Flow Cytometric Results Research Articles

Identification of Nrf2/STAT3 axis in induction of apoptosis through sub-G 1 cell cycle arrest mechanism in HT-29 colon cancer cells.

We investigated the role of stattic as an adjuvant molecule to increase the cytotoxicity of 5-fluorouracil (5-FU) through specific inhibition of molecular targets, signal transducer and activator of transcription 3 (STAT3) and nuclear factor erythroid 2-related factor 2 (Nrf2) in HT-29 colon cancer cells. Cytotoxicity and apoptotic effects were investigated by methylthiazolyldiphenyl-​tetrazolium bromide assay and flow cytometry analysis, respectively. Real-time polymerase chain reaction was applied to assess the messenger RNA (mRNA) level of STAT3, Nrf2, and apoptotic genes including Bax, Bcl-xl, and Bcl-2. The antitumor effect of 5-FU in combination with stattic induced synergistic effect in HT-29 cells with combination indexes (CIs) 0.49. Flow cytometric results related to apoptotic confirmed that there was up to 40% increase in the population of apoptotic cells in HT-29 colon cancer cells incubated with 5-FU and stattic compared with control groups. Our data from gene expression determined a substantial diminish in the mRNA levels of the Nrf2 and antiapoptotic gene Bcl-2 along with a noticeable increase in the level of the proapoptotic Bax in HT-29 colon cells that underwent cotreatment with 5-FU and stattic (P < 0.05). Moreover, the results exhibited that stattic can be used as adjuvant chemotherapy besides the 5-FU. This therapeutic approach in colon cancer could mediate 5-FU chemoresistance via modulating therapeutic targets (ie, STAT3 and Nrf2 pathways) and decreased 5-FU-related adverse effects.

Curcumin Induces Apoptosis by regulating the cell cycle proteins in MDA‐MB‐231 Breast CancerCells

BackgroundCurcumin, a natural polyphenolic compound found in turmeric, has potential cancertreatment efficacy, including apoptosis‐induction, in many cancer cell types. Cancer cells arecharacteristically anti‐apoptotic, highly proliferative andcontinue to live indefinitely. Controlof cell cycle progression and the initiation of apoptosis in breast cancercells is considered to be a potentially effective strategy for the prevention/treatment of the disease. The triple‐negative breast cancer cell line MDA‐MB‐231 does not express progesterone, estrogen, or human epidermal growth factor receptors, is highly aggressive and is difficult to treat. This study aims to elucidate the mechanism of curcumin‐induced apoptosis and its effects on cell cycle proteins in the MDA‐MB‐213 breast cancer cell line.MethodsApoptosis was measured by flow cytometric analysis of AnnexinV/Propidium Iodide expression. Cell cycle arrest was measured by Western Blot analyses of p21Waf‐1, p53 and retinoblastoma protein pRb, as well as the cyclin dependent kinases Cyclin B1, and Cyclin D1.ResultsFlow cytometry data suggest that curcumin induces both early and late stage apoptosis in MDA‐MB‐231 cells. Cell cycle inhibitory protein p21Waf‐1 was significantly increased in curcumin‐treated cells compared to untreated cells. Tumor suppressor protein p53 and Cyclin D1 were significantly down‐regulated (p=.0.0001 and p=0.014, respectively), pRb, another tumors uppressor protein, was unchanged, and Cyclin B1 was upregulated (p=0.0006).ConclusionGiven that upregulation of p21Waf‐1 can lead to apoptosis and can disrupt CDKs/cyclin complexes necessary for cell cycle progression, and that down‐regulation of Cyclin D1, which is required for progression through the G1/S phase and whose over expression is associated with most breast cancers, can lead to cell‐cycle arrest at G1, curcumin appears to be effective in both apoptosis induction, demonstrated also in the flow cytometric results, and in cell‐cycle arrest at G1. The upregulation of cyclinB1, which is involved in the G2/M phase transition, and the down‐regulation ofp53 by curcumin, indicate that additional mechanisms are involved with curcumin exposure. Our findings will aid in the development of future therapeutic strategies, including curcumin as a novel treatment supplement, to effectively induce both cell‐cycle arrest and apoptosis in breast tumors.Disclaimer: The views expressed in this manuscript are those of the author and do not reflect the official policy of the Department of Army, Department of Defense, or U.S. Government.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Development of flow cytometry based adherence assay for Neisseria gonorrhoeae using 5\u2032-carboxyfluorosceinsuccidyl ester

BackgroundNeisseria gonorrhoeae is an obligate human pathogen and its adherence to host cells is essential for its pathogenesis. Gonococcal adherence assays are based on the enumeration of bacteria attached to human cells on solid media. Because conventional adherence assays are based on bacterial counts, they are often time consuming to perform and prone to observer bias. A flow cytometry based method, using the cell-permeable fluorescent dye 5′-carboxyfluoroscein succidyl ester (CFSE), was developed to dramatically increase the number of adherent N. gonorrhoeae quantified per assay while improving repeatability and removing observer bias. Piliated N. gonorrhoeae F62 were stained with CFSE then the staining reaction was quenched with foetal bovine serum. Human cervical ME-180 cells were infected with CFSE-stained N. gonorrhoeae (multiplicity of the infection 100:1) for 2 h. Infected cells were washed to remove loosely adhered bacteria. Flow cytometry was used to quantify the percentage of ME-180 cells associated with CFSE-stained N. gonorrhoeae and a minimum of 30,000 events were recorded. Real time-PCR analysis targeting opa gene (encoding N. gonorrhoeae opacity associated gonococcal outer membrane protein) was performed on infected ME-180 cells to confirm the flow cytometric adherence assay results. A rabbit was immunized with heat-killed N. gonorrhoeaeF62 to generate hyperimmune serum. The functional compatibility of the assay was confirmed by studying the effect of N. gonorrhoeae F62 antiserum on blocking adherence/invasion of CFSE-stained bacteria to ME-180 cells.ResultsWe observed that 20.3% (+/− 1.0) ME-180 cells were associated with CFSE-stained N. gonorrhoeae. Heat-inactivated hyperimmune serum, at 1:10 to 1:80 dilutions, significantly inhibited gonococcal adherence by 6 and 3 fold, respectively. Real time-PCR analysis targeting opa gene confirmed that hyperimmune serum blocked adherence/invasion of N. gonorrhoeae to the ME-180 cells in a dilution-dependent manner.ConclusionsFlow cytometric analysis was amenable to quick, easy and high-throughput quantification of the association of N. gonorrhoeae with ME-180 cells and was functionally confirmed using PCR analysis. These approaches may be adapted for in vitro and in vivo adherence studies related to gonococcal pathogenesis.

Allicin-induced modulation of angiogenesis in lung cancer cells (A549)

Purpose: To investigate the effectiveness of allicin as a candidate for lung cancer therapy.Methods: Allicin solution at different concentrations was tested on A549 lung cancer cell line. Viability and proliferation of A549 cells were determined. The cytotoxic effects of allicin solution on A549 lung cancer cells were quantitatively determined using Alamar blue assay. Cell proliferation and cell cycle were measured by DAPI-flow cytometry analysis in order to investigate the possible cell signalling pathway targeted by allicin. The level of expression of VEGF-A protein was determined with confocal laser scanning microscopy.Results: Allicin inhibited lung cancer proliferation and down-regulated the protein expression of VEGF, but had no significant cytotoxic effects on A549 cells. Flow cytometric results showed that allicin induced cell cycle arrest of A549 cells at the G1 phase.Conclusion: These results indicate that allicin exerts anti-proliferative effects on A549 lung cancer cells. Thus allicin, an active component of garlic, might be a promising therapy against lung cancer metastasis.Keywords: A549 cells, Allicin, qPCR Taqman assay, VEGF-A protein expression, Garlic, Lung cancer

Abstract A021: Vaccination with autologous, nonattenuated, live glioblastoma cells induces potent peripheral and intratumoral anti-tumoral responses: A first-in-human study

Abstract Glioblastoma (GBM), which grows uninhibited in the brain, almost never metastasizes outside of it. The rare occurrence (&amp;lt;0.5%) of overt GBM extracranial metastasis is paradoxically associated with significantly enhanced patient survival. We previously reported that the highly malignant F98 Fischer-rat GBM model, which grows aggressively in the brain, spontaneously regresses when injected subcutaneously as live unattenuated cells. The peripheral growth and regression of live glioma cells markedly enhanced the survival of all rats, and cured (survival &amp;gt;9 months) approximately 50% of rats challenged with F98 cells intracranially. The results in the F98 model were corroborated in the CNS1 astrocytoma model in Lewis rats. The fraction of cured rats in both models were the highest reported in published literature. We found that the spontaneous rejection of live glioma cells inoculated in peripheral sites was immune-mediated. This location-biased immune response was termed “Split Immunity”—a tumor that thrives in an immune-privileged site (e.g., the brain) may be inhibited by injecting live unmodified tumor cells in a site that is not privileged, generating protective immunity that spreads back to the privileged site.To evaluate the applicability of the “Split Immunity” approach in humans, we ran a first-in-human (FIH) study on two recurrent GBM patients. The patients were vaccinated subcutaneously, initially with autologous irradiated tumor cells, and then with autologous, non-attenuated, live tumor cells. The treatment caused no serious adverse events. The injected live tumor cells did not grow at the inoculation site, nor did they spread metastatically, as evaluated by choline 18F PET-CT. Standard biochemical and hematologic blood tests showed no treatment-related adverse effects. Both patients’ quality of life (QoL) questionnaires demonstrated subjective improvements in self-evaluated “global health status.” The treatment had demonstrated several signs indicative of efficacy. MRI evaluation using “treatment response assessment maps” (TRAM) demonstrated strong increases in the “treatment-response” component following live-cell vaccination, compatible with “pseudo-progression-like” responses in both patients. Using elaborate 8-color flow cytometric panels, we detected potent tumor-cell specific polyfunctional activation in 2.5% and 5% of peripheral cytotoxic T-cell (CTL) and T helper cells (Th), respectively, 3 days following live-cell vaccination. Flow cytometric immune mapping of all intratumoral immune cell subsets showed major changes from baseline that had occurred shortly after live-cell vaccinations. We observed major increases (8 to 200 fold) in both patients of all monitored intratumoral dendritic cell (DC) subsets (CD1c+ and CD141+ myeloid DC, CD123+ plasmacytoid DC). Patient-1 showed the highest frequency of intratumoral CD56brt natural killer (NK) cells from all previously fully mapped brain tumor patients. Patient-2’s intratumoral CTL and Th were increase by 6- and 17-fold, respectively. Patient-2’s CD141+ and CD1c+ myeloid DC had exhibited upregulated CD86 maturation marker following treatment. Overall, the immune infiltrate following treatment in Patient-2 exceeded the immune infiltrates found in 42 previously fully-mapped brain tumor patients. The flow cytometric results were corroborated by the patients’ immunohistochemistry data. Patient-2, who had been treated by a tighter vaccination protocol than Patient-1, had survived 19 months after GBM recurrence, which is considerably longer than the median 6- to 8-month survival of recurrent GBM patients. Taken together, the accumulated FIH results are suggestive of safety and efficacy of the “Split Immunity” approach. Based on the these results, we have recently received an ethical approval for a phase IIa clinical trial on 8 recurrent- and 4 newly-diagnosed GBM patients. Citation Format: Ilan Volovitz, Nati Shapira, Rachel Grossman, Zvi Ram. Vaccination with autologous, nonattenuated, live glioblastoma cells induces potent peripheral and intratumoral anti-tumoral responses: A first-in-human study [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A021.

Detection and Characterization of the Cyanobacteria and Cyanophages of Barnegat Bay, New Jersey

Cyanobacterial harmful algal blooms (CHAB), caused by eutrophication, are known to threaten both wildlife and human health. Due to urbanization and land use changes, an increase of CHAB’s at a more frequent rate within Barnegat Bay has been observed. In order to detect possible CHAB causing cyanobacteria, water samples were collected from 12 different locations within Barnegat Bay. Each sample was filtered through a 30- and 0.4-μm polycarbonate filter sequentially. Flow cytometry was carried out for the filtrate collected between 0.4- and 30-μm. Chelex DNA extraction, polymerase chain reaction (PCR), and gel electrophoresis were then performed for all sites using four primer sets (27F/785R, PSF/UR, CYA359F/CYA781R and MSF/MSR) designed to detect cyanobacteria. Flow cytometric results indicated the water samples contained a wide range of cyanobacteria, including M. aeruginosa, Cylindrospermum spp., Anabaena spp., and Synechococcus sp. IU 625 ranges from 3.16 to 8.17 × 107 cells·L-1. PCR-based assays suggest that general cyanobacteria as well as phytospecific species were present for all sites, but no toxin-producing Microcystis aeruginosa was detected. Plaque assays demonstrated the presence of cyanophages for S. IU 625, Anabaena spp., and M. aeruginosa at all sites, up to 105 PFU·ml-1.

Switching Towards Monocytic Lineage and Discordancy between Flow Cytometric and PCR Minimal Residual Disease Results Is a Hallmark Feature of DUX4 Rearranged B-Cell Precursor Acute Lymphoblastic Leukemia

Introduction:Recently we described a subgroup of pediatric patients with B cell precursor acute lymphoblastic leukemia (BCP ALL) with switching from B to monocytic lineage in early phase of the therapy (Slamova et al., 2014). In a limited cohort of patients with switching ALL (swALL), we observed inferior response to treatment with discrepancy of minimal residual disease level (MRD) assessed by flow cytometry (FC) and quantitative polymerase chain reaction (qPCR) of Immunoglobulin-T cell receptor (Ig-TCR) rearrangements. In current Berlin-Frankfurt-Münster (BFM) treatment protocols, FC MRD value at day 15 (d15) and PCR MRD value at day 33 (d33) and week 12 (w12) are used for stratification. Using an extended cohort of patients with available RNA sequencing data (cohort mainly focused on B other cases or swALLs) we aimed to answer following questions:What is the genetic background of swALL? What is the frequency among swALLs of the recently described DUX4 rearranged subgroup?How do B cell oriented FC and PCR MRD correlate in standard protocol timepoints, i.e. day 8 (d8) (peripheral blood, PB) d15, d33 and w12 (bone marrow, BM) of treatment?What is the characteristic MRD response to treatment in swALL?Results:We performed RNA sequencing in 177 patients (median age 6.1 years, range 0-18) treated by several treatment protocols (ALL BFM 95 n=5, ALL IC BFM 2002 n=14, ALL AIEOP BFM 2000 n=17, ALL AIEOP BFM 2009 n=135, Interfant n=3, ALL IC/Interfant n=2, EsPhALL n=1). In 68 patients we observed switching phenomenon by appearance of B/monocytoid population coexpressing B lineage (CD19, CD34) and monocytic lineage (CD33, CD14) markers (median 0.98%, range 0.032-38%). In non swALLs median of this population was 0.059% (range 0.0025-1.1%) and the cells did not form a clear cluster. According to RNAseq data, majority of swALL patients (n=42/68) belong to DUX4 subgroup (chi square p< 0.00001). The distribution into other molecular genetic subtypes is summarized in table 1.Correlation coefficient (Spearman) of all included samples with both available values (n=552) was 0.82 (p<0.0001), Concordance in categorization of positivity and negativity with cut-off 1e-4 was 85%. We observed worse correlation between FC and PCR MRD in patients with swALL (d8 R=0.58, d15 R=0.6, d33 R=0.36, w12 n.s.) compared to non swALL (d8 R=0.83, d15 R=0.91, d33 R=0.69, w12 R=0.37). However, concordance in swALL in categorization of positivity and negativity with cut-off 1e-4 was still ≥80% in each analyzed timepoint apart from d33 with concordance only 44% showing significant discrepancy of both methods (Figure 1a). On the contrary, concordance in non swALL was ≥87% for each analyzed timepoint (d33 with concordance 87% shown in Figure 1b). Poor correlation between B-cell oriented FC MRD and PCR MRD at d33 was also obvious when analyzed DUX4 subgroup separately (R=0.31 (p=0.04), concordance 45%).We observed significantly higher MRD in swALLs compared to non swALLs at all analyzed timepoints: d8 (p=0.0021), d15 (p=0.0088, d33 (p<0.0001) and w12 (p=0.008). Higher MRD levels were also found in DUX4 patients when compared to non DUX4 (all timepoints p<0.05). Interestingly, when compared swALL and non swALLs pts in DUX4 subgroup only, the DUX4 swALLs are those with poorer treatment response (all timepoints p<0.05). With respect to protocolar cut-off values, FC MRD at d15 was above 10% in 18/67 swALL patients (in 13/52 DUX4 patients), d33 PCR MRD was above 0.1% in 33/57 swALLs (24/44 DUX4 pts) and at w12 PCR MRD was above 0.01% in 12/55 swALLs (10/44 DUX4 pts).Conclusions:DUX4 subgroup is the most prevalent genetic subtype among swALLs. SwALLs and/or DUX4 subgroup have poorer treatment response at d15, d33 and w12. However, it remains to be elucidated whether poor initial treatment response is eventually reflected in treatment outcome. In majority of swALL patients B cell phenotype of blasts is preserved at day 15 enabling correct classification. Prominent discrepancy between FC and PCR MRD is present especially at d33 and development of different FC MRD strategies focused on monocytic compartment is needed.Supported by Ministry of Health of the Czech Republic, grant nr. 15-28525A and NV18-03-00343; Czech Science Foundation nr.P302/12/G101, UNCE204012 [Display omitted] DisclosuresBrüggemann:Affimed: Research Funding; Regeneron: Research Funding; Amgen: Consultancy, Research Funding, Speakers Bureau; Roche: Speakers Bureau; Pfizer: Speakers Bureau; Incyte: Consultancy; PRMA: Consultancy. Ritgen:abbvie: Research Funding; Roche: Honoraria, Research Funding.

Central Nervous System Involvement Detected By Flow Cytometry Is a Risk Factor for Relapse in Childhood Acute Lymphoblastic Leukemia

Background: Central nervous system (CNS) involvement is common in childhood acute lymphoblastic leukemia (ALL). Today all patients receive intensive prophylactic CNS-directed therapy, which is associated with short- and long-term neurotoxicity. The conventional method for diagnosing CNS leukemia is by microscopic examination of cytospin preparations of cerebrospinal fluid (CSF). Both high (CNS3: ≥5 x 109 cells/L) and low level CNS disease (CNS2: <5 x 109 cells/L) indicate intensified CNS-directed therapy. The CNS is involved in 30-40% of relapses, and most occur in patients classified as CNS negative by cytospin (CNS1). We investigated if sensitive flow cytometric (FCM) analysis of CSF at diagnosis improves detection of CNS involvement and prediction of relapse risk.Methods: Cytospin results were collected from local oncology clinics for patients treated according to the Nordic Society of Pediatric Hematology (NOPHO) ALL2008 protocol from July 2008 to December 2017 (N=1841). CSF samples for FCM were obtained from patients enrolled from September 2012 to December 2017 at 17 participating centers (N=669). CSF samples were collected into Transfix® tubes (Intermedico Ltd., Hellerup, Denmark) and shipped to Rigshospitalet, Copenhagen, Denmark for centralized analysis as previously described (Levinsen at al., Pediatr Blood Cancer 2016). FCM positivity required detection of ≥10 cells with a leukemia-associated phenotype. CSF FCM results were blinded to the treating physician. Time to relapse was analyzed using Cox proportional hazards models with delayed entry at remission and with death and second malignant neoplasm as competing events.Results: At diagnosis, 241 of 1841 (13.1%; CNS2: 8.9% and CNS3: 4.2%) patients were positive by cytospin, whereas 167 of 669 (25.0%) patients were positive by FCM. FCM positivity was not associated with shipment duration (median: 2 days). In total, 191 of the 669 patients (28.6%) were CNS positive by cytospin and/or FCM. CNS positivity was associated with higher white blood cell (WBC) count at diagnosis, T-cell ALL, lack of t(12;21), and traumatic lumbar puncture (TLP) (all comparisons: p < 0.001). However, patients with CNS positivity did not differ in their end of induction bone-marrow minimal residual disease (MRD) levels compared to the remaining patients (cytospin: median MRD 1.2 x 10-4 vs. 0.9 x 10-4, p = 0.058; FCM: median MRD 2.0 x 10-4 vs. 1.7 x 10-4, p = 0.62). During follow-up, 30 patients relapsed with nine of the relapses involving the CNS and 16 relapses isolated to the bone-marrow. The 4-year cumulative incidence of any relapse was higher for patients who were CNS positive by cytospin (16.1% vs. 8.9%), FCM (18.6% vs. 6.0%), and combined cytospin/FCM (19.0% vs. 5.5%) (Figure 1a). Simple Cox regressions yielded relapse hazard ratio (HR) estimates of CNS positivity at diagnosis of 2.4 for cytospin (95% CI 1.0-5.7, p = 0.04), 3.5 for FCM (95% CI 1.7-7.2, p < 0.001), and 4.1 for combined cytospin/FCM (95% CI 2.0-8.7, p < 0.001). Cytospin and/or FCM was associated with increased risk of both isolated bone-marrow relapse (16 events, HR 3.1, 95% CI 1.2-8.4, p = 0.024) and of any CNS relapse (9 events, HR 4.9, 95% CI 1.2-19.5, p = 0.025). In a multiple Cox model stratified by immunophenotype and risk group, predictors of relapse were age (per year: HR 1.1, 95% CI 1.0-1.2, p = 0.003), WBC at diagnosis (per doubling: HR 1.3, 95% CI 1.1-1.6, p = 0.005), and cytospin and/or FCM (HR 3.2, 95% CI 1.4-7.2, p = 0.006) (Figure 1b). Cytospin and/or FCM was associated with a significantly higher risk of relapse for BCP-ALL (N=581; 23 relapses; HR 4.5, 95% CI 1.9-10.4, p = < 0.001). For T-cell ALL no significant association was observed (N=85; 7 relapses; HR 1.6, 95% CI 0.3-8.2, p = 0.58), however the number of T-cell ALL was too small to allow reliable conclusions. In a simple Cox model, TLP was associated with a significantly higher risk of relapse than no TLP (HR 2.7, 95% CI 1.3-5.9, p = 0.011), but this was only the case for patients who were positive by FCM (FCM pos: HR 2.7, 95% CI 1.0-7.0, p = 0.043; FCM neg: 0.7, 95% CI 0.1-5.0, p = 0.68).Conclusion: FCM markedly increases the detection rate for CNS involvement at diagnosis, and distinguish between patients at high and low risk of relapse. Diagnosis of CNS leukemia by combined cytospin and FCM analysis should be the standard for accurate classification of CNS status, which will enable better stratification of CNS-directed and systemic therapy. DisclosuresNo relevant conflicts of interest to declare.

Flow Cytometry in Neoplastic Hematology: Morphologic‐Immunophenotypic Correlation, 3rd ednW.GorczycaCRC Press, Boca Raton, 2017, ISBN 978‐1‐4987‐7502‐1

There are now a number of useful books available to help familiarise haematologists and haematology trainees with the principles and practice of flow cytometric immunophenotyping. Two others (Leach et al, 2013; Porwit & Béné (eds), 2018) have previously been reviewed in this journal. The third edition of this book is a valuable addition. Not surprisingly, given that it comprises 456 pages, it is comprehensive. In fact it is so informative that trainees may find it overwhelming. It is clearly written, well illustrated and supported by extensive references. The principles are thoroughly explained and there are 13 chapters dealing with individual diseases or groups of diseases. Flow cytometric immunophenotyping results are correlated with morphological characteristics and molecular/cytogenetic abnormalities. Limitations of the procedure are discussed. The text is up-to-date, incorporating the 2016 revision of the WHO classification of tumours of haemopoietic and lymphoid tumours. Since there is a great deal of detailed information, it will serve as a useful reference for immunophenotyping laboratories as well as being an educational tool for others. I have only a few minor quibbles. The term ‘granulocyte’ is not synonymous with ‘neutrophil’ and it is sometimes not clear whether ‘granulocyte’ is being used when ‘neutrophil’ is intended, or is being used more correctly to include eosinophils and basophils. There are some curious units; how does one interpret a white cell count of greater than ‘100,000k/μl’? There are occasional copy-setting errors, which can make a figure or a table confusing. Overall, a useful addition to your laboratory bookshelf.

Diagnostic Approach of New-Onset Pancytopenia: Study from A Tertiary Care CenterPancytopenia 2 Years Retrospective Study

Background: Pancytopenia is a disorder associated with decrease in number of all three major formed elements of blood (red blood cells, white blood cells and platelets). It is a triad of findings resulting from many disease processes. This may be associated with various hematopoietic and non-hematopoietic condition involving the bone marrow. The management and prognosis of patients with pancytopenia is based on the severity of pancytopenia and the underlying disease process. The present study helps in highlighting the role of various laboratory parameters in conjunction with clinical findings in evaluation of these patients. Methods: The present retrospective study was done for 2 years from April 2015 to April 2017 in Department of Pathology. A total of 210 patients with pancytopenia were included in the study. A detailed history and hematological findings including the bone marrow findings and flow cytometric results were noted in these patients, wherever available. Result: Out of 210 patients, 136 were adults and 74 were children. The mean age of the patients was 42 years. The male to female ratio was 2.7:1. The most common clinical feature in our study was pallor (in all cases) followed by fatigue (70.9%). Most of the cases had severe anemia (57.8%). Megaloblastic anemia was found to be the most common cause, constituting 61.9% of all cases, followed by hypersplenism (25.7%), aplastic anemia (9%), leukemia (1.9%). Occasional cases of kala-azar, typhoid and malaria was also noted. Conclusion: A proper clinico-hematological examination aids in making a proper diagnosis in patient presenting with pancytopenia. Sometimes, specific clinical features point towards a certain disease process. Various laboratory parameters can help in diagnosing the underlying disorder leading to pancytopenia. This diagnostic approach will facilitate to highlight the significance of various diagnostic modalities.

Real-time imaging tracking of a dual-fluorescent drug delivery system based on doxorubicin-loaded globin- polyethylenimine nanoparticles for visible tumor therapy.

Elucidation of the pathway of drug delivery and carrier degradation in vivo is essential for the fabrication of a feasible drug delivery system. Multispectral fluorescence imaging can track a drug delivery system simultaneously based on the separation of fluorescence at multiple wavelengths without interference. In this study, the Doxorubicin loaded genipin-crosslinked globin and PEI nanoparticles (Gb-G-PEI/Dox NPs) were designed as a dual fluorescent drug delivery system to track the drug release and carrier degradation process. Both confocal laser scanning microscopy and flow cytometric results showed that Gb-G-PEI/Dox NPs had a higher cellular uptake efficiency than Doxorubicin in the Bel-7402 cell line due to its small size and positively charged surface. The in vivo metabolism experiments tracked by the multispectral fluorescence imaging system elucidated the sustained drug release and carrier degradation process of the Gb-G-PEI/Dox NPs. Furthermore, the in vivo antitumor effects study by the bioluminescence imaging showed that Gb-G-PEI/Dox NPs reduced the tumor size most significantly on hepatoma-bearing nude mice. As a result, Gb-G-PEI/Dox NPs were demonstrated to be a highly efficient, feasible and visible drug delivery system for tumor therapy.

Immune responses to an early lytic cytomegalovirus antigen in systemic lupus erythematosus patients: T-cell responses, cytokine secretions and antibody status.

We investigated immune responses to a lytic cytomegalovirus antigen (CMVpp52), and to a lytic human herpes virus (HHV) 6 antigen (HHV6p41), in systemic lupus erythematosus (SLE) patients and healthy controls (HCs), in order to clarify if the previously established impaired responses to Epstein-Barr virus (EBV) in SLE patients is a general defect in their responses against (all) HHVs. Multiplex Luminex technology results showed a normal induction of five quantified cytokines (interferon γ, interleukin(IL)12, IL17, IL10, and tumor necrosis factor α) in SLE patients compared to HCs upon stimulation with CMVpp52 and HHV6p41. However, flow cytometric results showed a reduced upregulation of the activation marker CD69 on T-cells from SLE patients (n = 17) compared to HCs (n = 17) upon stimulation with CMVpp52, indicating limited or defective CMVpp52-specific T-cells and/or poor antigen-presentation in SLE patients, and thereby possibly decreased control of the CMV infection. In conclusion, the dysfunctional immune response against EBV previously established in SLE patients does not seem to apply to the same degree regarding the immune responses against CMV or HHV6. Results designate that the main contributing HHV agent in development or exacerbation of SLE (in genetically predisposed individuals) is the previously determined uncontrolled EBV infection, and to a lesser extent CMV infection, and probably with no involvement of HHV6 infection.

Copper-induced immunomodulation in mussel (Perna canaliculus) haemocytes.

Copper is a common contaminant in aquatic environments, which may cause physiological dysfunction in marine organisms. However, the toxicity mechanisms of copper in marine bivalves is not fully understood. In this study, we applied an integrated approach that combines flow cytometry and Gas Chromatography-Mass Spectrometry (GC-MS)-based metabolomics to characterize cellular and molecular mechanisms of copper immunotoxicity in New Zealand Greenshell™ mussel (Perna canaliculus) haemolymph. Flow cytometric results showed significant increases in haemocyte mortality, production of reactive oxygen species and apoptosis (via alteration of caspase 3/7 and mitochondrial membrane potential) of haemocytes exposed to increasing total concentrations of Cu2+ (62.5, 125.0 and 187.5 μM) compared to a low Cu2+ concentration (25.0 μM) and control (0.0 μM). In addition to flow cytometric data, our metabolomics results showed alterations of 25 metabolites within the metabolite profile of Cu2+-exposed haemolymph (125 μM) compared to those of control samples. Changes in levels of these metabolites may be considered important signatures of oxidative stress (e.g., glutathione) and apoptosis processes (e.g., alanine, glutamic acid). This study provides insights into the cellular and molecular mechanisms of oxidative stress and apoptosis in marine bivalves and highlights the applicability and reliability of metabolomic techniques for immunotoxicological studies in marine organisms.

Comparison of CD9 & CD146 markers in endometrial stromal cells of fertile & infertile females.

Background & objectives:CD9 and CD146 are important adhesion molecules that play a role in the implantation of an embryo. This study was undertaken to correlate the expression of these markers in fertile and infertile women's endometrial stromal cells.Methods:Human endometrial stromal cell culture from endometrial biopsies of fertile (n=50) and infertile females (n=50) was performed and primary cell lines were established. Expression of CD9 and CD146 was studied for all the 100 cell lines with the help of flow cytometry. Gene expression of CD9 and CD146 was performed by real-time polymerase chain reaction.Results:There was a significant difference in endometrial stromal cells of fertile and infertile females. Flow cytometric results revealed significantly lower expression of CD9 (P=0.0126) and CD146 (P=0.0006) in the infertile endometrial stromal cells as compared to fertile endometrial stromal cells. These results were comparable with real-time data.Interpretation & conclusions:This study showed that endometrial stromal cells from infertile females had lower expression of adhesion molecules, CD9 and CD146. Our findings suggest that CD9 and CD146 may have a role in infertility. Infertile female's endometrial stromal cells have decreased expression of CD9 and CD146 which can be the cause of infertility related to implantation failure.

The in vitro study of Her-2 targeted gold nanoshell liquid fluorocarbon poly lactic-co-glycolic acid ultrasound microcapsule for ultrasound imaging and breast tumor photothermal therapy

Antibody-mediated targeting therapy has been successful in treating patients with breast cancer by improving the specificity and clinical efficacy. In this study, we constructed the human epidermal growth factor receptor-2 (Her2) antibody-conjugated ultrasound contrast agent with lactic-co-glycolic acid (PLGA) as film forming and perfluorocty bromide (PFOB) as internal material, which was coated by gold nanoshell (Her2-PFOB@PLGA@Au), to realize the integration of diagnosis and treatment. The contrast agent was spherical, with the diameter was 256.8 ± 53.4 nm, and had a good dispersion; Ultrasound imaging experiments in vitro showed that the gold nanoshell polylactic acid microcapsule was suitable for ultrasound contrast imaging with the exquisite and uniform dot intensive high echo. The agent had a great photothermal effect under the near-infrared (NIR) with no obvious biological toxicity for both Her2-positive and negative tumor cells; Moreover, both the results of laser scanning confocal microscope (LSCM) and flow cytometer (FCM) demonstrated the great specificity of Her2-PFOB@PLGA@Au conjugating with Her2 positive breast cancer cells (SKBR3). In conclusion, the successful synthesis of the Her2-PFOB@PLGA@Au microcapsule, offered a new therapeutic strategy of combining diagnosis with therapy for fighting against the breast cancer.

Polyphyllin D inhibits proliferation of K562 on human leukemia cells by inducing apoptosis and differentiation

Objective To investigate the inhibitory effect of polyphyllin D on the proliferation of human chronic myelogenous leukemia (CML) cell line K562 cells and its mechanism. Methods Methyl thiazolyl thiazolium (MTT) assay was used to evaluate the inhibition effect of polyphyllin D on the proliferation of K562 cells. Flow cytometry was used to determine the effect of polyphyllin D on the apoptosis of K562 cells. The related proteins were analyzed by Western blotting. Results The MTT assay showed that polyphyllin D obviously inhibited the growth of K562 cells. The hemi-inhibitory concentration (IC50) of 24 hours for the K562 cells was (0.8±0.1)μmol/L. Flow cytometric results demonstrated that polyphyllin D significantly induced apoptosis in a dose-dependent manner, and the apoptosis rates of 0, 0.4, 0.8, 1.2 μmol/L polyphyllin D were (1.73±0.33)%, (14.47±2.57)%, (24.10±3.52)%, (31.51±4.09)% after 24 hours, with a significant difference (F=58.231, P<0.001). Western blotting showed that polyphyllin D significantly down-regulated the expression of Bcl-2, and this was accompanied by up-regulation of Bax, decrease of the Bcl-2/Bax ratio, up-regulation of cytochrome c and activation of caspase-3. Otherwise, polyphyllin D obviously induced monocyte differentiation by increasing CD14 expression on the surface of K562 cells. Conclusion Polyphyllin D can significantly inhibit the proliferation of K562 cells, and the mechanism may be realized by inducing apoptosis and promoting monocyte differentiation. Key words: Leukemia; Apoptosis; Cell differentiation; Polyphyllin D

P214 Significance and limitations of sera treated with molecular weight centrifugal column

Aim Our lab routinely treats high PRA patient serum with molecular weight centrifugal columns (SC) to remove high molecular weight interference from testing, especially in cases where prozone effect is suspected. Treated serum is used in Luminex single antigen bead (SAB) testing and/or flow cytometric crossmatching and results are correlated with test results using untreated sera. In this study our goal is to define the limitations and significance of sera treated by molecular weight centrifugal column. Methods 124 patients had complement dependent cytotoxicity (CDC) and SAB/SC testing performed between January 2015 and February 2017. Protocol treats 250 μl of serum using the PALL 300 K Nanosep Centrifugal Device. Samples were centrifuged at 14,000 g for 15 min and collected from the reservoir for testing. Specificities determined using One Lambda LABScreen single antigen bead (SAB) Luminex assay and the GEN TRAK 60 well cell tray (CDC: 3 W plus AHG). Analysis included correlation of SAB/SC and CDC including evaluation of regraft status and donor specific antibody (DSA). Results 100/124 patients were CDC positive. All CDC specificities were also identified by SAB/SC testing. 46 were regrafts, and 38 (83%) had DSA identified by both CDC and SAB/SC. 9 of these regraft patients had the prozone effect eliminated in SAB/SC testing with CDC specificities matching. 24/124 patients were CDC negative. Eight of these were regrafts, four had DSA identified by SAB/SC, three were also negative by SAB (SC); all had some reactivity on SAB, but it was weak or against putative cryptic antigens. Conclusions Our initial idea for use for the 300 K Nanosep centrifugal column (SC) was to treat sera to remove interference of high molecular weight substances including the IgM pentamers and eliminate any potential prozone effect. The concern is that IgG specific HLA antibody might also be lost in the process. Results confirm that all allo HLA antibody and DSA identified by CDC were preserved and positively identified by SAB/SC. 9 patients had prozone effect alleviated with SC treatment. One regraft patient had flow crossmatch results using SC sera that confirmed DSA remained positive. SAB/SC results MFI do not correlate to standard SAB results MFI. Interpretation should include pattern and history.

P175 Flow cytometry using monoclonal antibody for detection of HLA-B27: Analysis of ‘Gray Zone’ results for cross reactive antigens

Aim HLA-B27 (associated with ankylosing spondylitis, juvenile rheumatoid arthritis, and Reiter’s syndrome) can be detected utilizing monoclonal antibodies. This assay is a simple qualitative two-color direct immunofluorescence flow cytometric method for rapid detection of the cell-surface glycoprotein expressed on nucleated human cells in whole blood without additional processing. This assay is known to cross-react with other HLA-B antigens, as reported with limited sampling, which yields a gray zone result. We present our observations of this cross-reactivity over approximately an eight and a half year period. Methods Whole blood samples (ACD) are tested with the BD (BD Biosciences, San Jose CA) HLA B27 kit utilizing monoclonal antibody GS145.2. Flow cytometric results falling in the gray zone were confirmed using SSO typing assay. Gray zone results were analyzed to identify cross reacting HLA-B antigens. Results In our laboratory, 311 samples fell into our defined gray zone. Analysis of this demonstrated 30 HLA-B27 cross-reactive antigens, including published HLA-B27 cross-reactive antigens and additional antigens not previously published. The 11 most common cross reactive HLA-B antigens in descending order of frequency are: HLA-B7, 44, 39, 8, 60, 51, 37, 35, 62, 18, and 64. Conclusions Our study expands on the previously published breadth of known HLA-B27 cross reactive antigens. An ongoing understanding & confirmation of gray zone results are important for accurate patient diagnosis.

Enantioselective apoptosis and oxidative damage induced by individual isomers of profenofos in primary hippocampal neurons

ABSTRACTThe purpose of this study was to investigate the apoptosis-related cytotoxic effects and molecular mechanisms of individual isomers of profenofos (PFF) on primary hippocampal neurons at 1.0 to 20 mg L−1. The cell viability and lactate dehydrogenase (LDH) efflux indicated that (−)­PFF exposure was associated with more toxic effects than (+)­PFF above the concentration of 5 mg L−1 (P < 0.5). Flow cytometric results showed that the percentages of apoptotic cells incubated with 20 mg L−1 (−)­PFF, (+)­PFF and rac-PFF for 24 h reached 23.4%, 9.2% and 14.2% (P < 0.01), respectively. Hippocampal neurons incubated with (−)­PFF, (+)­PFF and rac-PFF exhibited a dose-dependent accumulation of intracellular reactive oxygen species (ROS) and malondialdehyde (MDA) and a dose-dependent inhibition of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) activity, implying that the defense system of the tests induces oxidative damage. A statistically significant difference was observed between the two enantiomers at 5 mg L−1 and above. Moreover, the results showed that (−)­PFF exposure caused a significant loss in mitochondrial transmembrane potential (MMP), an upregulation of Ca2+ and Bax protein expression, a downregulation of Bcl-2 protein expression, and the activation of caspase-3 and caspase-9 in a dose-dependent manner; (+)­PFF and rac-PFF exhibited these effects to a lesser degree. All results suggest that PFF induced apoptosis in rat hippocampal neurons via the mitochondria-mediated pathway, and oxidative stress is one of the factors of PFF-induced apoptosis. In addition, (−)­PFF appears to play an important role in oxidative stress and apoptosis, indicating that enantioselectivity should be considered when assessing ecotoxicological effects and health risks of chiral pesticides.

Secretion metabolites of probiotic yeast, Pichia kudriavzevii AS-12, induces apoptosis pathways in human colorectal cancer cell lines

There is a common agreement on the important role of the gastrointestinal microbiota in the etiology of cancer. Benign probiotic yeast strains are able to ameliorate intestinal microbiota and regulate the host metabolism, physiology, and immune system through anti-inflammatory, antiproliferative, and anticancer effects. We hypothesized that Pichia kudriavzevii AS-12 secretion metabolites possess anticancer activity on human colorectal cancer cells (HT-29, Caco-2) via inhibiting growth and inducing apoptosis. This study aimed to assess the anticancer effect of P. kudriavzevii AS-12 secretion metabolites and the underlying mechanisms. The cytotoxicity evaluations were performed via 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide assay; 4′,6-diamidino-2-phenylindole staining; and FACS-flow cytometry tests. Also, the effects of P. kudriavzevii AS-12 secretion metabolites on the expression level of 6 important genes (BAD, Bcl-2, Caspase-3, Caspase-8, Caspase-9 and Fas-R) involved in the extrinsic and intrinsic apoptosis pathways were studied by real-time polymerase chain reaction method. P. kudriavzevii AS-12 secretion metabolites showed significant (P < .0001) cytotoxic effects on HT-29 cells (57.5%) and Caco-2 (32.5%) compared to KDR/293 normal cells (25%). Moreover, the cytotoxic effects of examined yeast supernatant on HT-29 cells were comparable with 5-fluorouracil, as a positive control (57.5% versus 62.2% respectively). Flow cytometric results showed that the induction of apoptosis is the main mechanism of the anticancer effects. Also, according to the reverse transcriptase polymerase chain reaction results, the expression level of proapoptotic genes (BAD, Caspase-3, Caspase-8, Caspase-9, and Fas-R) in treated HT-29 and Caco-2 cells was higher than untreated and normal cells, whereas the antiapoptotic gene (Bcl-2) was downregulated. P. kudriavzevii AS-12 secretion metabolites exert its anticancer effects by inhibiting cell proliferation and inducing intrinsic and extrinsic apoptosis in colon cancer cells.