Flow Cytometry Assessment Research Articles

Flow Cytometry Assessment of Microalgae Physiological Alterations under CO2 Injection.

Growth rate (GR), esterase activity (EA), membrane potential (MP), and DNA content were measured by flow cytometry to test if this powerful tool could be included in risk assessment and monitoring programs. This study tests a battery of endpoints that were measured on Scenedesmus (Acutodesmus) obliquus as model species, under high levels of injected CO2 to be proposed as biomarkers of effect. New technologies such as carbon capture and storage (CCS) bring positive consequences on CO2 mitigation strategies but also could have negative consequences if a CO2 leakage occurs during injection. Under this scenario, pH might drop down to 4 pH units around the pipe where the leakage happens. The study focus on to ascertain if the endpoints fulfill the requirements such as sensitiveness, be timely and cost-effective, be easy to measure and interpret, and be nondestructive, valuable attributes in effective biomarkers. CO2 injected at high levels significantly affects the EA, the membrane polarization, as well as GRs. However the DNA content did not shown a clear response under this condition. In conclusion, the GR, the EA, and the MP analyzed by flow cytometry fulfilled the attributes mentioned above and are proposed as biomarkers of effect in CCS monitoring programs. © 2020 International Society for Advancement of Cytometry.

Preferential Killing of Tetraploid Colon Cancer Cells by Targeting the Mitotic Kinase PLK1.

Chromosomal instability is a well-known factor in the progression of different types of cancer, including colorectal cancer. Chromosomal instability results in severely rearranged karyotypes and aneuploidy. Tetraploidy constitutes an intermediate phase during the polyploidy/aneuploidy cascade in oncogenesis, and tetraploid cells are particularly resistant to chemotherapy. Whether inhibition of the mitotic protein polo-like kinase 1 (PLK1) prevents the survival of tetraploid colon cancer cells is unknown. Diploid and tetraploid cells were transfected with siPLK1 or treated with PLK1 inhibitor Bi2536 in combination with spindle poison. Cell toxicity was assessed via crystal violet staining and clonogenic assay. Flow cytometry assessment analyzed numerous cell apoptotic parameters and cell cycle phases. Synergistic activity between Bi2536 and paclitaxel, vincristine or colchicine was calculated using the CompuSyn software. Inhibition or abrogation of PLK1 prevented the survival of colon cancer cells, specifically tetraploid cells. The cell death induced by PLK inhibition was due to mitotic slippage, followed by the activation of the intrinsic pathway of apoptosis. We further demonstrated that co-treatment of the tetraploid colon cancer cells with a PLK1 inhibitor and the microtubule polymerisation inhibitor vincristine or colchicine, but not the microtubule depolymerisation inhibitor paclitaxel, provoked a lethal synergistic effect. PLK1 inhibition together with microtubule-targeting chemicals, serve as a potent therapeutic strategy for targeting tetraploid cancer cells.

IL-27 impact on NK cells activity: Implication for a robust anti-tumor response in chronic lymphocytic leukemia

Interleukin 27 (IL-27) belongs to IL-12 cytokine family, has shown anti-tumor potential in several solid tumors, as well as hematologic malignancies. IL-27 can inhibit tumor growth and progression through direct and indirect mechanisms, such as inhibition of proliferation, angiogenesis, induction of apoptosis in tumor cells, and anti-tumor immune response. B-CLL is characterized by remarkable immune perturbation, which leads to disease complications and reduced effectiveness of the treatment. Natural killer cells (NK) are considered as an important arm for the elimination of transformed cells. However, NK cells have shown significant impairment in patients with CLL. Here we analyzed the activity of recombinant human (rh) IL-27-stimulated NK cells in bone marrow (BM) and peripheral blood (PB) of CLL patients using cell surface flow cytometry assessment, and cytotoxicity assay. We showed that rhIL-27 can increase CD69 on NK cells both in BM and PB. Interestingly, BM-NK cells treated with rhIL-27 exhibited a significant increase in degranulation and NK cell-mediated cytotoxicity as compared with untreated NK cells, whereas it did not improve NK cell activity of PB. These observations added further explanation to the anti-tumor activity of IL-27 and also could pave the way to adoption immunostimulatory adjuvant for therapies in CLL.

Cellular Enumeration of Tregs and CD3+ Cells in Cryopreserved Umbilical Cord Blood Units

Background Allogeneic haematopoietic cell transplantation (HCT) is a curative therapy for severe haematological disorders, including acute leukaemia. However, it carries significant morbidity and mortality due to infection, graft-versus-host disease and relapse. The cellular content of HCT grafts, in particular the ratio between regulatory T cells (Treg) and conventional T cells, is an important factor influencing the severity of these complications. Whilst previous studies have been performed in fresh adult peripheral blood mobilised grafts, it has not been applied in umbilical cord blood (CB) HCTs. CB enumerations are more challenging as they must be performed from small cryopreserved segments stored alongside the CB units used for HCT. Study and design methods Fresh CB units and thawed segments were analysed for their Treg and T cell content using both flow cytometry (the benchmark technique) and an epigenetic, DNA-based methodology. The two methods were compared for their agreement, consistency and susceptibility to error when assessing Treg and CD3+ cell numbers in both fresh and cryopreserved samples. Results Epigenetic enumeration gave consistent results in both fresh and frozen samples, providing Treg/CD3 estimates that were similar. Assessment of Tregs and CD3+ cells by flow cytometry and epigenetic assessments in fresh samples showed that these two methods were correlated. Conversely, significant cell death was observed in the thawed segments which affected Treg and CD3 cell estimates by flow cytometry. Conclusion Epigenetic assessments offer significant advantages over flow cytometry for analysing cryopreserved CB. Unlike with flow cytometry assessments of thawed segments, similar cell numbers were observed in fresh and frozen samples, with material requirements not being limiting and being unaffected by high cell death. With this method, multiple epigenetic assessments can be performed from extracted DNA, to provide statistical confidence and confirm observed results. Finally, the method raises the possibility of retrospective studies of historical samples where only DNA is available.

Effect of micro-aerobic process on improvement of anaerobic digestion sewage sludge treatment: flow cytometry and ATP assessment.

Micro-aeration as a pretreatment method improves the efficiency of anaerobic digestion of municipal sewage sludge and consequently promotes the methane production. In this study, adenosine triphosphate (ATP) and flow cytometry (FCM) were employed to monitor the performance of the micro-aerobic process and investigate the survival of bacterial cells within the process. At first, the effect of air flow rate (AFR) (0.1, 0.2, 0.3 and 0.5 vvm) on hydrolysis of mixed sludge in 5 aeration cycles (20, 30, 40, 48 and 60 hours) was examined. Then, the effects of the micro aerobic process on methane (CH4) production in anaerobic digestion were surveyed. The highest VSS reduction was 30.6% and 10.4% for 40 hours in the reactor and control, respectively. Soluble COD also fluctuated between 40.87 and 65.14% in micro-aerobic conditions; the highest SCOD was achieved at the time of 40 h. Microbial activities were increased by 597%, 170% and 79.4% for 20, 30 and 40 h pretreatment with the micro-aerobic process, respectively. Apoptosis assay showed that micro-aerobic pre-treatment at 20, 30 and 40 h increased the percentage of living cells by 57.4, 62.8 and 67.9%, respectively. On the other hand, FCM results showed that the highest percentage of viable bacteria (i.e., 67.9%) was observed at 40 h pretreating which was approximately 40% higher the ones for the control. Variation in cumulative methane production shows that methane production was increased by 221% compared to anaerobic digestion (control group). Therefore, ATP and FCM can be employed as two appropriate, accurate, relatively specific indicators for monitoring the process and bacteria viability.

Trial in Progress: Phase I Dose-Escalation and Dose-Expansion Trial of a Novel Glutaminase Inhibitor (CB-839 HCl) in Combination with Carfilzomib and Dexamethasone in Relapsed and/or Refractory Multiple Myeloma

Background: c-MYC activation is an early event of myeloma pathogenesis. It upregulates the expression of the glutaminase 1 (GLS1) enzyme which converts glutamine to glutamate in the mitochondria. Glutamate is required for the biosynthesis of various molecules in the tricarboxylic acid (TCA) cycle (i.e., glutamine anaplerosis). CB-839 HCl is a first-in-class, orally available, selective, noncompetitive inhibitor of GLS1. This inactivation of GLS1 results in an increase of glutamine and a decrease of glutamate and several TCA cycle intermediates within cancer cells, leading to a decrease in their proliferation and/or an increase in cell death. In the phase 1 study, CX-839-002, the safety and tolerability of CB-839 HCl was evaluated in patients with hematological tumors (multiple myeloma (MM) and non-Hodgkin's lymphoma), either as monotherapy or in combination with pomalidomide and dexamethasone or with dexamethasone alone. It was determined to be well tolerated and the maximal tolerated dose (MTD) was not reached. Proteasome inhibitors (PI) are the cornerstone agents in the treatment of myeloma. They disrupt normal protein homeostasis causing an induction of cellular proteotoxic stress, thus, making it an effective strategy against myeloma plasma cells, which naturally mass-produce large quantities of immunoglobulin proteins. PI-resistant MM cells are associated with changes in cellular bioenergetics that favor the increased use of mitochondrial respiration for energy production. Given the increased reliance of PI-resistant MM cells on mitochondrial respiration, and the critical role of glutamine for cellular respiration, inhibition of glutamine metabolism is a rational molecular strategy for the treatment of PI-resistant MM. Furthermore, pre-clinical studies demonstrate the in vitro and ex vivo synergism of CB-839 HCl with carfilzomib (CFZ) in terms of its cytotoxicity and anti-proliferation capacity in various primary human myeloma cell lines and primary patient myeloma cells respectively. As a result, this novel combination of glutaminase inhibition with proteasome inhibition appears promising as a therapeutic combination in MM and warrants further clinical investigation. Methods: This study is a phase 1, multicenter clinical trial of CB-839 HCl in combination with carfilzomib and dexamethasone for patients with relapsed and/or refractory myeloma. Part A of this trial is a 3+3 dose escalation design and Part B is a dose expansion cohort at the RP2D determined in Part A. Up to a maximum of 42 patients will be enrolled at participating ETCTN sites. CFZ will be administered in its usual weekly dosing schedule of days 1, 8 and 15 of a 28 day schedule along with dexamethasone on days 1, 8, 15 and 22. CB-839 will be started at a dose level of 400 mg twice daily and will be investigated to a maximum dose of 800 mg twice daily. Prior to day 1 of Cycle 1, we will administer a 7 day lead in of CB-839 monotherapy before combining it with CFZ. Key inclusion criteria are having relapsed/refractory myeloma with at least 2 prior lines of therapy and prior exposure to PIs, immunomodulators and Anti-CD38 monoclonal antibodies, having measurable disease, adequate hematologic reserve, kidney function and liver function. Key exclusion criteria are being refractory or intolerant to CFZ, adverse cardiac history, central nervous system disease and AL amyloidosis. The primary objective of this trial is to determine the MTD or recommended phase II dosing (RP2D) of CB-839 HCl in combination with carfilzomib and dexamethasone. The secondary objective is to evaluate the safety and tolerability as well as the overall response rate (ORR) associated of CB-839 HCl in combination with carfilzomib and dexamethasone. Correlative objectives will evaluate plasma pharmacokinetic profiles of CB-839 HCl and carfilzomib when used in combination. They will also evaluate potential predictive and prognostic biomarkers as well as resistance mechanisms using genomic DNA, RNA, flow cytometry, immunohistochemistry and metabolomics-based assessment platforms. Recruitment is ongoing and this trial is registered on clinicaltrials.gov: NCT03798678. Disclosures Baz: Bristol-Myers Squibb: Research Funding; Sanofi: Research Funding; Karyopharm: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Research Funding; Merck: Research Funding. Neparidze:Janssen Scientific Affairs, LLC: Research Funding; Eidos Therapeutics: Other: Member of Independent Diagnostic Committee; MMRF/Synteract: Membership on an entity's Board of Directors or advisory committees. Kumar:Janssen: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Takeda: Research Funding.

Correlation of Flow Cytometric Aberrations with Cytogenetic, Molecular Genetic, and Morphology in Patients with Unexplained Cytopenias

Introduction: Whereas morphological assessment and cytogenetics have been the cornerstone for the diagnosis of myelodysplastic syndrome (MDS), flow cytometry and mutational analyses are novel, evolving techniques. These diagnostic procedures play a role in identifying pre-MDS cases including idiopathic cytopenia of undetermined significance (ICUS) and clonal cytopenia of undetermined significance (CCUS). Aim: To assess flow cytometric findings in ICUS and CCUS and their correlation with progression to MDS or myeloid neoplasm (MN), concurrent genetic aberrations, and underlying pathological evolution. Methods: Patients (Pts) who had undergone evaluation to rule out MDS and had retrospectively undergone flow cytometry, cytogenetic, mutational and morphological assessment were included. Flow cytometry results were classified as normal (no abnormality or 1 equivocal result), abnormal (2 abnormalities or at least 1 abnormality and 1 equivocal result), or atypical (1 abnormality or 2-3 equivocal results). Next generation sequencing included mutation analysis of: ASXL1, BCOR, BRAF, CALR, CBL, CEBPA, CSF3R, DNMT3A, ETV6, EZH2, FLT3, GATA1, GATA2, IDH1, IDH2, JAK2, KIT, KRAS, MPL, MYD88, NOTCH1, NPM1, NRAS, PHF6, PTPN11, RUNX1, SETBP1, SF3B1, TERT, TET2, TP53, U2AF1, WT1, ZRSR2. Results: A) Characteristics: 229 pts were assessed (median age 60 and 69% were males). Based on pathologist interpretation, 87 (38%) pts met diagnostic criteria of MDS, 106 (46%) pts had normal/reactive bone marrow (BM), and 36 (16%) pts had BM findings suspicious for MDS. Median time to follow-up was 24 months for MDS pts, and 32 months for pts with cytopenias. B) Morphology: Pts who had a BM biopsy that was suspicious for MDS by pathologist interpretation were more likely to develop MDS compared to pts with normal/reactive pathology report with an incidence rate (IR) of 16% versus 0.9% (p= .0005). Pts who were suspicious for MDS based on the BM biopsy were more likely to have underlying mutations compared to pts with normal/reactive BM (IR 83% versus 69%, p= .2). C) Flow cytometry: In the non-MDS cohort, 96 (67%) pts had normal flow results, 29 (20%) pts had atypical flow, and 17 (12%) pts had abnormal flow results compared to 16 (18%) pts, 18 (21%) pts, and 53 (61%) pts in the MDS cohort, respectively. Of the pts who had normal flow result (n=112), 16 were MDS, 16 were suspicious for MDS/MN, and 80 were normal/reactive. Pts who had biopsy interpretation suspicious for MDS/MN were more likely to have abnormal flow cytometry compared to normal/reactive BM biopsy (IR 55% versus 24%, p=.0003). IR of MDS was 4% in normal flow, 3% in atypical flow, and 12% in abnormal flow (p= .5). Likewise, IR of MN was 4% in normal flow, 20% in atypical flow, and 30% in abnormal flow (p= .1). D) Genetics (cytogenetics and mutation profiling): Cytogenetics was normal in 123 (86%) pts and abnormal in 19 (13%) pts. Pts who had abnormal cytogenetics were more likely to develop MN (IR 57% versus 5%, p= .002). Out of the 50 non-MDS pts tested for NGS, 38 (76%) were positive for mutations. 15 (39%) pts had 1 mutation, 8 (21%) pts had 2, 3 (8%) pts had 3, 6 (16%) pts had 4, 2 (5%) pts had 5, 1 (3%) pt had 7, and 1 (3%) pt had 8 mutations (61% had >1 mutation). Pts with positive mutations were more likely to develop MDS during follow-up compared to pts who did not have mutations (IR 16% versus 0%, p=.06). Additionally, flow cytometric aberrations significantly correlate with the number of mutations (p= .03). MN correlated significantly with BCOR mutation and RUNX1 (IR 60% (p= .008) and 50% (p=.02), respectively. E) Interaction between variables: In pts with positive mutations, flow cytometry did not correlate with progression to MDS with an IR of 17% of MDS in normal flow, 11% in atypical flow, and 17% in abnormal flow, (p= .9). There was no impact on overall survival neither in pts with abnormal flow results vs normal nor in pts with positive mutations vs wild type. Conclusion: Conventional diagnostic techniques remain the cornerstone of MDS diagnosis with flow cytometry and NGS arising as novel techniques that correlate significantly with progression of pre-MDS pts to MDS. Flow had an impact on pathology final interpretation but did not add value in pt with a positive mutation analysis. Larger studies are needed to confirm our findings. Disclosures Patnaik: Stem Line Pharmaceuticals.: Membership on an entity's Board of Directors or advisory committees. Al-Kali:Astex Pharmaceuticals, Inc.: Research Funding.

Potential of the green microalga Chlorella vulgaris to fight against fluorene contamination: evaluation of antioxidant systems and identification of intermediate biodegradation compounds

Polycyclic aromatic hydrocarbons (PAHs) have high risks for human and living organisms due to their mutagenic and carcinogenic properties. Here, the effect of different concentrations of fluorene as a persistent toxic PAH on growth parameters and antioxidant systems in the green microalga Chlorella vulgaris was investigated. Intriguingly, cell number as well as dry and fresh weight of the alga were raised at 2 ppm fluorene compared to the control sample. However, with the increasing levels of fluorene from 10 to 50 mg L−1, the growth parameters gradually decreased. Accordingly, cells of C. vulgaris were found to enhance the activity of ROS scavenging enzymes after 7 days of exposure to fluorene in a concentration-dependent manner. Exposure to 25 and 50 mg L−1 fluorene was led to a significant decrease at chlorophyll content, whereas the concentration of carotenoids was not changed. Total phenol and flavonoid contents were markedly raised in 50 mg L−1 of fluorene compared to the control. Although flow cytometry assessment showed no substantial reduction in the viability at 50 mg L−1 fluorene-treated samples for 24 h, chlorophyll fluorescence was noticeably reduced. The results of SEM analysis revealed that the 50 mg L−1 fluorene treatment clearly damaged the algal cells after 24 h. The ability of the alga for biodegradation of fluorene was assessed by GC-MS. Consequently, a number of produced intermediate compounds were identified. These findings displayed that C. vulgaris had not only notable resistance against fluorene but also noteworthy potential for its degradation.

Immunophenotypic Detection of Measurable Residual (Stem Cell) Disease Using LAIP Approach in Acute Myeloid Leukemia.

Half of the patients with acute myeloid leukemia (AML), who achieve complete remission after chemotherapy treatment, will ultimately experience a relapse. Measurable residual disease (MRD) is an important post‐treatment risk factor in AML, because it gives additional information about the depth of the remission. Within MRD, the small population of leukemic stem cells (LSCs) is thought to be at the base of the actual relapse. In this protocol, the flow cytometric detection of MRD and LSCs herein is outlined. We give a detailed overview of the sampling procedures for optimal multiparameter flow cytometry assessment of both MRD and LSC, using leukemia associated immunophenotypes (LAIPs) and LSC markers. Moreover, an overview of the gating strategies to detect LAIPs and LSC markers is provided. This protocol serves as guidance for flow cytometric detection of measurable residual (stem cell) disease necessary for proper therapeutic decision making in AML patients. © 2019 The Authors. Basic Protocol 1: Immunophenotypic LAIP detection for measurable residual disease monitoring Basic Protocol 2: Immunophenotypic detection of CD34+CD38− leukemic stem cells

Oleanolic Acid Mitigates 6-Hydroxydopamine Neurotoxicity by Attenuating Intracellular ROS in PC12 Cells and Striatal Microglial Activation in Rat Brains.

Oleanolic acid (OA), a biologically active pentacyclic triterpenoid compound, has been implicated in a number of clinical benefits including antioxidant, and anti-inflammatory properties. OA has been previously shown to ameliorate the toxic effects of 6-hydroxydopamine (6-OHDA), however, the mechanism by which this effect is exhibited is not clearly understood. In the present study, we investigated the role of OA in attenuation of microglial activation in 6-OHDA induced Parkinsonian rat model. We also explored the ability of OA to attenuate 6-OHDA-induced intracellular reactive oxygen species (ROS), and thus prevent cell death in PC12 cells. We accessed the utility of immunohistochemistry to assess striatal microglial activation, where shape descriptors such as area, perimeter, Feret’s diameter, aspect ratio and solidity were determined using the Fiji ImageJ software. Intracellular ROS and cell viability were assessed in PC12 cells using the OxiSelectTM Intracellular ROS Assay Kit and MTT assay, respectively. We found that microglial activation was decreased in rats pre-treated with OA prior to 6-OHDA insult as well as in rats treated with OA 1 day post 6-OHDA exposure when compared to untreated rats, as determined by shape descriptors. This finding was in correlation with significantly improved motor symptoms and increased striatal dopamine in treated rats as compared to non-treated rats. Flow cytometry assessment of PC12 cells revealed a decreased amount of intracellular ROS in cells pre-treated with OA 6 h prior to 6-OHDA exposure and cells treated with OA 1 h post 6-OHDA exposure, suggesting that OA provides neuroprotection in PC12 cells by removing intracellular ROS, thereby reducing oxidative stress. Our finding suggest that OA exhibits its neuroprotective effect by attenuating striatal microglial activation, which results in neuroinflammation that is implicated in Parkinson’s disease pathology. Further studies detailing the mechanism by which OA interacts with microglia may be useful in understanding the role of OA in attenuating neuroinflammation.

MicroRNA-204-3p represses colon cancer cells proliferation, migration, and invasion by targeting HMGA2.

Colon cancer is a detrimental neoplasm of the digestive tract. MicroRNAs (miRNAs) as central regulators have been discovered in colon cancer. Nonetheless, the impact of miR-204-3p on colon cancer remains indistinct. The research attempted to uncover the impacts of miR-204-3p on colon cancer cells growth, migration, and invasion. miR-204-3p expression level in colon cancer tissues and diverse colon cancer cell lines were testified by the quantitative real-time polymerase chain reaction. Exploration of the impacts of miR-204-3p on cell growth, migration, invasion, and their associated factors through assessment of CCK-8, flow cytometry, Transwell, and western blot, respectively. High mobility group AT-hook 2 (HMGA2) expression was then detected in Caco-2 cells after miR-204-3p mimic and inhibitor transfection, additionally dual-luciferase activity was implemented to further uncover the correlation between HMGA2 and miR-204-3p. The impact of HMGA2 on Caco-2 cell growth, migration, and invasion was finally assessed. We found that repression of miR-204-3p was discovered in colon cancer tissues and HCT116, SW480, Caco-2, HT29 and SW620 cell lines. MiR-204-3p overexpression mitigated Coca-2 cell viability, facilitated apoptosis, simultaneously adjusted CyclinD1 and cleaved caspase-3 expression. Cell migration, invasion, and the associated factors were all suppressed by miR-204-3p overexpression. Reduction of HMGA2 was presented in Caco-2 cells with miR-204-3p mimic transfection, and HMGA2 was predicated to be a target gene of miR-204-3p. Besides, HMGA2 silence showed the inhibitory effect on Caco-2 cells growth, migration, and invasion. In conclusion, miR-204-3p repressed colon cancer cell growth, migration, and invasion through targeting HMGA2.

Abstract LB-246: Apoptotic mechanisms of Vernonia amydalina delile in the prevention of prostate cancer

Abstract Prostate cancer is one of the common cancers in males and its incidence keeps increasing globally. Approximately 81% of prostate cancer is diagnosed during the early stage of the disease. The treatment options for prostate care include surgery, radiotherapy, and chemotherapy, but these treatments often have side effects that may result in a poor quality of life such as impotence or decreased bowel function. Our central goal is to test the apoptotic mechanisms of Vernonia amydalina Delile (an edible medicinal plant that is relatively inexpensive, non-toxic, and virtually without side effects) for the prevention of prostate cancer using human adenocarcinoma (PC-3) cells as a test model. To address our specific goal, PC-3 cells were treated with Vernonia amydalina Delile (VAD). Cell viability was determined by the MTT assay and cell morphology was analyzed by acridine orange and propidium iodide (AO/PI) dye using the fluorescent microscope. Cell cycle arrest and cell apoptosis were evaluated by Flow Cytometry assessment. Nucleosomal DNA fragmentation was detected by DNA laddering. Data obtained from the AO/PI dye assessment indicated that VAD significantly reduced the number of live cells in a dose-dependent manner, showing a gradual increase in the loss of viability in VAD-treated cells. A similar result was obtained by the MTT assay. We observed a significant increase in DNA damage in VAD-treated cells compared to the control group. Flow cytometry data demonstrated that VAD induced apoptosis in treated cells compared to the control cells. These results suggest that induction of cell death, cell cycle arrest, and cell apoptosis are involved in the therapeutic efficacy of VAD as anticancer candidate towards the prevention and/or treatment of prostate cancer. Citation Format: Clement G. Yedjou, Solange S. Tchounwou, William K. Johnson, Sylvianne Njiki, Paul B. Tchounwou. Apoptotic mechanisms of Vernonia amydalina delile in the prevention of prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-246.

Abstract 511: An assessment of pre-conditioning regimens for optimal hCD34+ hematopoietic stem cell humanization of NSGTMmice

Abstract Humanized immune system (HIS) mice - immunocompromised Nod.Cg-Prkdcscid Il2rgtm1Wjl/SzJ animals in which the immune system has been humanized by the engraftment of CD34+ hematopoietic stem cells (HSC), peripheral blood mononuclear cells (PBMC), or other human cells/tissues - represent an important tool in pre-clinical oncology research. Here we investigated several pre-conditioning regimens (Busulfan pre-treatment or total body irradiation (TBI)) to determine which would yield maximum human leukocyte engraftment and lineage diversity. Animals were pre-treated either one or two doses of Busulfan (25 mg/kg) or varying doses of TBI (1.75, 2.0, or 2.5 Gy). One to two days later, animals received an adoptive transfer of native hCD34+ HSCs. Engraftment was monitored by flow cytometry assessment of peripheral blood four weeks after adoptive-transfer, and every four weeks thereafter through experiment termination at Week 16. Upon termination, spleen and bone-marrow were assessed for various leukocyte populations by flow cytometry. Animals undergoing pre-conditioning displayed increased human CD45+ cell engraftment at all timepoints as compared to animals without pre-conditioning, with animals treated with two doses of Busulfan demonstrating the greatest human CD45+ engraftment at all timepoints and reaching statistical significance at endpoint (vs no pre-conditioning; p<0.05). Diversity of leukocyte cell types (B cells, T cells, NK cells, and other hCD45+) in the bone-marrow was similar across pre-treatment paradigms, however for some lineages, representation in the bone-marrow was significantly altered between pre-conditioning type. The absolute number of T or B cells were increased in TBI and Busulfan treated groups, respectively, whereas NK cells as a percentage of singlets were increased in TBI treated groups. The diversity of leukocyte cell types in the spleen was likewise similar across groups, with B cells dominating. These data provide important insights into the utility of various pre-conditioning regimens for the humanization of NSG mice for pre-clinical oncology research, and demonstrate that the optimum pre-conditioning regimens should be selected based on treatment target. Citation Format: Caitlin S. Parello, Benjamin G. Cuiffo, Alexandria Kury, Kasey Reardon, Brett Van Dam, Sallyann Vu, Catarina Costa, Veronica Ritchie, Gavin Gagnon, Timothy Bateson, Samantha Rogers, Gregory D. Lyng, Stephen T. Sonis. An assessment of pre-conditioning regimens for optimal hCD34+ hematopoietic stem cell humanization of NSGTMmice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 511.

Sex differences in the inflammatory immune response to multi-walled carbon nanotubes and crystalline silica

Background: Respiratory disease is a leading cause of death and disability worldwide. These diseases frequently present with a sex bias in occurrence and severity, yet the mechanisms responsible for these sex biases is a critically understudied area of basic research.Methods: Male and female C57BL/6 mice were exposed to multi-walled carbon nanotubes (MWCNTs) or crystalline silica (cSiO2) via oropharyngeal aspiration. Acute assessments were conducted 24 h and 7 days after a single exposure. In chronic experiments, mice were exposed to respective particles once per week for 4 weeks and sacrificed 8 weeks after the last exposure. Lung lavage fluid (LLF) was assessed for markers of injury and inflammation. Immune cell populations were analyzed by flow cytometry and histopathology assessment was performed on lung tissue from chronically exposed mice.Results: Female mice exposed to a single dose of MWCNTs generated a greater eosinophilic response than males 24 h and 7 days post-exposure. Eosinophilia was accompanied by elevated type 2 cytokine production in LLF. The exaggerated acute response in females was consistent with lung pathology observed in the chronic model: females had greater alveolitis and epithelial cell hyperplasia compared to males. There were no sex differences 24 h after cSiO2 exposure, but by 7-day post-exposure female mice had greater airspace neutrophilia and inflammatory cytokine levels compared to males. However, following repeated exposure to cSiO2, male mice had worse alveolitis and greater dendritic cell presence within the lungs.Conclusions: Female mice are more susceptible to acute and chronic MWCNT-induced inflammation, but male mice are more susceptible to chronic cSiO2-induced lung pathology.

Issue Highlights - May 2019.

Issue Highlights - May 2019.

Neoadjuvant atezolizumab in resectable NSCLC patients: Updated clinical and immunophenotyping results from a multicenter trial.

99 Background: Targeting PD-L1/PD-1 to activate anti-tumor immunity is associated with improved response and survival compared to chemo in NSCLC pts. We present a preliminary analysis of the clinical efficacy, safety and peripheral blood (PB) immunophenotyping from an ongoing multicenter atezolizumab (atezo) neoadjuvant immunotherapy study in resectable NSCLC. Methods: Pts received 2 cycles of atezo, 1200mg, days 1, 22 before resection. Tumor biopsies and PB were obtained pre-atezo & pre-surgery. The biomarker evaluable population (BEP) included pts with paired PB analyzed within 72 hrs by 10-color flow cytometry (IMMUNOME) and major pathological response (MPR) assessment (defined as ≤ 10% residual tumor). The primary endpoint was MPR. Secondary endpoints included safety, MPR by PD-L1, OS, and DFS. Immunophenotypic analyses were correlated with treatment, MPR and PD-L1 expression. Results: 116 patients have been enrolled as of October 31, 2018 and here we report on 54 of 180 planned pts with follow-up through surgery. 15 pts had Gr 3-4 AEs (3 treatment related), one Gr 5 AE (sudden death) was unrelated. By RECIST there were 3 PR, 49 SD, and 2 PD. 50/54 pts underwent the planned surgery, 47 pts had MPR assessment: 4 pts discontinued study preop (2 radiographic PD, 2 other reasons); 3 were unresectable. Excluding 5 pts with EGFR or ALK mutations, MPR rate was 10/45 (22%, 95% CI 11-37%). Baseline PD-L1 status was evaluable in 44/54 pts; BEP included 31 pts, 23 had tissue PD-L1 status: 16 PD-L1+. We observed significant increases in natural killer (NK) cells, CD8+ T-cells, Th1-response related dendritic cells (DC), and decreases in B-cells after atezo. Non-MPR pts showed significant increases in late activated NK cells, monocytic myeloid cells and Th2 and Th17-response–related DCs. PD-L1+ pts showed significant decreases of senescent T cells, monocytic myeloid cells, and increases of Th1-response–related DCs. We analyzed 22/54 tumor pairs, PD-L1+ cells increased in most pts after atezo treatment. Conclusions: Neoadjuvant atezo was well tolerated and the MPR rate is encouraging. Preliminary immunophenotyping data showed significant changes in PB with immunotherapy. Clinical trial information: NCT02927301.

Kidney Transplant Outcome Is Associated with Regulatory T Cell Population and Gene Expression Early after Transplantation.

Successful long-term kidney allograft survival with parallel reduction of complications resulting from prolonged immunosuppressive treatment is a goal in kidney transplantation. We studied the immune changes in cell phenotypes and gene expression induced by kidney transplantation. Our goal was to find a phenotypic and/or transcriptional pattern that might be considered prognostic for the kidney transplant outcome. The analysis was performed prospectively on 36 KTx recipients sampled during the first year and followed for five years after transplantation and on 40 long-term KTx recipients (7.9 ± 2.2 y. post-KTx). The research involved flow cytometry assessment of lymphocyte subpopulations (including Tregs and CD3+CD8+CD28− lymphocytes) and gene expression analysis of immune-related genes (CD4, CD8, CTLA4, GZMB, FOXP3, IL10, IL4, ILR2A, NOTCH, PDCD1, PRF1, TGFB, and TNFA). The analysis of patterns observed over the first post-KTx year was confronted with control, pretransplant, and long-term transplant results. Treg counts at months one and three post-KTx correlated positively with the current and future allograft function. FOXP3 gene expression at month one post-KTx was also associated with long-term allograft function. The KTx-induced CD3+CD8+CD28− population correlated with GZMB and PRF1 expression and suggested their cytotoxic properties. The size of the Treg population and regulatory FOXP3 gene expression in the early period after transplantation are associated with kidney transplant outcome. The outlined predictive power of the Treg population needs to be investigated further to be confirmed as one of the immune monitoring strategies that may help achieve the best long-term kidney allograft outcomes.

Flow cytometry in assessment of sperm integrity and functionality – a review

Spermatozoa must possess many attributes to fertilize an egg but few laboratory methods can assess all of these attributes simultaneously and objectively. Most laboratory methods used to assess the quality of semen in veterinary andrology can be inaccurate and time-consuming. Laboratory techniques which evaluate only one sperm attribute, often provide results that have a weak correlation with fertility. Therefore, semen used for artificial insemination should not be assessed based on the results of one method only but rather on the comprehensive results of several laboratory tests. Flow cytometry is a modern method of analysing diJ169fferent types of cells, including sperm cells. It is based on the scattering of light and fluorescence, the outcome of which yields results that are recorded and evaluated by computer technology which makes an objective assessment. Flow cytometry in combination with fluorescence microscopy and fluorescent probes provides a comprehensive, accurate, objective, and rapid analysis of the ejaculate. In a short time frame it allows us to test thousands of sperm for their structure and properties, even with a minimal amount of semen. This method enables the evaluation of several indicators simultaneously in a population of sperm as a whole or for each sperm individually. It informs us about the selected indicators of sperm quality in the sample by examining the membrane integrity, DNA, mitochondria, acrosome, oxidative stress, and other properties. Flow cytometry has become an important method of evaluating the functional and morphological properties of sperm and is used for research in veterinary science as well as for a routine assessment of the semen quality.

Flow Cytometry Assessment of Procoagulant Platelets Using a Dithiol-Reactive Probe.

Flow cytometry assessment of platelets using the combination of GSAO [4-(N-(S-glutathionylacetyl)amino)phenylarsonous acid], a dithiol-reactive probe, and P-selectin, a platelet activation marker, is a novel and powerful assay in the identification and quantification of the procoagulant subpopulation of platelets that has the capacity to support thrombin generation. In this chapter, we provide the flow cytometry protocols aimed at the study of procoagulant platelets under resting and agonist-stimulated conditions in whole blood and washed platelets of both human and murine (mouse) samples.

Thymol as a reciprocal regulator of T cell differentiation: Promotion of regulatory T cells and suppression of Th1/Th17 cells

Regulatory T cells (Tregs) are critical for maintaining immune response and enhancing their differentiation has therapeutic implications for autoimmune diseases. In this study, we investigated the effects of thymol a well-known monoterpene from Thyme on differentiation and function of Tregs. In vitro generation of Tregs from purified naïve CD4+CD25− T cells in the presence of thymol was carried out. Suppressor activity of generated Tregs was examined by changes in the proliferation of CFSE-labeled conventional T cells. Thymol promotes differentiation of naïve CD4+CD25− T cells to CD4+CD25+Foxp3+ Tregs [66.9–71.8% vs. control (47%)] and increased intensity of Foxp3 expression on Tregs (p < 0.01). In functional assay, an increased immune suppression by thymol-induced Tregs (≈2.5 times of untreated Tregs) was detected. For in vivo study, thymol was intraperitoneally administered to ovalbumin (Ova)-immunized mice. Flow cytometry assessment of spleens from thymol-treated Ova-immunized mice showed increased number of CD4+ Foxp3+ Tregs (>8%, p < 0.01(and decreased levels of CD4+T-bet+ Th1 and CD4+RORγt+ Th17 cells resulted in significant decreased Th1/Treg and Th17/Treg ratios. In ex vivo Ova challenge of splenocytes from thymol-treated Ova-immunized mice, similarly higher levels of CD4+ Foxp3+ Tregs, and also elevated TGF-β expression in CD4+Foxp3+ population (48.1% vs. 18.9% in untreated Ova-immunized group) and reduced IFN-γ-producing CD4+T-bet+ T cells and IL-17-producing CD4+RORγt+ T cells were detected. This led to marked decreased ratios of IFNγ/TGF-β and IL-17/TGF-β expressions. In conclusion, this study revealed thymol as a compound with enhancing effects on Treg differentiation and function, which may have potential benefits in treatment of immune-mediated diseases with Th1/Th17 over-activation.