Flow Cytometry Procedure Research Articles

Involvement of intrinsic mitochondrial pathway in neosergeolide-induced apoptosis of human HL-60 leukemia cells: The role of mitochondrial permeability transition pore and DNA damage

Context: Quassinoids are biologically active secondary metabolites found exclusively in the Simaroubaceae family of plants. These compounds generally present important biological properties, including cytotoxic and antitumor properties.Objective: In the present study, the cytotoxic effects of neosergeolide, a quassinoid isolated from Picrolemma sprucei Hook. f., were evaluated in human promyelocytic leukemia cells (HL-60).Materials and methods: Cytotoxicity and antiproliferative effects were evaluated by the MTT assay, May-Grünwald-Giemsa’s staining, BrdU incorporation test, and flow cytometry procedures. The comet assay and micronuclei analysis were applied to determine the genotoxic and mutagenic potential of neosergeolide.Results: After 24 h exposure, neosergeolide strongly inhibited cancer cell proliferation (IC50 0.1 µM), and its activity seemed to be selective to tumor cells because it had no antiproliferative effect on human peripheral blood mononuclear cells (PBMC) at tested concentrations. Apoptosis was induced at submicromolar concentrations (0.05, 0.1, and 0.2 µM) as evidenced by morphological changes, mitochondrial depolarization, phosphatidylserine externalization, caspases activation, and internucleosomal DNA fragmentation. Additionally, neosergeolide effects were prevented by cyclosporine A (CsA), an inhibitor of the mitochondrial permeability transition (MPT) pore, which reinforced the participation of intrinsic pathways in the apoptotic process induced by this natural quassinoid. Direct DNA damage was further confirmed by comet assay and cytokinesis-block micronucleus test.Discussion and conclusion: The present study provided experimental evidence to support the underlying mechanism of action involved in the neosergeolide-mediated apoptosis. In addition, no antiproliferative effect or DNA damage effect of neosergeolide was evident in PBMC, highlighting its therapeutic potential.

Genome size and base composition of Bromeliaceae species assessed by flow cytometry

Flow cytometry (FCM) has been used to estimate the nuclear DNA content of Bromeliaceae species, which constitutes relevant information for studies of taxonomy, evolution, genetic diversity, and reproductive biology in bromeliads. Nevertheless, C values have only been estimated for 58 out of the 3,140 existing Bromeliaceae species. Aiming to contribute to the genome database of Bromeliaceae, the current study was carried out to measure the nuclear DNA content and base composition of Bromelioideae and Tillandsioideae species occurring in the Atlantic Rainforest. The most adequate FCM procedure provided histograms exhibiting G0/G1 peaks with coefficients of variation below 5%, so that these histograms were used to measure the mean 2C and AT% values for all collected Bromelioideae and Tillandsioideae species. These values were statistically compared, and dendrograms were plotted. A second comparison was performed among all mean 2C values reported for Pitcairnioideae, Tillandsioideae, and Bromelioideae species. In accordance with previous statistical comparisons, two groups were formed: cluster 1, composed by Tillandsia loliacea, Tillandsia usneoides, and Tillandsia cyanea, and cluster 2, gathering other 69 species. Based on these results, we concluded that FCM was a rapid, accurate, and reliable technique to assess genome size and base composition. Furthermore, the FCM data reported here will contribute to the Monocot and Bromeliaceae database, which still displays several ongoing gaps, especially for endemic species.

Fluorescent In Vivo Detection Reveals that IgE+ B Cells Are Restrained by an Intrinsic Cell Fate Predisposition

IgE antibodies may be protective in parasite immunity, but their aberrant production can lead to allergic disease and life-threatening anaphylaxis. Despite theimportance of IgE regulation, few studies have directly examined the B cells that express IgE, because these cells are rare and difficult to detect. Here, we describe fluorescent IgE reporter mice and validate a flow cytometry procedure to allow sensitive and specific identification of IgE-expressing B cells invivo. Similar to IgG1(+) cells, IgE(+) B cells differentiated into germinal center (GC) B cells and plasma cells (PCs) during primary immune responses toa Tcell-dependent hapten-protein conjugate and the helminth Nippostrongylus brasiliensis. However, the participation of IgE(+) B cells in GCs was transient. IgE(+) B cells had an atypical propensity to upregulate the transcription factor Blimp-1 and undergo PC differentiation. Most IgE(+) PCs were short lived andshowed reduced affinity maturation, revealing intrinsic mechanisms that restrict the IgE antibody response.

POTENTIAL ROLE of 8-COLOR FLOW CYTOMETRY for CHARACTERIZATION of MDS with Del(5q) ABNORMALITIES

Abstract 5020Four-colour based flow cytometry (FCM) of bone marrow (BM) samples has become a valuable tool in the diagnostics of patients (pts) with myelodysplastic syndrome (MDS). It is still unknown, whether aberrations detected by FCM differ between certain cytogenetic subgroups. By using an 8-color (5-tube) FCM diagnostic panel we aimed to investigate the antigen patterns of MDS pts with del(5q) abnormalities and compared these to MDS pts with non-del(5q) MDS.BM samples of healthy donors (n=19) and 57 MDS pts including 28 pts (15 RCMD; 3 RAEB-1; 8 RAEB-2; 1 RAEB-t; 1 CMMoL-1) with del(5q) MDS (single n=15) and 29 pts (1 RCUD; 2 RA±RS; 17 RCMD; 2 RAEB-2; 7 sAML including 1 RAEB-t) with non-del(5q) MDS were investigated. FCM procedure was performed at a FACS Canto II (200,000 events) and standardized including the analysis of all samples after overnight storage at room temperature and erythrocytes lysis prior to antibody staining. Thresholds were set according to median ± 2SD and/or ½ log differences compared to healthy BM. Aberrations were scored according to the FCSS by Wells et al. 2003 and modified by Van de Loosdrecht et al. 2008.In del(5q) MDS several phenotypic characteristics differed to non-del(5q) pts. Of note, lineage infidelity (CD2, CD5, CD7, CD19, CD56) was detectable in significantly fewer del(5q) pts compared to non-del(5q) MDS (in granulocytes: 8% vs. 33%, p=0.040 and in monocytes: 24% vs. 56%, p=0.027). Thus, especially aberrant CD56 expression contributed to these results with only small subpopulations being positive in del(5q) vs. often more than 50% of aberrant granulocytes/monocytes in non-del(5q) MDS pts. Additionally, the lymphoid-to-granulocytes/monocytes-ratios were higher in del(5q) (p=0.029 and 0.025) and hypogranularity was more pronounced in those pts (p=0.05). Intensity of expression of CD14 on monocytes as well as CD71 on granulocytes showed significant differences (2478 vs. 4402, p=0.035 and 2098 vs. 1552, p=0.007). Most of the above mentioned differences could also be confirmed if only RCMD pts of both groups were considered (e.g. lineage infidelity in granulocytes: 0% vs. 36%, p=0.041). Furthermore, trends were seen for a lower CD45-lymphocytes-to-blasts-ratio (4.8 vs. 5.4; p=0.09), higher CD33 intensity on blasts (2416 vs. 1673, p=0.08), and higher qualitative CD36 expression on granulocytes (3.2% vs. 2.5%, p=0.0619) as well as quantitative expression on monocytes (1940 vs. 663, p=0.06). Finally, monitoring of aberrations by FCM allowed for minimal residual disease monitoring in del(5q) MDS pts. Interestingly, the decreased CD19 expression on blasts seemed to be stable independent from the extent of treatment response.Eight-colour based FCM is a valuable tool in the diagnostics of MDS. It might also be able to distinguish del(5q) MDS from other MDS subtypes. Disclosures:No relevant conflicts of interest to declare.

Isolation of CD133+ Liver Stem Cells for Clonal Expansion

Liver stem cell, or oval cells, proliferate during chronic liver injury, and are proposed to differentiate into both hepatocytes and cholangiocytes. In addition, liver stem cells are hypothesized to be the precursors for a subset of liver cancer, Hepatocellular carcinoma. One of the primary challenges to stem cell work in any solid organ like the liver is the isolation of a rare population of cells for detailed analysis. For example, the vast majority of cells in the liver are hepatocytes (parenchymal fraction), which are significantly larger than non-parenchymal cells. By enriching the specific cellular compartments of the liver (i.e. parenchymal and non-parenchymal fractions), and selecting for CD45 negative cells, we are able to enrich the starting population of stem cells by over 600-fold.The proceduresdetailed in this report allow for a relatively rare population of cells from a solid organ to be sorted efficiently. This process can be utilized to isolateliver stem cells from normal murine liver as well as chronic liver injury models, which demonstrate increased liver stem cell proliferation. This method has clear advantages over standard immunohistochemistry of frozen or formalin fixed liver as functional studies using live cells can be performed after initial co-localization experiments. To accomplish the procedure outlined in this report, a working relationship with a research based flow-cytometry core is strongly encouraged as the details of FACS isolation are highly dependent on specialized instrumentation and a strong working knowledge of basic flow-cytometry procedures. The specific goal of this process is to isolate a population of liver stem cells that can be clonally expanded in vitro.

Isolation of CD133+ Liver Stem Cells for Clonal Expansion

Liver stem cell, or oval cells, proliferate during chronic liver injury, and are proposed to differentiate into both hepatocytes and cholangiocytes. In addition, liver stem cells are hypothesized to be the precursors for a subset of liver cancer, Hepatocellular carcinoma. One of the primary challenges to stem cell work in any solid organ like the liver is the isolation of a rare population of cells for detailed analysis. For example, the vast majority of cells in the liver are hepatocytes (parenchymal fraction), which are significantly larger than non-parenchymal cells. By enriching the specific cellular compartments of the liver (i.e. parenchymal and non-parenchymal fractions), and selecting for CD45 negative cells, we are able to enrich the starting population of stem cells by over 600-fold.The proceduresdetailed in this report allow for a relatively rare population of cells from a solid organ to be sorted efficiently. This process can be utilized to isolateliver stem cells from normal murine liver as well as chronic liver injury models, which demonstrate increased liver stem cell proliferation. This method has clear advantages over standard immunohistochemistry of frozen or formalin fixed liver as functional studies using live cells can be performed after initial co-localization experiments. To accomplish the procedure outlined in this report, a working relationship with a research based flow-cytometry core is strongly encouraged as the details of FACS isolation are highly dependent on specialized instrumentation and a strong working knowledge of basic flow-cytometry procedures. The specific goal of this process is to isolate a population of liver stem cells that can be clonally expanded in vitro.

Phagocytic Activity Is Impaired in Type 2 Diabetes Mellitus and Increases after Metabolic Improvement

Objective1) To evaluate whether peripheral blood mononuclear cells (PBMCs) from type 2 diabetic patients present an impairment of phagocytic activity; 2) To determine whether the eventual impairment in phagocytic activity is related to glycemic control and can be reversed by improving blood glucose levels.Methods21 type 2 diabetic patients and 21 healthy volunteers were prospectively recruited for a case-control study. In addition, those patients in whom HbA1c was higher than 8% (n = 12) were hospitalized in order to complete a 5-day intensification treatment of blood glucose. Phagocytic activity was assessed by using a modified flow cytometry procedure developed in our laboratory based on DNA/RNA viable staining to discriminate erythrocytes and debris. This method is simple, highly sensitive and reproducible and it takes advantage of classic methods that are widely used in flow cytometry.ResultsType 2 diabetic patients showed a lower percentage of activated macrophages in comparison with non-diabetic subjects (54.00±18.93 vs 68.53±12.77%; p = 0.006) Significant negative correlations between phagocytic activity and fasting glucose (r = −0.619, p = 0.004) and HbA1c (r = −0.506, p = 0.019) were detected. In addition, multiple linear regression analyses showed that either fasting plasma glucose or HbA1c were independently associated with phagocytic activity. Furthermore, in the subset of patients who underwent metabolic optimization a significant increase in phagocytic activity was observed (p = 0.029).ConclusionsGlycemic control is related to phagocytic activity in type 2 diabetes. Our results suggest that improvement in phagocytic activity can be added to the beneficial effects of metabolic optimization.

Male germ cells of the Pacific oysterCrassostrea gigas: flow cytometry analysis, cell sorting and molecular expression

A technique was developed for dissection and isolation of male germ cells in the oyster Crassostrea gigas. This procedure can provide cells for the exploration of processes involved in the reproductive physiology of bivalves. Spermatogonia were chosen because of their essential role in spermatogenesis and the impact of gonia proliferation on reproductive effort. A non lethal method for determining sex and reproductive cycle stage was first validated in oysters. This first step was essential in order to constitute a homogeneous pool of oysters at the same stages of gametogenesis. Germ cell fractions were then obtained from a density gradient, and enrichment of each fraction was ratified by electron microscopy and by means of a 2-parameter flow cytometry procedure (DNA and mitochondrial staining). A significant enrichment in spermatogonia and spermatocytes was confirmed by the increased expression of markers of proliferative cells (proliferative cell nuclear antigen, PCNA) and early germ cells (oyster vasa-like gene). A preliminary cell sorting procedure is also reported, which was applied to fractions enriched in spermatogonia.

Circulating perivascular progenitors: A target of PDGFR inhibition

Cancer blood vessels consist of two interacting types of cells: inner lining endothelial cells (ECs) and surrounding perivascular cells (pericytes, vascular smooth muscle cells or mural cells). PDGFRbeta(CD140b)+ progenitor perivascular cells (PPC) can differentiate into pericytes and regulate vessel stability and vascular survival in tumors. Similarly to what we have done with circulating ECs and progenitors, we developed a flow cytometry procedure for the enumeration of circulating PPCs and the study of their viability in murine models of cancer and in cancer patients. DNA+CD45-CD31-CD140b+ cells were enumerated by six-colour flow cytometry, their morphology was studied by electron microscopy, PPC specificity confirmed by reverse trascription-PCR (RT-PCR) expression of CD140b mRNA, and viability assessed by Syto16 and 7AAD. In preclinical marrow transplantation studies, 9 ± 4% of circulating PPCs were derived from the marrow donor. PPCs were increased in cancer-bearing mice and in patients affected by some types of cancer. At variance with the kinetic of circulating endothelial progenitors, high-dose cyclophosphamide reduced the number of viable PPCs. The administration of sunitinib, a drug known to inhibit PDGFR, was associated in murine models and in cancer patients with an increase of apoptotic/necrotic circulating PPC, suggesting a direct targeting of these cells. PPC enumeration might be studied as a tool for the definition of the optimal biologic dose of anti-PDGFR drugs and investigated clinically as a possible predictive/prognostic tool in patients receiving anti-PDGFR drugs.

Comparison of Conventional Cytology Vs. High Sensitivity Flow Cytometry for the Diagnosis of Leptomeningeal Involvement by Hematological Lymphoid Malignancies

AbstractAbstract 3112 Objective:To compare the utility of high sensitivity (5-colour) flow cytometry (FCM) vs. conventional cytology (CC) for detecting cerebrospinal fluid (CSF) involvement in patients with hematological lymphoid malignancies. Methods:The results of diagnostic evaluations on all CSF samples analyzed for involvement by neoplastic lymphoid cells between January 2005 and February 2010 were reviewed retrospectively. Cases were identified by reviewing logs of all FCM procedures performed during that time period. FCM was performed on the CSF using a 5-antibody panel (“high sensitivity”). Result:108 patients (62M/46F) diagnosed with non-Hodgkin lymphoma or acute lymphoblastic leukemia underwent a total of 609 lumbar punctures (LP). The 359 samples that were sent for both CC and FCM form the basis of this analysis. The majority of the LP's (312/359, 87%) were negative for malignant cells by both FCM and CC (FCM-/CC-). 47 samples showed infiltration by tumor cells; of these, 25 (7%) were FCM+/CC+, and 22 (6%) were FCM+/CC-. No cases (0%) were FCM-/CC+. Using FCM as the gold standard, CC had a specificity of 100%, but a sensitivity of only 53%. Conclusion:High-sensitivity FCM has superior sensitivity to CC for diagnosing leptomeningeal involvement by lymphoid malignancies. CC failed to identify any additional cases that were not seen on FCM. This raises the question of whether performing CC on CSF samples to search for neoplastic lymphoid cells is of any additional diagnostic value. Disclosures:No relevant conflicts of interest to declare.

Pilot study to relate clinical outcome in pancreatic carcinoma and angiogenic plasma factors/circulating mature/progenitor endothelial cells: Preliminary results

Circulating endothelial cells (CEC) and bone marrow-derived endothelial progenitors (ECP) play important roles in tumor growth and have been proposed as non-invasive markers of angiogenesis. However, CEC and ECP levels have not been investigated in pancreatic carcinoma patients. Using four-color flow cytometry procedures, we evaluated the count of resting (rCEC) and activated (aCEC) endothelial cells and ECP in the peripheral blood of pancreatic carcinoma patients before and after chemotherapy, consisting of gemcitabine (GEM) alone or in combination with oxaliplatin (OX), or with 5-fluorouracil (5-FU). We also correlated CEC and ECP levels with plasma levels of relevant angiogenic factors, such as vascular endothelial growth factor (VEGF)-A, VEGF-D, angiopoietin (Angio)-1, and chemokine C-X-C motif ligand (CXCL)12, measured by ELISA, and with clinical features of pancreatic cancer. The aCEC, rCEC, ECP, and VEGF-A plasma levels were significantly higher in locally-advanced and metastatic patients than controls. Both ECP and VEGF-A levels correlated positively with disease stage and inversely with patient's overall survival. Measurements after the treatment course showed that VEGF-A plasma concentrations and ECP counts had decreased significantly. In particular, VEGF-A and rCEC were significantly down after treatment with GEM alone or in combination with OX. No significant differences in terms of circulating angiogenic factor or endothelial cell subtype levels were found between responders (patients entering partial remission or with stable disease) and non-responders (patients with progressive disease). The study provides insights into angiogenesis mechanisms in pancreatic carcinoma, for which anti-angiogenic targeting of VEGF-A and ECP could be of interest.

Computational study of scattering from healthy and diseased red blood cells

We present a comparative study of scattering from healthy red blood cells (RBCs) and diseased RBCs with deformed shapes. Scattering problems involving three-dimensional RBCs are formulated accurately with the electric and magnetic current combined-field integral equation and solved efficiently by the multilevel fast multipole algorithm. We compare scattering cross section values obtained for different RBC shapes and different orientations. In this way, we determine strict guidelines to distinguish deformed RBCs from healthy RBCs and to diagnose various diseases using scattering cross section values. The results may be useful for designing new and improved flow cytometry procedures.

Zinc fixation preserves flow cytometry scatter and fluorescence parameters and allows simultaneous analysis of DNA content and synthesis, and intracellular and surface epitopes

Zinc salt-based fixation (ZBF) has proved advantageous in histochemical analyses conducted on intact tissues but has not been exploited in flow cytometry procedures that focus on quantitative analysis of individual cells. Here, we show that ZBF performs equally well to paraformaldehyde in the preservation of surface epitope labeling and forward and side scatter parameters as measured by flow cytometry. ZBF-fixed mouse epithelial keratinocytes exhibit a staining pattern for the surface markers Sca-1, CD34 and alpha6 integrin that is highly analogous to live cells. Furthermore, ZBF also preserves DNA allowing subsequent quantitative PCR analysis or labeling for incorporation of the thymidine analog EdU following surface and intracellular epitope staining. Finally, ZBF treatment allows for long-term storage of labeled cells with little change in these parameters. Thus, we present a protocol for zinc salt fixation of cells that allows for the simultaneous analysis of DNA and intracellular and cell surface proteins by flow cytometry.

Abstract 3441: Circulating perivascular progenitors, a target of PDGFRbeta inhibitors

Abstract Cancer blood vessels consist of two interacting types of cells: inner lining endothelial cells and surrounding perivascular cells (pericytes, vascular smooth muscle cells or mural cells). Marrow-derived PDGFRbeta(CD140b)+ progenitor perivascular cells (PPC) can differentiate into pericytes and regulate vessel stability and vascular survival in tumours (Song et al, Nature Cell Biol 2005). Similarly to what we have done with circulating endothelial cells and progenitors (Mancuso et al, Clin Cancer Res 2009), we have developed and validated a flow cytometry procedure for the enumeration of circulating PPC and the study of their viability in murine models of cancer and in cancer patients. DNA+CD45-CD31-CD140b+ cells were enumerated by six-colour flow cytometry, their perivascular nature was confirmed by electron microscopy and RT-PCR expression of CD140b, and their viability was assessed by 7AAD. PPC were found to circulate in cancer-bearing mice, and to be increased (compared to age-matched healthy controls) in patients affected by some types of cancer such as metastatic breast and kidney cancer. In murine models of cancer and in cancer patients, at variance with the kinetcs of circulating endothelial progenitors (Bertolini et al, Cancer Res 2003; Shaked et al, Cancer Cell 2008), cyclophosphamide-based chemotherapy reduced the number of viable PPC, most likely because of an increase of PPC recruitment towards vessels damaged by cyclophosphamide administration (Bertolini et al, Lancet Oncology 2001). Conversely, the administration of the tyrosine kinase inhibitor (TKI) sunitinib, known to inhibit PDGFRbeta, was associated in murine models and human cancer patients with an increase of apoptotic circulating PPC, suggesting a direct targeting of these cells. Studies are ongoing to validate PPC enumeration and viability assessment as a surrogate marker of clinical efficacy in preclinical models and in cancer patients receiving multiple TKI inhibitors including an anti-PDGFRbeta activity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3441.

Flow Cytometry: A New Approach for Indirect Assessment of Sperm Protamine Deficiency

Background Flow cytometry (FCM) has been extensively used to study mammalian sperm in the areas of clinical andrology and reproductive toxicology. FCM provides a powerful advantage over microscopy technique in terms of rapid accurate and reproducible technology for the quantification of various cell characteristics including chromatin status. During spermiogenesis histones are replaced by protamines resulting in a very condensed structure of sperm chromatin. Infertile men have an increased sperm histone: protamine ratio than fertile counterparts. Chromomycin A3 (CMA3) staining represents a useful tool for assessing the packaging quality of sperm chromatin and allows indirect visualization of protamine deficiency. Routinely fluorescence microscope is used for evaluation of protamine deficiency by CMA3. Considering the advantages of FCM and increasing use of CMA3 in assessment of protamine deficiency in the literature and its possible use as a diagnostic test the aim of this study is to standardize this procedure for routine laboratory analysis. Materials and methods Semen samples were collected from 85 infertile men who referred to Isfahan Fertility and Infertility Center. A portion of semen sample was used for routine semen analysis according to WHO criteria and the remainder were evaluated to standardize CMA3 staining procedure for fixation the number of sperm and duration of exposure to CMA3. The results were compared with standard fluorescent microscopic procedure. Percentage CMA3 positive sperm were compared between flow cytometry and standard fluorescent microscopic procedure. Results Our results show that fixation the number of sperm and duration of exposure to CMA3 can affect on FCM outcomes. In addition we show that the samples can be fixed stained with CMA3 stores and then assessed for FCM. Conclusion The optimal conditions for FCM assessment of CMA3 are: fixation concentration of 0.25 mg/ml sperm density of 2 million/ml and exposure for 60 minutes.

Rat Monoclonal Antibodies Specific for LST1 Proteins

The LST1 gene is located in the human MHC class III region and encodes transmembrane and soluble isoforms that have been suggested to play a role in the regulation of the immune response and are associated with inflammatory diseases such as rheumatoid arthritis. Here we describe the generation and characterization of the first monoclonal antibodies against LST1. Two hybridoma lines secreting monoclonal antibodies designated 7E2 and 8D12 were established. The 7E2 antibody detects recombinant and endogenous LST1 by Western blot analysis while 8D12 reacts with recombinant and endogenous LST1 in immunoprecipitation and flow cytometry procedures. The newly established antibodies were used to survey LST1 protein expression in human cell lines, which was found to be tightly regulated, allowing the expression of transmembrane isoforms but suppressing soluble isoforms.

Circulating β1 Adrenergic Autoantibodies from Patients with Chronic Periodontitis Interact with Gingival Fibroblasts

To demonstrate the presence of circulating autoantibodies (Abs) from patients with chronic periodontitis (CP) that interacted with human gingival fibroblast membranes activating beta(1) adrenoceptors (beta(1)-AR). Sera and purified IgG from 25 patients with CP and 20 age-matched healthy subjects were studied by flow cytometry, ELISA and DNA synthesis. Human gingival fibroblast membranes and/or synthetic peptides with amino acid sequences identical to human beta(1)-AR were used as antigens. By flow cytometry and ELISA procedures, we proved that the serum-purified IgG fraction from patients with CP reacted with the fibroblast surface and to the beta(1) synthetic peptide. The corresponding affinity-purified anti-beta(1) peptide Abs displayed agonist-like activity associated with specific receptor activation, inhibiting the DNA synthesis of human gingival fibroblasts. This study demonstrates that beta(1)-AR autoantibodies are elevated in patients with CP. These autoantibodies were targeted to the fibroblasts, and specifically to the beta(1)-AR, and has receptor-like activity inhibiting DNA synthesis.

Copper-induced regression of cardiomyocyte hypertrophy is associated with enhanced vascular endothelial growth factor receptor-1 signalling pathway

Vascular endothelial growth factor (VEGF) has been well documented to stimulate cell proliferation and differentiation; however, we have observed that copper (Cu)-induced regression of heart hypertrophy was VEGF-dependent. The present study was undertaken to test the hypothesis that Cu causes alterations in the distribution of VEGF receptors (VEGFRs) in hypertrophic cardiomyocytes so that it switches the signalling pathway from stimulation of cell growth to reversal of cell hypertrophy. Primary cultures of neonatal rat cardiomyocytes were exposed to phenylephrine (PE) at a final concentration of 100 microM in cultures for 48 h to induce cell hypertrophy. The hypertrophic cardiomyocytes were exposed to copper sulfate at a final concentration of 5 microM in cultures for 24 h with a concomitant presence of PE. Flow cytometry, gene silencing, and ELISA procedures were used to analyse the changes in VEGFRs and their relationship with regression of cardiomyocyte hypertrophy. Cu did not change the concentration of VEGF in culture media, but increased the ratio of VEGFR-1 to VEGFR-2 two-fold. Gene silencing of VEGFR-2, in the absence of Cu addition, reversed PE-induced cardiomyocyte hypertrophy, which was suppressed by an anti-VEGF antibody. Gene silencing of VEGFR-1 blocked Cu-induced regression of cell hypertrophy and decreased the activity of cGMP-dependent protein kinase-1 (PKG-1). A PKG-1 antagonist, Rp-8-pCPT-cGMPS, blocked both Cu- and VEGFR-2 gene silencing-induced regression of cardiomyocyte hypertrophy. Enhanced VEGFR-1 signalling is involved in Cu regression of cardiomyocyte hypertrophy, and the PKG-1 pathway is likely associated with VEGFR-1.

A flow cytometry approach for quantitative analysis of cellular phosphatidylserine distribution and shedding

Phospholipids are asymmetrically distributed across the membrane of all cells, including red blood cells (RBCs). Phosphatidylserine (PS) is mainly localized in the cytoplasmic membrane leaflet, but during RBC ageing it flip-flops to the external leaflet—a process that is increased in certain pathological conditions (e.g., β-thalassemia). PS externalization in RBCs mediates their phagocytosis by macrophages and removal from the circulation. PS is usually measured by flow cytometry and is reported as the percentage of cells with external PS. In the current study, we developed a novel two-step flow cytometry procedure to quantitatively measure not only the external PS but also the intracellular and shed PS. In this method, PS is first bound to fluorescent annexin V, and then the residual nonbound annexin is quantified by binding to PS exposed on apoptotic cells. Using this method, we measured 1.1 ± 0.2 and 0.12 ± 0.04 μmol inner and external PS, respectively, per 10 7 normal RBCs. Thalassemic RBCs demonstrated increased PS externalization (1.7-fold) and shedding (11-fold) that was accompanied by lower intracellular PS (31%). These results suggest that quantitative flow cytometry of PS could have a diagnostic value in evaluating the pathology of RBCs in hemolytic anemias associated with increased PS externalization and shortening of the RBC life span.

Vitamin E protects against the mitochondrial damage caused by cyclosporin A in LLC-PK1 cells

Cyclosporin A (CsA) has nephrotoxic effects known to involve reactive oxygen species (ROS), since antioxidants prevent the kidney damage induced by this drug. Given that mitochondria are among the main sources of intracellular ROS, the aims of our study were to examine the mitochondrial effects of CsA in the porcine renal endothelial cell line LLC-PK1 and the influence of the antioxidant Vitamin E (Vit E). Following the treatment of LLC-PK1 cells with CsA, we assessed the mitochondrial synthesis of superoxide anion, permeability transition pore opening, mitochondrial membrane potential, cardiolipin peroxidation, cytochrome c release and cellular apoptosis, using flow cytometry and confocal microscopy procedures. Similar experiments were done after Vit E preincubation of cells. CsA treatment increased superoxide anion in a dose-dependent way. CsA opened the permeability transition pores, caused Bax migration to mitochondria, and decreased mitochondrial membrane potential and cardiolipin content. Also CsA released cytochrome c into cytosol and provoked cellular apoptosis. Vit E pretreatment inhibited the effects that CsA induced on mitochondrial structure and function in LLC-PK1 cells and avoided apoptosis. CsA modifies mitochondrial LLC-PK1 cell physiology with loss of negative electrochemical gradient across the inner mitochondrial membrane and increased lipid peroxidation. These features are related to apoptosis and can explain the cellular damage that CsA induces. As Vit E inhibited these effects, our results suggest that they were mediated by an increase in ROS production by mitochondria.