Flow Cytometric Studies Research Articles

Protective Effects of Vitamin E against Methotrexate-Induced Testicular Damage in Rats: Histopathologic and flow cytometric study

Background: Methotrexate (MTX) can cause oxidative stress-related tissue damage.Vitamin E neutralizes lipid peroxidation arising from the effect of free oxygen radicals.In this study, the protective effect of vitamin E against possible MTX-related testicular damage was analyzed.
 Method: Thirty two mature male Spraque dawley rats were grouped as MTX, Vitamin E, MTX+Vitamin, Control groups. 20 mg/kg MTX intraperitoneal (i.p.) was applied to MTX Group in the first day; 100mg/kg i.p. vitamin E was applied to Vitamin E Group for 5 days; 20 mg/kg i.p. MTX in the first day and 100 mg/kg i.p. vitamin E for 5 days were applied to MTX+Vitamin E Group;2 ml physiological saline solution (i.p.) was applied to Control Group for 5 days. Histopathology, flow cytometry and apoptosis were evaluated on testicular tissue. 
 Result: Apoptotic Index (%) and testicular damage were highest for MTX Group, and significant decrease was observed for MTX+Vitamin E Group compared to MTX Group.Seminiferous tubule size significant decreased in MTX Group and it increased in MTX+Vitamin E Group compared to MTX Group. No significant difference was found between MTX and MTX+Vitamin E Groups regarding germinal epithelium thickness and testicle weights.
 Conclusion: The results show that MTX can cause structural disruptions in testicles and vitamin E can rehabilitate MTX-related testicular damage.

Permuted 2,4-thiazolidinedione (TZD) analogs as GLUT inhibitors and their in-vitro evaluation in leukemic cells

Cancer is a heterogeneous disease, and its treatment requires the identification of new ways to thwart tumor cells. Amongst such emerging targets are glucose transporters (GLUTs, SLC2 family), which are overexpressed by almost all types of cancer cells; their inhibition provides a strategy to disrupt tumor metabolism selectively, leading to antitumor effects. Here, novel thiazolidinedione (TZD) derivatives were designed, synthesized, characterized, and evaluated for their GLUT1, GLUT4, and GLUT5 inhibitory potential, followed by in-vitro cytotoxicity determination in leukemic cell lines. Compounds G5, G16, and G17 inhibited GLUT1, with IC50 values of 5.4 ± 1.3, 26.6 ± 1.8, and 12.6 ± 1.2 μM, respectively. G17 was specific for GLUT1, G16 inhibited GLUT4 (IC50 = 21.6 ± 4.5 μM) comparably but did not affect GLUT5. The most active compound, G5, inhibited all three GLUT types, with GLUT4 IC50 = 9.5 ± 2.8 μM, and GLUT5 IC50 = 34.5 ± 2.4 μM. Docking G5, G16, and G17 to the inward- and outward-facing structural models of GLUT1 predicted ligand binding affinities consistent with the kinetic inhibition data and implicated E380 and W388 of GLUT1 vs. their substitutions in GLUT5 (A388 and A396, respectively) in inhibitor preference for GLUT1. G5 inhibited the proliferation of leukemia CEM cells at low micromolar range (IC50 = 13.4 μM) while being safer for normal blood cells. Investigation of CEM cell cycle progression after treatment with G5 showed that cells accumulated in the G2/M phase. Flow cytometric apoptosis studies revealed that compound G5 induced both early and late-stage apoptosis in CEM cells.

Correction of glycemic and oxidative stress sensors in streptozotocin induced diabetic rat model: Impact of dose dependent study of n-butanol solvent fraction of hydro-methanol extract of banana flower (Musa balbisiana).

The experiment focused the most effective dose out of 2.5, 5, and 10mg/100g body weight/day for 4weeks of n-butanol fraction from the pretested promising hydro-methanol extract of Musa balbisiana flower to manage the streptozotocin-induced diabetes in rat and to minimize the bioburden of the body by excluding the inactive components from the said extract. Fraction treatment exhibited an outstanding recovery in fasting blood glucose, glycated hemoglobin, serum insulin levels, carbohydrate metabolic, antioxidant enzymes activities, expression of their marker genes, and apoptotic gene in diabetic rat. Flow-cytometric study of pancreatic β-cell population was significantly recovered in fraction-treated groups which was also supported by histological study. The moderate dose, that is, 5mg dose showed maximum efficacy in respect to other doses. The high dose, that is, 10mg dose is not significantly effective than 5mg dose may be due to the desensitization of the target organ's orphan receptor(s). PRACTICAL APPLICATIONS: The work includes the role of M. balbisiana on diabetes management. Result focused that M. balbisiana flower can able to rectify the diabetic condition and recovered the β cell population. M. balbisiana flower is a very common vegetable to people, use mostly in their daily meal. The experiment showed positive results to cure diabetes in the long-term. Moreover, this study is potential for drug development using nutraceutical(s) isolation. The pharmaceutical companies may develop an antidiabetic drug from the concerned plant part after successful clinical trial.

Diarylurea derivatives comprising 2,4-diarylpyrimidines: Discovery of novel potential anticancer agents via combined failed-ligands repurposing and molecular hybridization approaches

A series of diarylurea derivatives comprising 2,4-diarylpyrimidines were synthesized based on a combination of postulated molecular hybridization design and failed-ligands repurposing approaches, which enabled the discovery of novel potential antiproliferative agents. Towards credible biological evaluation, an in vitro anticancer activity assay was conducted employing a library of 60 cancer cell lines constituting nine panels representing blood, lung, colon, CNS, skin, ovary, renal, prostate, and breast cancers. The results revealed high effectiveness and broad-spectrum anticancer activity of compounds 4m and 4g. Five-dose assay of compounds 4m and 4g proved their high potency that surpassed that of four standard kinase inhibitors FDA-approved anticancer drugs against many cancer cells. Towards the identification of their molecular target, screening of kinase inhibitory profile employing a panel of 51 kinases involved in cancer revealed inhibition of several kinases from the platelet-derived growth factor/vascular endothelial growth factor receptor (PVR) kinase family, which might mediate, at least in part, the antiproliferative activity. Molecular docking of 4g into the crystal structure of the Feline McDonough Sarcoma (FMS) kinase predicted that it binds to a pocket formed by the juxtamembrane domain, the catalytic loop, and the αE helix, thus stabilizing the inhibited conformation of the kinase. Flow cytometric study of the cytotoxic effects of compound 4g in A549 cells showed it induces dose- and time-dependent apoptotic events leading to cell death. Collectively, this work presents compound 4g as a potential broad-spectrum anticancer agent against multiple cancer types.

Garcinone-E exhibits anticancer effects in HeLa human cervical carcinoma cells mediated via programmed cell death, cell cycle arrest and suppression of cell migration and invasion

Xanthones are an important class of natural compounds bearing huge bioactivity profiles. Garcinone-E is one among most active xanthones showing potential anticancer activity against various human cancer cell lines. Therefore, the current study was performed to explore the anticancer potency of naturally occurring garcinone-E xanthone against human HeLa cervical cancer cells. The underlying mechanism of action was also tried to be explored via testifying its induction of programmed cell death, arrest of cell cycle, suppression of cell migration, cell invasion and cell adhesion. MTT assay was implemented to estimate the viability of HeLa cells after garcinone-E exposure and clonogenic assay was used to analyze the effect on clonogenic potential. Acridine orange/ethidium bromine (AO/EB) staining assay was performed for monitoring of programmed cell death along with western blotting. Flow cytometric studies were carried out to analyze cell cycle check points. Transwell chambers assays were carried out for studying the impact of garcinone-E on migration and invasion potency of HeLa cells. Western blotting was used to study the expressions of apoptosis linked proteins in HeLa cells. Results indicated that garcinone-E remarkably decreased the viability to minimum in HeLa cells in both dose and time-reliant manner. The clonogenic capacity of HeLa cells was efficiently reduced by garcinone exposure. AO/EB staining showed that the anti-viability action of garcinone-E was apoptosis allied which was supported by western blotting as well. The cell cycle check points study indicated cell cycle arrest at G2/M-phase. HeLa cell migration and invasion were reduced efficiently after being subjected to garcinone-E treatment in a dose reliant fashion. In conclusion, garcinone-E has a remarkable potential to act as anti-cervical cancer chemopreventive provided further in vivo studies are required.

Danon disease is an underdiagnosed cause of advanced heart failure in young female patients: a LAMP2 flow cytometric study.

AimsDanon disease (DD) is a rare X‐linked disorder caused by mutations in the lysosomal‐associated membrane protein type 2 gene (LAMP2). DD is difficult to distinguish from other causes of dilated or hypertrophic cardiomyopathy (HCM) in female patients. As DD female patients regularly progress into advanced heart failure (AHF) aged 20–40 years, their early identification is critical to improve patient survival and facilitate genetic counselling. In this study, we evaluated the prevalence of DD among female patients with non‐ischemic cardiomyopathy, who reached AHF and were younger than 40 years.Methods and resultsThe study cohort comprised 60 female patients: 47 (78%) heart transplant recipients, 2 (3%) patients treated with ventricular assist device, and 11 (18%) patients undergoing pre‐transplant assessment. Aetiology of the cardiomyopathy was known in 15 patients (including two DD patients). LAMP2 expression in peripheral white blood cells (WBC) was tested by flow cytometry (FC) in the remaining 45 female patients. Whole exome sequencing was used as an alternative independent testing method to FC. Five additional female DD patients (two with different novel LAMP2 mutations) were identified by FC. The total prevalence of DD in this cohort was 12%. HCM phenotype (57% vs. 9%, * P = 0.022) and delta waves identified by electrocardiography (43% vs. 0%, ** P = 0.002) were significantly more frequent in DD female patients.ConclusionsDanon disease is an underdiagnosed cause of AHF in young female patients. LAMP2 expression testing in peripheral WBCs by FC can be used as an effective screening/diagnostic tool to identify DD in this patient population.

Electrostatically bound lanreotide peptide - gold nanoparticle conjugates for enhanced uptake in SSTR2-positive cancer cells.

Lanreotide peptide (LP) has high affinity to somatostatin receptors like SSTR2 and is commonly used in the treatment of neuro-endocrine tumors. The main objective of this study is to target gold nanoparticles (AuNPs) towards SSTR2-positive cancer cells using lanreotide peptide (LP) as the targeting agent for enhanced tumor uptake and antitumor activity. pH mediated changes in the surface potential of LP and AuNP is used to prepare electrostatically bound AuNP-LP complexes. AuNP-LP complex formation was demonstrated by UV-Visible spectroscopy, surface potential, dynamic light scattering (DLS), small angle X-ray scattering and HR-TEM. Confocal microscopy and flow cytometric studies show that AuNP-LP complex has higher cellular uptake in SSTR2 expressed cancer cells (MCF-7 and AR42J) than in CHO cells. The enhanced cellular uptake of LP coated AuNPs lead to ~1.5 to 2-fold GSH depletion and enhanced ROS generation in MCF-7 cells. The preferential cytotoxicity of the AuNP-LP complex towards MCF-7 and AR42J cells, as revealed by MTT assay, is consistent with the increased cellular uptake. Our studies demonstrate that LP coated AuNP can be used as an effective platform to selectively target SSTR2 positive cancer cells for combination therapy approaches involving gold nanoparticles.

Unexpected enhancement of FVIII immunogenicity by endothelial expression in lentivirus-transduced and transgenic mice

AbstractFactor VIII (FVIII) replacement therapy for hemophilia A is complicated by development of inhibitory antibodies (inhibitors) in ∼30% of patients. Because endothelial cells (ECs) are the primary physiologic expression site, we probed the therapeutic potential of genetically restoring FVIII expression selectively in ECs in hemophilia A mice (FVIIInull). Expression of FVIII was driven by the Tie2 promoter in the context of lentivirus (LV)-mediated in situ transduction (T2F8LV) or embryonic stem cell–mediated transgenesis (T2F8Tg). Both endothelial expression approaches were associated with a strikingly robust immune response. Following in situ T2F8LV-mediated EC transduction, all FVIIInull mice developed inhibitors but had no detectable plasma FVIII. In the transgenic approach, the T2F8Tg mice had normalized plasma FVIII levels, but showed strong sensitivity to developing an FVIII immune response upon FVIII immunization. A single injection of FVIII with incomplete Freund adjuvant led to high titers of inhibitors and reduction of plasma FVIII to undetectable levels. Because ECs are putative major histocompatibility complex class II (MHCII)-expressing nonhematopoietic, “semiprofessional” antigen-presenting cells (APCs), we asked whether they might directly influence the FVIII immune responses. Imaging and flow cytometric studies confirmed that both murine and human ECs express MHCII and efficiently bind and take up FVIII protein in vitro. Moreover, microvascular ECs preconditioned ex vivo with inflammatory cytokines could functionally present exogenously taken-up FVIII to previously primed CD4+/CXCR5+ T follicular helper (Tfh) cells to drive FVIII-specific proliferation. Our results show an unanticipated immunogenicity of EC-expressed FVIII and suggest a context-dependent role for ECs in the regulation of inhibitors as auxiliary APCs for Tfh cells.

Study of the proliferative activity of hemolymph cells in pulmonate molluscs

Hemocytes, the cell of the hemolymph, play a key role in the immune response of pulmonate molluscs to various pathogens including trematode infection. The number of hemocytes is known to increase after immunization but the mechanism of their multiplication remains debatable, proliferative capacity being the stumbling block. Some scientists consider that hemocytes may proliferate, while others think that their multiplication is only possible in special hematopoietic centres. In this work we studied the proliferative activity of hemocytes in five species of pulmonate molluscs: Biomphalaria glabrata, Planorbarius corneus, Planorbis planorbis, Lymnaea stagnalis and Succinea putris. ImageStream technique was used for the study of the hemocyte populations of these molluscan species for the first time. The hemocytes of all the studied species were represented by two main types, granular cells and hyalinocytes. Microscopic and flow-cytometric study of the hemocytes with the use of EdU revealed some EdU-positive cells. However, the analysis of the cell cycle of the hemocytes showed that the amount of DNA in these cells was not increased. Thus, it remains unclear whether the cells of the hemolymph retain the capacity to multiplication.

Green-lipped mussel (Perna canaliculus) hemocytes: A flow cytometric study of sampling effects, sub-populations and immune-related functions

Green-lipped mussels (Perna canaliculus) are a commercially and culturally important bivalve species in New Zealand (NZ). As the highest value export aquaculture product in NZ, understanding and safeguarding the health of this species is imperative. The identification and characterization of hemocytes can provide useful information regarding the health of this species. Using flow cytometry (FCM), the present study assessed for the first time the use of different antiaggregant solutions and storage times on the immune-related parameters of hemocytes from cultured adult P. canaliculus. In addition, characterization of the immune-related functions of hemocyte sub-populations within the hemolymph were assessed. The two antiaggregant solutions tested (Modified Alserver's, MAS, A and B) maintained similar numbers of hemocytes in circulation over a 60 min period but, reduced the viability (MAS A) and increased the ROS production (MAS B) of the hemocytes compared to hemocytes diluted in cold filtered seawater (FSW). Hemocytes diluted in FSW and kept on ice showed significant aggregation after 2 h and a reduction in viability from 4 h. Three different hemocyte sub-populations were identified, discernible by their relative size and internal complexity: blast-like cells, hyalinocytes and granulocytes, which accounted for approximately 4, 67 and 29% of the total hemolymph population respectively. Granulocytes showed significantly higher reactive oxygen species production, phagocytic capabilities and neutral lipid content compared to hyalinocytes and blast-like cells. Results indicate that maintaining extracted hemolymph in cold FSW, completing analysis of fresh samples within 2 h of extraction and FCM assay incubation times of no longer than 30 min are best to obtain accurate results. Formalin fixation can also be used for future determination of hemocyte sub-populations and internal structures. Results from this study will allow effective future study of the effects of various stressors on P. canaliculus health and lead to improved management and production strategies in this species.

Abstract B15: Tumor genotype dependency of checkpoint blockade therapy in EGFR-driven glioblastoma

Abstract Background and Rationale: The tumor microenvironment plays a significant role in tumor progression and how patients respond to therapy. Recent work suggests that tumor genotypes significantly influence the immune landscapes of tumors and guide the degree of immune suppression; however, detailed mechanistic insights remain scant. Glioblastoma multiforme (GBM) is an aggressive primary brain cancer with a poor median survival of approximately 15 months. Approximately 60% of GBMs harbor overexpression of EGFR, of which 50% contain a specific mutant of EGFR (EGFRvIII). Downstream signaling of EGFRvIII differs from EGFR. Utilizing genetically engineered mouse models of EGFR- and EGFRvIII-driven GBM, we determined 1) the two GBMs comprise distinct immune landscapes, 2) EGFR GBMs respond to checkpoint blockade immunotherapy whereas EGFRvIII GBMs do not, and 3) how the unique cellular composition of EGFRvIII GBM immune landscapes actively suppresses the effects of checkpoint blockade. Methods: Conditional transgenic EGFR or EGFRvIII, CDKN2A null, PTEN floxed mice with a floxed luciferase reporter were stereotactically injected intracranially with iCre lentivirus to initiate GBMs. Cohorts of mice were imaged using bioluminescence (BLI) to detect and stage GBMs for treatment studies. Mice were randomly enrolled in treatments with antibodies against murine PD-1, CTLA-4 or combination, or matching IgG controls every 3 days for 3 doses. Treated mice were monitored for overall survival and tumor response by BLI. Mice were also sacrificed at predetermined time points before and after therapy to determine the immune landscape by flow cytometry. Results: We identified that, strikingly, only combination blockade of PD-1/CTLA-4 was effective in prolonging survival in EGFR GBMs but not in EGFRvIII GBMs. Flow cytometric studies demonstrate that response to combination blockade is dependent on the balance of CD8 T-cells to immunosuppressive PMN-MDSC infiltrates. Interestingly, EGFRvIII GBMs have higher PMN-MDSC infiltrates, which are associated with insensitivity to checkpoint blockade. Depletion of PMN-MDSCs in EGFR tumors restores sensitivity to monotherapy checkpoint blockade whereas depletion of PMN-MDSCs in EGFRvIII tumors restores sensitivity to combination treatment. Furthermore, EGFRvIII GBMs have higher mRNA expression of ligands for the neutrophil chemokine CXCR2 receptor compared to EGFR GBMs, suggesting a mechanism for the observed higher infiltration of PMN-MDSCs and resistance to checkpoint blockade in EGFRvIII GBMs. Conclusions: We present that combination blockade of PD-1/CTLA-4 provides survival benefit in EGFR GBMs whereas monotherapies are ineffective. All treatment conditions are ineffective in EGFRvIII GBMs. Flow cytometry analysis and depletion studies demonstrate that PMN-MDSCs play a role by limiting the efficacy of PD-1/CTLA-4 response. Our data point to the use of CXCR2 inhibitors in combination with checkpoint blockade as a potential approach for treatment of GBM. Citation Format: Alan T. Yeo, Alain Charest. Tumor genotype dependency of checkpoint blockade therapy in EGFR-driven glioblastoma [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B15.

Genome size analysis of field grown and somatic embryo regenerated plants in Allium sativum L.

In the present study, an efficient in vitro propagation protocol has been developed from clove explants of Allium sativum L., one of the oldest vegetable and medicinal plant used worldwide. Garlic is propagated vegetatively as cross-fertilization is strictly precluded due to sterile flowers. Due to a low rate of multiplication, limited genetic improvement possibility and increased germplasm degradation, plant tissue culture becomes an efficient and preferred tool for quality and rapid propagation of garlic. Here, the clove explants were cultured on Murashige and Skoog basal medium amended with different concentrations of Plant Growth Regulators (PGRs) namely 2,4-dichlorophenoxy acetic acid (2,4-D), 6-benzyl amino purine (BAP), and 1-naphthalene acetic acid (NAA). Within 2weeks of inoculation, white compact callus was formed, maximum callus induction frequency (85.99%) was on 1.5mgl-1 2, 4-D added MS medium. Induced callus transformed into an embryogenic callus on 2, 4-D and BAP amended MS medium with highest embryogenic frequency (77.7%) was noted on 0.25mgl-1 2, 4-D and 1.0mgl-1 BAP added medium. Embryogenic callus differentiated into progressive stages of somatic embryos starting from globular, scutellar, and finally to coleoptilar stage of the embryo. Histological and scanning electron microscopic study of embryogenic callus was conducted, showing different stages of embryos, their origin and development, re-confirming somatic embryogenesis incidence in A. sativum. Green and mature somatic embryos were germinated and converted into plantlets on 0.5mgl-1 BAP amended MS medium. The in vitro regenerated plants were cultured separately in IBA and NAA supplemented media for root induction. The MS medium amended with 1.0mgl-1 IBA proved to be the best PGR treatment in inducing roots. The rooted plants were acclimatized and transferred ex vitro with about 87% survival rate. Cytological and flow cytometric analyses were performed to assess the genetic stability of in vitro regenerated plants. Cytological studies of in vitro regenerated plants showed 2n = 16 chromosome number and did not reveal any numerical variation in chromosomes. Flow cytometry was employed to measure the 2C DNA content of somatic embryo regenerated A. sativum plants and compared with in vivo grown garlic. The histogram peaks of relative 2C DNA content of in vitro regenerated plantlets were similar to the corresponding 2C DNA peak of in vivo grown plants. Flow cytometric 2C DNA content of embryo regenerated and field-grown A. sativum plants were the same, i.e., 33.45pg and 33.56pg, respectively, confirming genetic similarity. In conclusion, the present cytological and flow cytometric study suggest that the in vitro culture conditions are quite safe, did not encourage genetic alterations, and regenerants were "true to type."

Role of interleukin 6 in liver cell regeneration after hemi-hepatectomy, correlation with liver enzymes and flow cytometric study.

Aim of the studyLiver regeneration after hemi-hepatectomy may be affected by several growth factors and cytokines. The aim is to evaluate the importance of interleukin 6 (IL-6) in the induction of liver cell regeneration and find correlations with other parameters such as liver enzymes, and DNA analysis by flow cytometric studies.Material and methods80 adult male Sprague-Dawley rats were obtained and divided into two equal groups (n = 40 rats) to undergo 70% partial hepatectomy: group 1 – untreated (control) group; 40 rats not treated; and group 2 – treated group, 40 rats treated with IL-6 35 μg/100 gm body weight according to a lethality study for a period of 4 days, then hepatic resection was carried out according to the steps of Higgins and Anderson. Assessment of liver enzymes and bilirubin level was done. Flow cytometric study was done using a flow cytometer (FACSCalibur; Becton Dickinson) and DNA content was estimated with CellQuest software (Becton Dickinson).ResultsThe levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP) were significantly higher in the untreated group of rats with liver resection. A higher value of bilirubin was observed in the treated group. Rat weight at sacrification was significantly lower in the group of rats treated with IL-6 than those without treatment, p < 0.001. Liver weight at sacrification was significantly higher in the group of rats treated with IL-6 (p < 0.001). The percentage of apoptotic cells with hypodiploid DNA content was determined from DNA histograms. Untreated rat resected liver showed a peak pattern that represented liver damage with high damage of 73.4%.ConclusionsInterleukin 6 is of value in induction of liver cell regeneration after seventy percent hemi-hepatectomy as evident by increased liver cell mass, liver enzymes and flow cytometric analysis.

Impact of storage, leukofiltration, and ascorbic acid fortification on red cell-derived microparticles in stored packed red blood cells: A flow cytometric and procoagulant study

INTRODUCTION: During blood storage, red blood cells (RBCs) undergo changes termed “storage lesion” including shape modification and microparticles (MPs) production, as well as oxidative injury to lipids and proteins. OBJECTIVE: We studied the impact of storage on RBCs by quantification of red cell MPs and their potential procoagulant activity (PCA). The antioxidant effect of ascorbic acid (AA) was assessed, as a proposed additive, to improve stored RBC quality. METHODS: Forty randomly selected packed red cell units (filtered and nonfiltered) were sampled weekly over 35 days of storage. Flow cytometric and coagulation tests were performed on MPs isolated from erythrocyte concentrates. RBCs were either in citrate phosphate dextrose adenine-1 (CPDA1) only (standard storage) or CPDA1 with AA (Vitamin C) in three concentrations. RESULTS: Red cell MPs progressively increased over 35 days of storage. Filtration efficiently reduced the production of MPs (P < 0.001). PCA increased significantly on storage assessed by the shortened coagulation time (P < 0.001). A strong inverse correlation between coagulation time and Annexin V positive MPs was found, (r = −0.723, P < 0.001), and the dual-positive population. This highlights the strong correlation between the functional assay (PCA) and the direct quantitative assay (flow cytometry). The red cells fortified with AA demonstrated a reduction in the accumulation of RBC MPs by almost 50% compared to standard storage in CPDA-1 alone in an evident dose-dependent manner. CONCLUSION: We concluded that MPs increase on storage with increased PCA. AA reduced MPs production which can be possible additive for further research in the future

Bombesin receptor-targeted liposomes for enhanced delivery to lung cancer cells.

Background: Gastrin-releasing peptide is a member of the bombesin family of peptides. Its cognate receptor, gastrin releasing peptide receptor (GRPR), is widely expressed in cancers of the lung, pancreas and ovaries. Gastrin releasing peptide (GRP) is an autocrine growth factor in small cell lung cancer, which has very poor patient outcomes. High affinity antagonist peptides have been developed for in vivo cancer imaging. In this report we decorated pegylated liposomes with a GRPR antagonist peptide and studied its interaction with, and accumulation within, lung cancer cells.Results: An N-terminally cysteine modified GRPR antagonist (termed cystabn) was synthesised and shown to inhibit cell growth in vitro. Cystabn was used to prepare a targeted 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (DSPE-PEG2000) lipid conjugate that was formulated into liposomes. The liposomes displayed desirable colloidal properties and good stability under storage conditions. Flow cytometric and microscopic studies showed that fluorescently labelled cystabn-decorated liposomes accumulated more extensively in GRPR over-expressing cells than matched liposomes that contained no cystabn targeting motif.Conclusion: The use of GRPR antagonistic peptides for nanoparticle targeting has potential for enhancing drug accumulation in resistant cancer cells.

Anti-cancer potential of polysaccharide extracted from hawthorn (Crataegus.) on human colon cancer cell line HCT116 via cell cycle arrest and apoptosis

Abstract This study demonstrated that a homogeneous polysaccharide (HPS) extracted from Hawthorn exerted anti-cancer effects on colon cancer cells. Human colon cancer cell line HCT116 were treated with 125–1000 µg/mL HPS for 12 h and subsequent analysis was performed on proliferation and signaling pathways. HPS significantly inhibited proliferation of HCT116 cells when analyzed using WST-1 assay and immunofluorescence staining of Ki-67. Flow cytometric studies revealed that HPS could arrest the cell cycle in the S and G2/M phases and increase the rate of apoptosis. HPS downregulated the expression of Cyclin A1/D1/E1 and CDK-1/2. HPS induced apoptosis on HCT116 cells through upregulation of caspase3, 7, 8, 9 and Fas, FADD, TNF-R1, TRADD, upregulation of caspase3 protein was also observed. HPS has an anti-cancer potential in the treatment of human colon cancer which makes it a potential candidate in functional foods for cancer patients.

Clinical Utility of Flow Cytometry Immunophenotyping in Acute Myeloid Leukemia

INTRUDUCTION: Acute myeloid leukemia (AML) is a heterogeneous group of clonal disease of hematopoietic tissue, characterized by proliferation of abnormal myeloid progenitor cells, suppressing the normal hematopoietic activity and constituting a great diagnostic challenge. With the advent of immunophenotyping by flow cytometry, the diagnosis of these neoplasms became more faithful, facilitating the treatment and follow-up of the patients. METHODS: In this study a flow cytometric immunophenotyping study was performed in bone marrow aspirate and / or peripheral blood samples from 38 patients with AML, using a panel of monoclonal antibodies specific for acute leukemias, also investigating clinical, laboratory and demographic data of these patients. RESULTS AND DISCUSSION: Of the 38 subjects studied, 23 were male and 15 females. In relation to the age, we found a higher number of cases in adult patients. According to clinical data, splenomegaly and hepatomegaly were present in most cases. The immunophenotyping demonstrated a characteristic profile of AML with expression of CD13 and CD33 in all cases and CD34 and CD117 in most cases. The CD14 was reactive in monocytic leukemia (M4 and M5-AML). Were observed negativity for lymphoid antigens CD19, CD10 and CD3 and positivity to CD7, CD56 and CD2, present in 10, 5 and one cases respectively. Regarding cytomorphology, there was a direct correlation with FAB classification, with the prevalence of type myeloid-monocytic (AML-M4). CONCLUSION: These data demonstrate the importance of immunophenotyping in the differential diagnosis of AML and monitoring of these neoplasms. Disclosures No relevant conflicts of interest to declare.

Clinical Utility of B- and T-Cell Gene Rearrangement Studies in Blood and Bone Marrow Samples

In the evaluation of bone marrow (BM) and peripheral blood (PB) for hematologic malignancy, positive immunoglobulin heavy chain (IG) or T-cell receptor (TCR) gene rearrangement results may be detected despite unrevealing results from morphologic, flow cytometric, immunohistochemical (IHC), and/or cytogenetic studies. The significance of positive rearrangement studies in the context of otherwise normal ancillary findings is unknown, and as such, we hypothesized that gene rearrangement studies may be predictive of an emerging B- or T-cell clone in the absence of other abnormal laboratory tests. Data from all patients who underwent IG or TCR gene rearrangement testing at the authors' affiliated VA Hospital between January 1, 2013 and July 6, 2018 were extracted from the electronic medical record. Date of testing; specimen source; and morphologic, flow cytometric, IHC, and cytogenetic characterization of the tissue source were recorded from pathology reports. Gene rearrangement results were categorized as true positive, false positive, false negative, or true negative. Lastly, patient records were reviewed for subsequent diagnosis of hematologic malignancy in patients with positive gene rearrangement results with negative ancillary testing. A total of 136 patients, who had 203 gene rearrangement studies (50 PB and 153 BM), were analyzed. In TCR studies, there were 2 false positives and 1 false negative in 47 PB assays, as well as 7 false positives and 1 false negative in 54 BM assays. Regarding IG studies, 3 false positives and 12 false negatives in 99 BM studies were identified. Sensitivity and specificity, respectively, were calculated for PB TCR studies (94% and 93%), BM IG studies (71% and 95%), and BM TCR studies (92% and 83%). Analysis of PB IG gene rearrangement studies was not performed due to the small number of tests (3; all true negative). None of the 12 patients with false positive IG/TCR gene rearrangement studies later developed a lymphoproliferative disorder, although two patients were later diagnosed with acute myeloid leukemia. Of the 14 false negatives, 10 (71%) were related to a diagnosis of plasma cell neoplasms. Results from the present study suggest that positive IG/TCR gene rearrangement studies are not predictive of lymphoproliferative disorders in the context of otherwise negative BM or PB findings. As such, when faced with equivocal pathology reports, clinicians can be practically advised that isolated positive IG/TCR gene rearrangement results may not indicate the need for closer surveillance. Disclosures No relevant conflicts of interest to declare.

Assessment of intermittent exposure of zinc oxide nanoparticle (ZNP)-mediated toxicity and biochemical alterations in the splenocytes of male Wistar rat.

Nanoparticles are being used extensively and found in applications to various fields ranging from agriculture to electronic devices, diagnosis to drug delivery, and cosmetics to food packaging. Increasing usage of engineered nanomaterials (ENM) raises potential concern for human health as well as to the environment. The present study aims to explore the effects of intermittent intraperitoneal exposure of ZNP on the spleen of male Wistar rat. Animals were divided into three groups, control and ZNP-treated groups (50 mg/kg and 250 mg/kg body weight), six in each group. Experimental animals were treated with different doses of ZNP once a week for 4 weeks, whereas control groups received water. After 28 days of exposure, animals were sacrificed, spleen tissue was excised, and various parameters such as hematological, genotoxicity, antioxidants, and histopathological were studied for changes in spleen if any. Results showed that ZNP exposure manages to induce alteration in various studied hematological parameters like neutrophils, platelets, and eosinophils which are found to increase significantly after the last treatment compared with the first treatment of ZNP. However, hemoglobin content, PCV, and MCV decrease with increasing dose of ZNP significantly in last treatment, when compared with the first treatment. DNA damage was observed in rats treated with a high dose of ZNPs compared with that in the control when analyzed through comet assay. Flow cytometric study was performed for better understanding of the underlying mechanism of the ZNP-mediated toxicity. From the present investigation, an increase in ROS production, a decrease in MMP, and increased apoptosis were exhibited. Further, altered antioxidant level (SOD, CAT, LDH, CYT P450, and CYT b5 r) has been observed in the studied splenic tissue, also histopathological changes observed in the rats exposed with high doses of ZNP. Therefore, ZNP may have the potential to induce a toxic effect even when exposed intermittently.

Diagnostic workup in front of an adult monocytosis

The discovery of a monocytosis is a frequent phenomenon, requiring confirmation by reading under a microscope by an experimented biologist, to overcome usual cytological traps such as the presence of hairy cells, promonocytes or monoblasts. In the vast majority of cases the secondary origin is very easily found by the context and/or the presence of a biological inflammatory syndrome. More rarely the diagnosis is directed towards an eosinophilic pathology or an acute leukemia. In other cases, CMML, MPN or MDS with monocytosis may be highlighted. In the absence of any pathognomonic element and the presence of "borderline" forms the differential diagnosis between these 3 entities is not always straightforward, requiring, according to WHO, molecular investigations and elimination of any reactive cause of monocytosis. Although histological, immunohistochemical and phenotypic flow cytometric studies are not currently recommended by WHO, these investigations could be of interest in the evaluation of difficult cases.