleen immune cells were detected. Results Forty days after the injection,serious inflammatory cell infiltration and demyelination occurred in the brain and spinal cord of EAE group,whereas demyelination and axonal injury were improved in ADMSC group and ADMSC-SPK1 group.Compared with EAE group,the ADMSC group and ADMSC-SPK1 group had significantly improved levels of IL-17A(Z=1.021,P=0.017;Z=1.193, P=0.009)and tumor necrosis factor-α(TNF-α)(Z=2.540,P=0.004; Z=3.005, P=0.006).The expression level of IL-17A in mouse brain and spinal cord tissues was significantly reduced in the ADMSC group(t=10.323,P=0.013;t=7.422,P=0.008),and it was significantly lower in the ADMSC-SPK1 group than in the ADMSC group (t=14.244, P=0.017; t=16.865,P=0.006).The expression level of Foxp3 in the ADMSC group was significantly increased(t=14.544, P=0.008;t=9.420,P=0.002),and it was significantly higher in the ADMSC-SPK1 group than ADMSC group(t=9.654,P=0.005; t=11.535, P=0.009).The proportion of IL-17+IFN-γ+T cells in spleen decreased in ADMSC-SPK1 group,while that of CD25+Foxp3+Treg cells increased. Conclusions ADMSC transfected with SPK1 has an ideal therapeutic effect on EAE mice,and the effect is superior to ADMSC without SPK1 transfection.The mechanism may be that ADMSC-SPK1 can markedly reduce the Th17/Treg cell ratio and decrease the release of related inflammatory cytokines in EAE mice.

Residual red cells in blood components: A multisite study of fully automated enumeration using a hematology analyzer.

BackgroundManufacture of platelet concentrates (PCs) and plasma may fail to remove all residual red blood cells (rRBCs). Measuring rRBCs for compliance to guidelines has proven challenging, leading to an absence of a consensus methodology. Sysmex hematology analyzers with the Blood Bank mode (BB mode) analysis option offer the potential for automated rRBC counting. We therefore performed a two‐site appraisal of the system.Study Design and MethodsPerformance characteristics were determined using platelet and plasma samples spiked with RBCs. Sample stability (n = 47) and the impact of sample type were also assessed. Components (platelets, n = 1474; plasma, n = 77) prepared using different routine manufacturing methods were tested to assess variation in rRBC concentration.ResultsLinearity studies up to 19 000 RBCs/μL demonstrated good correlation between expected and observed results (R2 ≥ 0.9731), and flow cytometric results also correlated well with BB mode (R2 = 0.9400). Precision analysis gave a limit of quantitation of 6 to 7 RBCs/μL, and carryover was 0.03%. Ethylenediaminetetraacetic acid and plain tube results were not significantly different (P ≥ 0.10), and samples were stable up to 24 hours. Apheresis PCs produced at two sites had lower rRBC concentrations (medians, 17 and 13 RBCs/μL) than those produced with the buffy coat method either manually (median, 681 RBCs/μL) or with the automated Terumo Automated Centrifuge and Separator Integration process (median, 81 RBCs/μL). All PCs failing visual inspection as having RBCs ≥4000 RBCs/μL were also detected by the BB mode.ConclusionThe BB mode had acceptable performance characteristics and has the potential for integration into a fully automated process control system for rRBC enumeration in plasma and PCs.

Circumstantial Overdose Management of an Efficient Cancer Cell Photosensitizer with Preclinical Evidence: A Biophysical Study.

In this article, pharmacological management of circumstantial overdose of an anticancer drug, Harmine (HM), under in vitro and in vivo conditions is described and further validated by employing in silico methods. HM, an efficient cancer cell photosensitizer, interacts extensively with nontoxic β-cyclodextrin (β-CD). Steady-state fluorescence studies and molecular docking analysis established differential nature of molecular inclusion depending on the relative concentrations of β-CD. Presently, β-CD is commonly used as a standard drug-delivery vehicle but its application for controlled drug withdrawal is rarely explored. Flow cytometric results and in vivo investigations on a zebrafish model showed that conditional overdose of preadministered drug molecules can be efficiently removed by encapsulating successfully within nontoxic β-CDs, albeit by controlled application of the same. This is an approach to manage the cytotoxicity of a drug in a safe way that is already administered. We believe that this β-CD-mediated withdrawal of drugs may find possible applications in controlled capturing of excess or unused drug inside living systems and reducing the unwanted toxicity associated with chemotherapeutics.

Mechanism by which the combination of SjCL3 and SjGAPDH protects against Schistosoma japonicum infection.

A vaccine is an important method to control schistosomiasis. Molecules related to lung-stage schistosomulum are considered potential vaccine candidates. We previously showed that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and cathepsin L3 (CL3) displayed differential expression in the lung-stage schistosomula of Schistosoma japonicum cocultured with host cells. In the present study, we prepared the two proteins and detected the protective effects of SjGAPDH by immunizing mice with this protein alone and in combination with SjCL3 with or without Freund's adjuvant. Then, we investigated the possible mechanisms underlying S. japonicum infection. The results showed that vaccination of adjuvanted SjGAPDH decreased the worm burden (37.8%) and egg load (38.1%), and the combination of adjuvanted SjGAPDH and SjCL3 further decreased the worm burden (65.6%) and egg load (70.9%) during Schistosoma japonicum infection. However, the immunization of a combination of adjuvant-free SjGAPDH and SjCL3 displayed a lower protective effect (< 15%) than those of the adjuvanted SjCL3, the adjuvanted SjGAPDH, and a combination of adjuvanted SjGAPDH and SjCL3. Flow cytometric results showed that the frequency of regulatory T cells (Tregs) was lower (P < 0.05) in the group with adjuvanted SjGAPDH and SjCL3 (2.61%) than the remaining groups. The enzyme-linked immunosorbent assay (ELISA) results indicated that except for the uninfected and infected control groups, the remaining groups displayed a Th1-type shift in immune responses. These results showed the immunization of SjGAPDH resulted in partial protection (approximately 38%); inoculation with a combination of SjCL3 and SjGAPDH in Freund's adjuvant resulted in a high immunoprotective effect (> 65%) against Schistosoma japonicum infection in mice, which was possibly caused by the reduced percentage of Tregs and a Th1-type shift in immune responses; and SjCL3 has no adjuvant-like effect, dissimilar to SmCL3.