MEASURING THE CELLULAR RESPONSE OF MACROPHAGES TO SOIL COMPONENTS BY FLOW CYTOMETRY: DETERMINING THE TOXICITY OF PODOCONIOSIS-ASSOCIATED SOILS

2016 ◽  
Author(s):  
Jamey N. Cooper ◽  
◽  
Andy Nguyen ◽  
Nathan H. Lee ◽  
Kerby C. Oberg ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cellular Response ◽  
Soil Components

scholarly journals Cellular Response of the Amoeba Acanthamoeba castellanii to Chlorine, Chlorine Dioxide, and Monochloramine Treatments

2011 ◽  
Vol 77 (14) ◽  
pp. 4974-4980 ◽  
Author(s):  
Emerancienne Mogoa ◽  
Charles Bodet ◽  
Franck Morel ◽  
Marie-Hélène Rodier ◽  
Bernard Legube ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Flow Cytometry ◽  
Chlorine Dioxide ◽  
Mode Of Action ◽  
Cellular Response ◽  
Acanthamoeba Castellanii ◽  
Size Reduction ◽  
Resistant Bacteria ◽  
Free Living ◽  
Content Type

ABSTRACTAcanthamoeba castellaniiis a free-living amoebae commonly found in water systems. Free-living amoebae might be pathogenic but are also known to bear phagocytosis-resistant bacteria, protecting these bacteria from water treatments. The mode of action of these treatments is poorly understood, particularly on amoebae. It is important to examine the action of these treatments on amoebae in order to improve them. The cellular response to chlorine, chlorine dioxide, and monochloramine was tested onA. castellaniitrophozoites. Doses of disinfectants leading to up to a 3-log reduction were compared by flow cytometry and electron microscopy. Chlorine treatment led to size reduction, permeabilization, and retraction of pseudopods. In addition, treatment with chlorine dioxide led to a vacuolization of the cytoplasm. Monochloramine had a dose-dependent effect. At the highest doses monochloramine treatment resulted in almost no changes in cell size and permeability, as shown by flow cytometry, but the cell surface became smooth and dense, as seen by electron microscopy. We show that these disinfectants globally induced size reduction, membrane permeabilization, and morphological modifications but that they have a different mode of action onA. castellanii.

200 TRANSPLANTATION AND IN VIVO DIFFERENTIATION OF ADIPOSE-DERIVED STEM CELLS IN SWINE

2006 ◽  
Vol 18 (2) ◽  
pp. 208 ◽  
Author(s):  
A. S. Lima ◽  
S. A. Malusky ◽  
M. R. B. Mello ◽  
S. J. Lane ◽  
J. R. Rivera ◽  
...  
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
Stem Cell ◽  
Cellular Response ◽  
Agricultural Research ◽  
Primary Concern ◽  
Adipose Derived Stem Cells ◽  
Subcutaneous Adipose

A primary concern in stem cell biology is that observations made in vitro may be an artifact of the in vitro culture environment. In vitro derived stem cells can be implanted into the environment from which they are derived so that their response to physiological conditions may be observed. Several important cellular characteristics need to be examined following the cell's reintroduction to the in vivo environment, including the potential for differentiation, proliferative ability, and life span. Studying implanted stem cells will assist in determining the potential for stem cell use in clinical therapies and provide further understanding of the role adult stem cells have in the adult body. Currently, the scientific literature is lacking a detailed description of the cellular response of adipose-derived stem cells (ADSCs) reintroduced to their exact tissue of origin. Thus, the aim of this study was to evaluate porcine ADSC growth in vivo and to analyze cell differentiation in vivo following injection of undifferentiated ADSCs into subcutaneous fat. Subcutaneous adipose tissue was isolated from the back fat of male pigs (11 months of age) and digested with 0.075% collagenase at 37�C for 90 min. The digested tissue was centrifuged at 200g for 10 min to obtain a cell pellet. The pellet was re-suspended with DMEM and the ADSCs were plated onto 75 cm2 flasks (5000-10 000 cells per cm2) and cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% gentamicin. Passage 3 ADSCs were labeled with fluorescent dye (PKH26; Sigma, St. Louis, MO, USA) and sorted by flow cytometry. After sorting, positive cells were washed and re-suspended in culture medium. For transplantation, 100 �L of cell suspension in DMEM containing one of four cell concentrations (0 (control); 30 000; 300 000; and 900 000 cells) were placed in a 1-mL syringe and injected into the subcutaneous back fat of recipient pigs (n = 2). Each pig had previously been tattooed with 12 13 � 13 squares to mark injection sites. The treatments were replicated three times within each animal. Two and three weeks after transplantation, animals were euthanized, the back fat containing the transplantation site was harvested, and the cells were disaggregated as described above. The buoyant adipocytes and pelleted ADSCs cells were then analyzed by flow cytometry. The results indicated that there were dose- and time-dependent increases in labeled ADSCs and labeled adipocytes in the fat samples with increasing cell number (from 0 to 300 000 cells). There was, however, a decrease in labeled ADSCs at the 900 000-cell dose, which is likely due to excess cells being transplanted or an immune reaction. Both of these aspects are currently being evaluated. In conclusion, undifferentiated ADSCs from swine can be isolated from and returned to the subcutaneous adipose layer and differentiate into mature adipocytes. This work was supported by the Council for Food and Agricultural Research (C-FAR) Sentinel Program, University of Illinois.

Early Clearance of Mycobacterium tuberculosis Is Associated With Increased Innate Immune Responses

2019 ◽  
Author(s):  
Ayesha J Verrall ◽  
Marion Schneider ◽  
Bachti Alisjahbana ◽  
Lika Apriani ◽  
Arjan van Laarhoven ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Mycobacterium Tuberculosis ◽  
Immune Responses ◽  
Cellular Response ◽  
Ex Vivo ◽  
Interferon Γ ◽  
Innate Immune ◽  
Innate Immune Responses ◽  
Cytokine Responses ◽  
Protection Against Infection

AbstractBackgroundA proportion of tuberculosis (TB) case contacts do not become infected, even when heavily exposed. We studied the innate immune responses of TB case contacts to understand their role in protection against infection with Mycobacterium tuberculosis, termed “early clearance.”MethodsIndonesian household contacts of TB cases were tested for interferon-γ release assay (IGRA) conversion between baseline and 14 weeks post recruitment. Blood cell populations and ex vivo innate whole blood cytokine responses were measured at baseline and, in a subgroup, flow cytometry was performed at weeks 2 and 14. Immunological characteristics were measured for early clearers, defined as a persistently negative IGRA at 3 months, and converters, whose IGRA converted from negative to positive.ResultsAmong 1347 case contacts, 317 were early clearers and 116 were converters. Flow cytometry showed a resolving innate cellular response from 2 to 14 weeks in persistently IGRA-negative contacts but not converters. There were no differences in cytokine responses to mycobacterial stimuli, but compared to converters, persistently IGRA-negative contacts produced more proinflammatory cytokines following heterologous stimulation with Escherichia coli and Streptococcus pneumoniae.ConclusionsEarly clearance of M. tuberculosis is associated with enhanced heterologous innate immune responses similar to those activated during induction of trained immunity.

Oral inoculation of probioticsLactobacillus acidophilusNCFM suppresses tumour growth both in segmental orthotopic colon cancer and extra-intestinal tissue

2011 ◽  
Vol 107 (11) ◽  
pp. 1623-1634 ◽  
Author(s):  
Chien-Chang Chen ◽  
Wei-Chuan Lin ◽  
Man-Shan Kong ◽  
Hai Ning Shi ◽  
W. Allan Walker ◽  
...  
Keyword(s):  
Colon Cancer ◽  
Flow Cytometry ◽  
Mhc Class I ◽  
Cellular Response ◽  
Tumour Growth ◽  
Class I ◽  
Structural Abnormality ◽  
Intestinal Tissue ◽  
Cxcr4 Mrna ◽  
The Mean

Modulation of the cellular response by the administration of probiotic bacteria may be an effective strategy for preventing or inhibiting tumour growth. We orally pre-inoculated mice with probioticsLactobacillus acidophilusNCFM (La) for 14 d. Subcutaneous dorsal-flank tumours and segmental orthotopic colon cancers were implanted into mice using CT-26 murine colon adenocarcinoma cells. On day 28 after tumour initiation, the lamina propria of the colon, mesenteric lymph nodes (MLN) and spleen were harvested and purified for flow cytometry and mRNA analyses. We demonstrated thatLapre-inoculation reduced tumour volume growth by 50·3 %, compared with untreated mice at 28 d after tumour implants (2465·5 (sem1290·4)v. 4950·9 (sem1689·3) mm3,P < 0·001). Inoculation withLareduced the severity of colonic carcinogenesis caused by CT-26 cells, such as level of colonic involvement and structural abnormality of epithelial/crypt damage. Moreover,Laenhanced apoptosis of CT-26 cells both in dorsal-flank tumour and segmental orthotopic colon cancer, and the mean counts of apoptotic body were higher in mice pre-inoculated withLa(P < 0·05) compared with untreated mice.Lapre-inoculation down-regulated the CXCR4 mRNA expressions in the colon, MLN and extra-intestinal tissue, compared with untreated mice (P < 0·05). In addition,Lapre-inoculation reduced the mean fluorescence index of MHC class I (H-2Dd, -Kd and -Ld) in flow cytometry analysis. Taken together, these findings suggest that probioticsLamay play a role in attenuating tumour growth during CT-26 cell carcinogenesis. The down-regulated expression of CXCR4 mRNA and MHC class I, as well as increasing apoptosis in tumour tissue, indicated thatLamay be associated with modulating the cellular response triggered by colon carcinogenesis.

Dexamethasone Accumulation in Dexamethasone Sensitive and Resistant Acute Lymphoblastic Leukemia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5140-5140
Author(s):  
Rosanna K Jackson ◽  
Ali Alhammer ◽  
Zach Dixon ◽  
Gareth J Veal ◽  
Julie Irving
Keyword(s):  
Flow Cytometry ◽  
Acute Lymphoblastic Leukemia ◽  
Annual Meeting ◽  
Cellular Response ◽  
Lymphoblastic Leukemia ◽  
Annual Meeting Abstract ◽  
Meeting Abstract ◽  
The Mean ◽  
Reh Cells ◽  
Resistant Cells

Abstract Introduction: Glucocorticoids (GC) have been at the forefront of acute lymphoblastic leukemia (ALL) treatment for a number of decades. However, there is heterogeneity of response, both in terms of GC-related toxicity and leukemia cell sensitivity. Contributing factors include marked pharmacokinetic variability observed in children (Yang et al. JCO, 2008; Jackson et al.AACR annual meeting abstract CT115, 2016) and a several hundred fold range of GC cellular response. Cellular resistance can be caused by deletion of the glucocorticoid receptor (GR) but is more commonly downstream of the GR. One neglected upstream parameter relates to GC accumulation, which may be an important factor in ALL GC response, given the evidence for drug transporters in ALL cells. Therefore, this study aimed to determine whether variation in intracellular dexamethasone (dex) levels is a determinant of dex sensitivity in an ALL setting. Methods: A number of cell lines including PreB697, GC resistant PreB697 sub-lines and REH cells, along with primagraft material from 9 patients and 6 primary patient samples (5 presentation and 1 relapse) were studied. The relative sensitivity of cells to dex was assessed using Alamar Blue drug sensitivity assay. Two methods were developed to assess intracellular dex accumulation; a liquid-chromatography mass spectrometry (LC/MS) method and a flow cytometry method, using dex conjugated to the fluorochrome FITC analysed on a FACSCalibur flow cytometry machine. GR status of the cells was confirmed by western blotting. Results: Dex GI50 values (concentration giving 50% growth inhibition) ranged from 37nM in PreB697 cells to >1000nM in GC resistant sub-lines and REH cells. Dex GI50 values in patient ALL cells ranged from 2 to >1000nM. Dex resistant cells were defined as having a dex GI50 of >500nM. The mean GI50 of the dex sensitive cells was 3.8nM. Western blotting suggested wildtype GR status in all samples, with R3D11 and REH serving as hemizygous deleted and GR negative controls, respectively. The mean dex accumulation was measured in cells using an LC/MS assay developed from a fully validated assay measuring plasma dex concentrations (Jackson et al. NCRI annual meeting abstract BACR9, 2014). Dex was stable in RF10 media for at least 8 hours and there was no matrix effect of RF10 media on dex chromatograms compared to dex in plasma. An incubation concentration of 500nM was chosen as this is the observed median value of dex cell exposure clinically. Dex concentrations were quantifiable in cell numbers of 1 x 106after incubation with 500nM dex, allowing measurement of patient samples where limited numbers of cells are available. Dex accumulation in cell lines after incubation with 500nM dex for 4h was 2.1 and 1.8 pmol/million cells in PreB697 and dex resistant sub-lines, respectively (range for resistant subclones 1.2 - 2.1pmol/million cells). There was greater variability in patient cells with a 40-fold range seen, but dex accumulation was not significantly different between sensitive (mean, 1.0 pmol/million; range, 0.1-2.3) and resistant cells (1.4 pmol/million; range, 0.4-4.4) (unpaired students t-test, p=0.17). To assess intra-leukemia heterogeneity in terms of dex accumulation, a flow based assay was established using dex-FITC. Incubation conditions of 500nM dex-FITC at 37°C for 45 minutes were optimal. Dex-FITC accumulation did not differ significantly between sensitive and resistant cells; mean fluorescence intensity of 4.2 (range 1.5-5.9) versus 4.1 (range 2.0 - 9.1) in sensitive and resistant cells, respectively (p=0.97, unpaired students t test). Dex-FITC accumulation appeared uniform within the ALL samples examined. Conclusions: These data suggest that variations in dex accumulation are unlikely to play a role in dex resistance in ALL, at least in vitro. Advancement of the flow-based dex accumulation assay to include leukaemia-associated immunophenotype markers will allow measurement in dex-resistant MRD in vivo. Given that 35% of patients do not achieve plasma concentrations of 200nM dex (Jackson et al. AACR annual meeting abstract CT115, 2016), a combined approach incorporating pharmacokinetic assessments, drug accumulation and cellular response in ALL cells, may allow a comprehensive understanding of dex pharmacology in order to optimise its clinical utility. Disclosures No relevant conflicts of interest to declare.

scholarly journals Clostridium difficile recurrence is characterized by pro-inflammatory peripheral blood mononuclear cell (PBMC) phenotype

2014 ◽  
Vol 63 (10) ◽  
pp. 1260-1273 ◽  
Author(s):  
Mary B. Yacyshyn ◽  
Tara N. Reddy ◽  
Lauren R. Plageman ◽  
Jiang Wu ◽  
Amy R. Hollar ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Clostridium Difficile ◽  
Peripheral Blood ◽  
Cellular Response ◽  
Mononuclear Cells ◽  
Treg Cells ◽  
Sample Collection ◽  
Peripheral Blood Mononuclear ◽  
History Of ◽  
Ifn Γ

Clostridium difficile infection (CDI) is a prevalent nosocomial and increasingly community-acquired problem. Little is known about the productive cellular response in patients. We used flow cytometry to define inflammatory (Th1 and Th17) and regulatory [Foxp3+ T-regulatory (Treg)] cells present in circulating peripheral blood mononuclear cells (PBMC) from CDI patients. We consented 67 inpatients that tested either positive or negative for CDI and 16 healthy controls and compared their PBMC phenotypes. PBMC were collected, isolated, and stained for CD3, CD8 and either IL17 (Th17), IFN-γ (Th1) or Foxp3 (Treg) and analysed using flow cytometry. Twenty thousand events were collected in the lymphocyte gate (gate 1) and T-cell phenotypes were defined. CDI patients who clear the primary initial infection have greater numbers of non-CD3 PBMC. CDI patients who develop recurrence of CDI have a greater percentage of CD3+CD8+, CD3+CD4+Foxp3 and fewer low granular CD3−Foxp3+ PBMC. These patients have greater numbers of IFN-γ-producing lymphocytes, as well as PBMC phenotypes represented by increased IFN-γ- and IL17-co-expressing CD4+CD3+. This initial pro-inflammatory phenotype decreases with repeated recurrence, demonstrating importance of timing of sample collection and history of symptoms. Patients with a history of recurrence had increased Foxp3+CD3+CD4+ and IL17+CD3+CD4+ populations. Hence, CDI recurrence is hallmarked by greater numbers of circulating CD3+ lymphocytes skewed towards a Th1/Th17 inflammatory population as well as possible immune plasticity (Th17/Treg).

scholarly journals IN VITRO STUDY ACTIVITY PROLIFERATIVE FIBROBLAST HUMAN SUBMITTED TO TREATMENT ZINCUM METALLICUM AND CALENDULA OFFICINALIS IN ULTRA DILUTIONS HOMEOPATHIC

2021 ◽  
Vol 16 (1) ◽  
pp. 7-19
Author(s):  
Victor Cavalaro
Keyword(s):  
Cell Cycle ◽  
Flow Cytometry ◽  
Connective Tissue ◽  
Proliferative Activity ◽  
Cellular Response ◽  
In Vitro Study ◽  
Calendula Officinalis ◽  
Study Activity ◽  
Skin Healing

Cicatrization can be divided into three phases: inflammation, fibroblastic, maturation and remodeling [1]. The extracellular matrix may be replaced by a stronger and more elastic connective tissue. In a scar collagen is the major component of the mature connective tissue [2]. In homeopathic area, the greater is the investigated segment ultra dilutions [6]. However, little research has been done to explore the effect of dynamized drugs in in vitro cell culture [4]. Using the Zincum metallicum 6CH and Calendula officinalis 6CH applied independently in different concentrations in fibroblast cultures sought to determine the increase in proliferative activity using techniques such as IC50, MTT, flow cytometry and quantification of collagen. As expected from the literature, ie both homeopathic according to the literature are used for treatments that Require skin healing, both showed increased proliferative activity, having Calendula most cellular response, presenting as cell cycle stimulating checked via flow cytometry.

Caveolin-1, galectin-3 and lipid raft domains in cancer cell signalling

2015 ◽  
Vol 57 ◽  
pp. 189-201 ◽  
Author(s):  
Jay Shankar ◽  
Cecile Boscher ◽  
Ivan R. Nabi
Keyword(s):  
Plasma Membrane ◽  
Lipid Rafts ◽  
Cancer Cell ◽  
Cellular Response ◽  
Spatial Organization ◽  
Essential Feature ◽  
Cell Signalling ◽  
Coated Pits ◽  
Signalling Molecules ◽  
Raft Domains

Spatial organization of the plasma membrane is an essential feature of the cellular response to external stimuli. Receptor organization at the cell surface mediates transmission of extracellular stimuli to intracellular signalling molecules and effectors that impact various cellular processes including cell differentiation, metabolism, growth, migration and apoptosis. Membrane domains include morphologically distinct plasma membrane invaginations such as clathrin-coated pits and caveolae, but also less well-defined domains such as lipid rafts and the galectin lattice. In the present chapter, we will discuss interaction between caveolae, lipid rafts and the galectin lattice in the control of cancer cell signalling.

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