scholarly journals Brucella abortus Rough Mutants Are Cytopathic for Macrophages in Culture

2004 ◽  
Vol 72 (1) ◽  
pp. 440-450 ◽  
Author(s):  
Jianwu Pei ◽  
Thomas A. Ficht
Keyword(s):  
Brucella Abortus ◽  
Cytopathic Effect ◽  
Apoptotic Cell ◽  
Morphological Changes ◽  
Annexin V ◽  
Intracellular Survival ◽  
Intracellular Killing ◽  
Vitro Assay ◽  
O Antigen

ABSTRACT Rough mutants of Brucella spp. are attenuated for survival in animal models. However, conflicting in vitro evidence has been obtained concerning the intracellular survival of rough mutants. Transposon-derived rough mutants isolated in our laboratory were previously shown to exhibit small but significant reductions in intracellular survival in a 12-h in vitro assay. Several recent publications report that rough mutants exhibited increased macrophage uptake relative to their smooth parental strains, and a reduction in numbers at the end of the assay has been interpreted as intracellular killing. In an effort to explore the role of O antigen in the interaction between Brucella abortus and macrophages, we have monitored the uptake of rough mutants and survival in vitro by using the murine macrophage cell line J774.A1. The results confirm a 10- to 20-fold-increased uptake of rough mutants over that of smooth organisms under standard conditions. Recovery of the rough mutants persisted up to 8 h postinfection, but at the point when intracellular replication of the smooth organisms was observed, the number of rough organisms recovered declined. Fluorescence microscopy revealed the intracellular multiplication of both smooth and rough organisms, and assays performed in the absence of antibiotic confirmed the replication of the rough organisms. Examination by phase-contrast microscopy revealed the lytic death of macrophages infected with the rough mutants, which was confirmed by the release of lactate dehydrogenase (LDH) from the cell cytoplasm. Thus, the decline in the number of rough organisms was the result of the lysis of macrophages and not from intracellular killing. The cytopathic effect is characterized as necrotic rather than apoptotic cell death based on early LDH release, annexin V and propidium iodide staining, morphological changes of infected cells and nuclei, and glycine protection. The cytopathic effect was observed with macrophages at multiplicities of infection (MOIs) of as low as 20 and was not observed with epithelial cells at MOIs of as high as 2000. These findings suggest a role for O antigen during the early stages of host-agent interaction that is essential in establishing an intracellular niche that maintains and supports persistent intracellular infection resulting in disease.

scholarly journals Multiorganelle Localization of Metallated Phthalocyanine Photosensitizer in Colorectal Cancer Cells (DLD-1 and CaCo-2) Enhances Efficacy of Photodynamic Therapy

2014 ◽  
Vol 2014 ◽  
pp. 1-10 ◽  
Author(s):  
Palesa Rose Sekhejane ◽  
Nicolette Nadene Houreld ◽  
Heidi Abrahamse
Keyword(s):  
Colorectal Cancer ◽  
Cell Death ◽  
Photodynamic Therapy ◽  
Cancer Cells ◽  
Apoptotic Cell ◽  
Morphological Changes ◽  
Annexin V ◽  
Zinc Phthalocyanine ◽  
Cell Death Pathway

Colorectal cancer is the third most commonly diagnosed cancer. Amongst treatments that have been explored, photodynamic therapy (PDT) is a treatment that is of interest as it poses ideal advantages such as affinity for cancer cells. This study aimed to determine the correlation between the localization site of a sulfonated zinc phthalocyanine (ZnPcSmix) photosensitizer (PS) and its associated cell death pathwayin vitroin colorectal cancer cell lines (DLD-1 and CaCo-2). Visible morphological changes were observed in PDT treated cells after 24 h. Reactive oxygen species (ROS) were detected and visualized 1 h after PDT.ZnPcSmixwas predominantly localized in lysosomes and partially in the mitochondria. FITC Annexin V staining showed a significant decrease in the percentage of viable DLD-1 and CaCo-2 cells 24 h after PDT, with an increase in apoptotic cell population. Moreover, there was a significant increase in both cathepsin D and cytochrome C at 1 and 24 h. In conclusion,ZnPcSmixshowed the ability of inducing apoptotic cell death features in PDT treated cells.

scholarly journals Effect of Lignocaine Concentration on Human Fibroblasts Growth in Eyes Undergoing Trabeculectomy: An in vitro Study

2018 ◽  
Vol 3 (3) ◽  
pp. 1-10 ◽  
Author(s):  
Madhuravasal Krishnan Janani ◽  
Venkatakrishnan Jaichandran ◽  
Hajib Narahari Rao Madhavan ◽  
Lingam Vijaya ◽  
Ronnie Jacob George ◽  
...  
Keyword(s):  
Culture Medium ◽  
In Vitro Model ◽  
Morphological Changes ◽  
Human Fibroblasts ◽  
Annexin V ◽  
In Vitro Study ◽  
Tenon’S Capsule ◽  
Tenon's Capsule ◽  
Vitro Model

Purpose: To evaluate the effect of lignocaine on growth and apoptosis indication of primary human Tenon’s capsule fibroblast (HTFs) in an in vitro model. Patients and Methods: Tenon’s capsule tissue obtained from patients undergoing trabeculectomy were grown in cell culture medium. The effect of different concentrations of lignocaine (0.5, 1.0, 1.5, and 2%) on the morphology and growth of the fibroblasts was studied using microscopy, cell viability, and proliferation assay, and apoptosis was detected using the FITC Annexin V Apoptosis Kit. Results: Morphological changes similar to those of apoptotic cells, including cytoplasmic vacuolation, shrinkage, and rounding were visualized in the cells treated with concentrations greater than 1.0% (i.e., 1.5, 2.0%). Though proliferation inhibition was found with all four concentrations (0.5–2.0%), the viability of cells decreased from 1.0% lignocaine. Conclusion: 0.5% lignocaine prevents proliferation of fibroblasts without causing apoptosis in vitro.

Biochemical Characterisation of an In Vitro Model of Hepatocellular Apoptotic Cell Death

2009 ◽  
Vol 37 (2) ◽  
pp. 209-218 ◽  
Author(s):  
Mathieu Vinken ◽  
Elke Decrock ◽  
Elke De Vuyst ◽  
Luc Leybaert ◽  
Tamara Vanhaecke ◽  
...  
Keyword(s):  
Cell Death ◽  
In Vitro Model ◽  
Caspase 3 ◽  
Fas Ligand ◽  
Apoptotic Cell ◽  
Annexin V ◽  
Apoptotic Cell Death ◽  
Biochemical Characterisation ◽  
Vitro Model

This study was set up to critically evaluate a commonly-used in vitro model of hepatocellular apoptotic cell death, in which freshly isolated hepatocytes, cultured in a monolayer configuration, are exposed to a combination of Fas ligand and cycloheximide for six hours. A set of well-acknowledged cell death markers was addressed: a) cell morphology was studied by light microscopy; b) apoptotic and necrotic cell populations were quantified by in situ staining with Annexin-V, Hoechst 33342 and propidium iodide (PI); c) apoptotic and necrotic activities were monitored by probing caspase 3-like activity and measuring the extracellular leakage of lactate dehydrogenase (LDH), respectively; and d) the expression of apoptosis regulators was investigated by immunoblotting. The initiation of apoptosis was evidenced by the activation of caspase 8 and caspase 9, and increased Annexin-V reactivity. Progression through the apoptotic process was confirmed by the activation of caspase 3 and Bid, the enhanced expression of Bax, and the occurrence of nuclear fragmentation. Late transition to a necrotic appearance was demonstrated by an increased number of PI-positive cells and augmented extracellular release of LDH. Thus, the in vitro model allows the study of the entire course of Fas-mediated hepatocellular apoptotic cell death, which is not possible in vivo. This experimental system can serve a broad range of in vitro pharmaco-toxicological purposes, thereby directly assisting in the reduction of animal experimentation.

scholarly journals Curcuma longa Is Able to Induce Apoptotic Cell Death of Pterygium-Derived Human Keratinocytes

2017 ◽  
Vol 2017 ◽  
pp. 1-9
Author(s):  
Silvia Sancilio ◽  
Silvio Di Staso ◽  
Stefano Sebastiani ◽  
Lucia Centurione ◽  
Nick Di Girolamo ◽  
...  
Keyword(s):  
Cultured Cells ◽  
Therapeutic Potential ◽  
Apoptotic Cell ◽  
Curcuma Longa ◽  
Annexin V ◽  
Surgical Excision ◽  
Human Keratinocytes ◽  
Alcoholic Extract ◽  
Treatment And Prevention

Pterygium is a relatively common eye disease that can display an aggressive clinical behaviour. To evaluate the in vitro effects of Curcuma longa on human pterygium-derived keratinocytes, specimens of pterygium from 20 patients undergoing pterygium surgical excision were collected. Pterygium explants were put into culture and derived keratinocytes were treated with an alcoholic extract of 1.3% Curcuma longa in 0.001% Benzalkonium Chloride for 3, 6, and 24 h. Cultured cells were examined for CAM5.2 (anti-cytokeratin antibody) and CD140 (anti-fibroblast transmembrane glycoprotein antibody) expression between 3th and 16th passage to assess cell homogeneity. TUNEL technique and Annexin-V/PI staining in flow cytometry were used to detect keratinocyte apoptosis. We showed that Curcuma longa exerts a proapoptotic effect on pterygium-derived keratinocytes already after 3 h treatment. Moreover, after 24 h treatment, Curcuma longa induces a significant increase in TUNEL as well as Annexin-V/PI positive cells in comparison to untreated samples. Our study confirms previous observations highlighting the expression, in pterygium keratinocytes, of nuclear VEGF and gives evidence for the first time to the expression of nuclear and cytoplasmic VEGF-R1. All in all, these findings suggest that Curcuma longa could have some therapeutic potential in the treatment and prevention of human pterygium.

Serum deprivation induces apoptotic cell death in a subset of Balb/c 3T3 fibroblasts

1994 ◽  
Vol 107 (5) ◽  
pp. 1169-1179 ◽  
Author(s):  
G.V. Kulkarni ◽  
C.A. McCulloch
Keyword(s):  
Cell Death ◽  
Apoptotic Cell ◽  
Morphological Changes ◽  
Time Dependent ◽  
Cell Diameter ◽  
Adherent Cells ◽  
Serum Withdrawal ◽  
3T3 Fibroblasts ◽  
Mean Diameter

Little is known about the regulation of apoptosis in fibroblasts although several model systems including serum deprivation and treatment with staurosporine or topoisomerase inhibitors have been used to induce apoptosis in vitro. To validate a reproducible in vitro model for the study of apoptosis in fibroblasts, we cultured density-inhibited monolayer cultures of Balb/c 3T3 fibroblasts in Dulbecco's modified essential medium plus 15% fetal calf serum and then withdrew serum. Time-lapse video microscopy demonstrated that within minutes of serum withdrawal, cells lost substrate attachment and floated to the top of the liquid growth medium. There was a time-dependent increase in the number of non-adherent cells. Some of these cells regained attachment and spread momentarily, but they eventually rounded up and lost attachment permanently. In contrast to serum-containing cultures in which similar morphological changes were followed by mitosis, in serum-free cultures repeated attempts at mitosis were followed by permanent attachment loss and presumably cell death. To assess whether all the non-adherent cells were in fact dead, the percentages of cells that continued to proliferate upon return to serum-supplemented conditions was computed. After various periods of serum starvation a decreasing proportion (approx. 75% at 30 minutes; < 2% at 24 hours) of the non-adherent cells could be rescued by addition of serum. Transmission electron microscopy of cells 3 hours after serum withdrawal showed that the majority (approximately 60%) of non-adherent cells exhibited marked intranuclear chromatin condensation but maintained integrity of cell and nuclear membranes and cell organelles, morphological changes consistent with those of apoptotic cell death. Scanning electron microscopy of cultures 3 hours following serum withdrawal showed rounded cells with marked surface blebbing. Fluorescence and confocal microscopy revealed increased intensity of nuclear staining with DAPI while actin filaments became indistinct or collapsed around the nucleus. After cycloheximide treatment to inhibit protein synthesis, there was no reduction of apoptosis. Gel electrophoresis of DNA from both control and 3 hour-serum-deprived cells showed intact DNA with no oligonucleosomal length fragmentation. After serum withdrawal, intracellular calcium was reduced by about 32% over 5 minutes as measured by fura2 ratio fluorimetry in single cells. Serum-starved cells showed a time-dependent shrinkage in mean cell diameter compared to trypsinized, adherent control cells (at 0 hours, mean diameter = 18.0 microns--viable; at 4 hours, mean diameter = 15.5 microns--apoptotic). Flow cytometric analysis showed increased propidium iodide staining and reduced fluorescein diacetate uptake over 3 hours, changes that were contemporaneous with the reduction of cell diameter.(ABSTRACT TRUNCATED AT 400 WORDS)

Synergistic Induction of Cell Death and Apoptosis by the Histone Deacetylase Inhibitor SAHA (Vorinostat) and the Demethylating Agent DAC in Multiple Myeloma.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2393-2393
Author(s):  
Edgar Jost ◽  
Claudia Schubert ◽  
Tim Bruemmendorf ◽  
Oliver Galm
Keyword(s):  
Cell Lines ◽  
Apoptotic Cell ◽  
Hdac Inhibitors ◽  
Annexin V ◽  
Rt Pcr ◽  
Advisory Committees ◽  
Dnmt Inhibitors ◽  
Induction Of Apoptosis ◽  
Rpmi 8226

Abstract Abstract 2393 Poster Board II-370 Introduction: Hypermethylation of CpG islands in the promoter region of genes is a well characterized epigenetic modification associated with transcriptional silencing of cancer related genes and plays a crucial role in carcinogenesis. In addition, acetylation of core histones is necessary for the maintenance of transcriptional activity of genes. DNA methylation and histone deacetylation are reversible and can be influenced by DNA methyltransferase (DNMT) inhibitors such as 5-aza-2`-deoxycytidine (DAC) or 5-azacytidine (AZA) and histone deacetylase (HDAC) inhibitors such as suberoylanilide hydroxamic acid (SAHA), respectively. Clinical trials using a strategy based on the modification of epigenetic changes with DAC or AZA in combination with HDAC inhibitors have been promising and may help to generate new strategies in treatment of hematopoietic malignancies including multiple myeloma (MM). In MM however, only limited data are published about the possible synergistic effects between DNMT inhibitors and the highly potent pan-HDAC inhibitor SAHA. Material and Methods: To assess the in vitro effects of SAHA on the MM cell lines U266, LP-1, RPMI8226 or OPM-2 and the possible interactions with DNMT inhibitors, cells were first incubated with DAC in a final concentration of 0.1 or 0.2 mM for 72 hours. After exposure to DAC, cells were incubated for 72 or 96 hours with SAHA in a final concentration between 0.1 and 20 mM. The toxic effect of the treatment was assessed by an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The drug concentration inducing a 50 % killing of the cells compared to control cell survival was calculated from the dose-response curve (IC50). Induction of apoptosis was analysed by flow cytometry with annexin V-binding. In addition, the expression of the epigenetically silenced tumor suppressor genes SFRP-2 and DAB2 was determined by real time RT-PCR before and after exposure to DAC and SAHA. Results: In LP-1 and U-266 cells, no relevant enhancement in the cytotoxic effect of SAHA was observed after previous exposure to DAC. In contrast, in OPM-2 and RPMI-8226 cells, a significant increase in cytotoxicity of SAHA was observed, when the cells were first incubated with DAC with a decrease of the IC50 from 6.5 μM to 2.43 μM and 10.37 μM to 4.5 μM, respectively. We further analysed a possible synergism between SAHA and DAC for the induction of apoptosis by flow cytometry. After sequential exposure of the cells with DAC for 72 hours and with SAHA for 72 hours, no change in the apoptotic cell fraction was observed for the cell lines OPM-2 and RPMI-8226. However, for U-266 and LP-1, a significant increase in apoptotic cells was observed after incubation with SAHA, when the cells were previously exposed to DAC with a increase in the apoptotic cell fraction of 39.5 % to 55.4 % and 2.5 % to 14.4 %, respectively. By real-time RT-PCR, corresponding transcriptional silencing for SFRP-2 and DAB2 was demonstrated in untreated cells, and exposure of cell lines to DAC and SAHA resulted in reexpression. A synergism for the induction of reexpression of these genes was observed when cells were incubated with DAC and SAHA sequentially. Discussion: After treatment with SAHA, we observed a dose-dependent induction of cell death and apoptosis as assessed by MTT and annexin V assay, respectively. In the different MM cell lines, we observed a synergism between SAHA and DAC both for cytotoxic effects and the induction of apoptosis. A synergism was also observed for the reexpression of epigenetically silenced genes after exposure to DAC and SAHA. These in vitro data can be considered as a basis for further in vitro studies and preclinical models with SAHA in combination with demethylating agents such as DAC in order to improve treatment response and survival in MM patients. Disclosures: Jost: MSD: Research Funding. Bruemmendorf:Genzyme: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees.

Catechol cytotoxicity in vitro: Induction of glioblastoma cell death by apoptosis

2010 ◽  
Vol 29 (3) ◽  
pp. 199-212 ◽  
Author(s):  
DM de Oliveira ◽  
BPS Pitanga ◽  
MS Grangeiro ◽  
RMF Lima ◽  
MFD Costa ◽  
...  
Keyword(s):  
Cell Death ◽  
Morphological Changes ◽  
Public Health Problem ◽  
Annexin V ◽  
Toxic Effects ◽  
Cytotoxic Effects ◽  
The Central Nervous System ◽  
Hematopoietic Toxicity ◽  
In Cells

The exposure to benzene is a public health problem. Although the most well-known effect of benzene is hematopoietic toxicity, there is little information about the benzene and its metabolites effects on the central nervous system (CNS). This study examined the toxic effects of 1,2-dihydroxybenzene (catechol), a benzene metabolite, to human glioblastoma GL-15 cells. GL-15 cell cultures were used as a model to provide more information about the toxic effects of aromatic compounds to the CNS. Catechol induced time- and concentration-dependent cytotoxic effects. Morphological changes, such as the retraction of the cytoplasm and chromatin clumping, were seen in cells exposed to 200 μM catechol for 48 hours. In cells exposed to 600 μM catechol for 48 hours, 78.0% of them presented condensed nuclei, and the Comet assay showed DNA damage. The percentage of cells labeled with annexin V (apoptotic cells) was greater in the group exposed to catechol (20.7%) than in control cells (0.4%). Exposure to catechol at concentrations greater than 100 μM enhanced Bax levels, and a decrease in Bcl-2 level was observed after the exposure to 600 μM catechol for 48 hours. Furthermore, catechol depleted reduced glutathione. Hence, catechol induced cell death mainly by apoptosis.

scholarly journals Mechanism of Asp24 Upregulation in Brucella abortus Rough Mutant with a Disrupted O-Antigen Export System and Effect of Asp24 in Bacterial Intracellular Survival

2014 ◽  
Vol 82 (7) ◽  
pp. 2840-2850 ◽  
Author(s):  
Mingxing Tian ◽  
Jing Qu ◽  
Xiangan Han ◽  
Chan Ding ◽  
Shaohui Wang ◽  
...  
Keyword(s):  
Brucella Abortus ◽  
Intracellular Survival ◽  
Raw264.7 Cells ◽  
Double Knockout ◽  
Content Type ◽  
O Antigen ◽  
Reverse Transcription Pcr ◽  
Raw264.7 Macrophages ◽  
Export System ◽  
Rough Mutant

ABSTRACTWe previously showed thatBrucella abortusrough mutant strain 2308 ΔATP(called the ΔrfbEmutant in this study) exhibits reduced intracellular survival in RAW264.7 cells and attenuated persistence in BALB/c mice. In this study, we performed microarray analysis to detect genes with differential expression between the ΔrfbEmutant and wild-type strain S2308. Interestingly, acid shock protein 24 gene (asp24) expression was significantly upregulated in the ΔrfbEmutant compared to S2308, as confirmed by quantitative reverse transcription-PCR (qRT-PCR) and Western blotting. Further studies using additional strains indicated that the upregulation ofasp24occurred only in rough mutants with disrupted O-antigen export system components, including the ATP-binding protein generfbE(bab1_0542) and the permease generfbD(bab1_0543), while the ΔwboArough mutant (which lacks an O-antigen synthesis-related glycosyltransferase) and the RB51 strain (a vaccine strain with the rough phenotype) showed no significant changes inasp24expression compared to S2308. In addition, abolishing the intracellular O-antigen synthesis of the ΔrfbEmutant by deleting thewboAgene (thereby creating the ΔrfbEΔwboAdouble-knockout strain) recoveredasp24expression. These results indicated thatasp24upregulation is associated with intracellular O-antigen synthesis and accumulation but not with the bacterial rough phenotype. Further studies indicated thatasp24upregulation in the ΔrfbEmutant was associated neither with bacterial adherence and invasion nor with cellular necrosis on RAW264.7 macrophages. However, proper expression of theasp24gene favors intracellular survival ofBrucellain RAW264.7 cells and HeLa cells during an infection. This study reveals a novel mechanism forasp24upregulation inB. abortusmutants.

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