Exposure of Mycobacterium avium subsp. homonissuis to Metal Concentrations of the Phagosome Environment Enhances the Selection of Persistent Subpopulation to Antibiotic Treatment

Mycobacterium avium subspecies hominissuis (MAH) is an opportunistic intracellular pathogen causing infections in individuals with chronic lung conditions and patients with immune-deficient disorders. The treatment of MAH infections is prolonged and outcomes many times are suboptimal. The reason for the extended treatment is complex and reflects the inability of current antimicrobials to clear diverse phenotypes of MAH quickly, particularly, the subpopulation of susceptible but drug-tolerant bacilli where the persistent fitness to anti-MAH drugs is stimulated and enhanced by the host environmental stresses. In order to enhance the pathogen killing, we need to understand the fundamentals of persistence mechanism and conditions that can initiate the drug-tolerance phenotype in mycobacteria. MAH can influence the intracellular environment through manipulation of the metal concentrations in the phagosome of infected macrophages. While metals play important role and are crucial for many cellular functions, little is known how vacuole elements influence persistence state of MAH during intracellular growth. In this study, we utilized the in vitro model mimicking the metal concentrations and pH of MAH phagosome at 1 h and 24 h post-infection to distinguish if metals encountered in phagosome could act as a trigger factor for persistence phenotype. Antibiotic treatment of metal mix exposed MAH demonstrates that metals of the phagosome environment can enhance the persistence state, and greater number of tolerant bacteria is recovered from the 24 h metal mix when compared to the viable pathogen number in the 1 h metal mix and 7H9 growth control. In addition, bacterial phenotype induced by the 24 h metal mix increases MAH tolerance to macrophage killing in TNF-α and IFN-γ activated cells, confirming presence of persistent MAH in the 24 h metal mix condition. This work shows that the phagosome environment can promote persistence population in MAH, and that the population differs dependent on a concentration of metals.

Download Full-text

Antioxidant and Hypolipidemic Activity of Açai Fruit Makes It a Valuable Functional Food

Antioxidants ◽

10.3390/antiox10010040 ◽

2020 ◽

Vol 10 (1) ◽

pp. 40

Author(s):

Anna Virginia Adriana Pirozzi ◽

Paola Imbimbo ◽

Antonella D’Agostino ◽

Virginia Tirino ◽

Rosario Finamore ◽

...

Keyword(s):

Functional Food ◽

Subcutaneous Fat ◽

Cell Models ◽

Alcoholic Fatty Liver ◽

Hypoxic Conditions ◽

Tnf Α ◽

Different Types ◽

Vitro Model ◽

Alcoholic Fatty Liver Disease

Several plant extracts are acquiring increasing value because of their antioxidant activity and hypolipidemic properties. Among them, great interest has been recently paid to açai fruit as a functional food. The aim of this study was to test the ability of açai extract in reducing oxidative stress and modulating lipid metabolism in vitro using different cell models and different types of stress. In fact, lipid peroxidation as evaluated in a HepG2 model was reduced five-fold when using 0.25 µg/mL of extract, and it was further reduced (20-fold) with the concentration increase up to 2.5 µg/mL. With the non alcoholic fatty liver disease (NAFLD)in vitro model, all concentrations tested showed at least a two-fold reduced fat deposit. In addition, primary adipocytes challenged with TNF-α under hypoxic conditions to mimic the persistent subcutaneous fat, treated with açai extract showed an approximately 40% reduction of fat deposit. Overall, our results show that açai is able to counteract oxidative states in all the cell models analysed and to prevent the accumulation of lipid droplets. No toxic effects and high stability overtime were highlighted at the concentrations tested. Therefore, açai can be considered a suitable support in the prevention of different alterations of lipid and oxidative metabolism responsible for fat deposition and metabolic pathological conditions.

Download Full-text

Effect of the proinflammatory cytokine TNF-α on intestinal riboflavin uptake: inhibition mediated via transcriptional mechanism(s)

AJP Cell Physiology ◽

10.1152/ajpcell.00295.2018 ◽

2018 ◽

Vol 315 (5) ◽

pp. C653-C663 ◽

Cited By ~ 5

Author(s):

Kasin Yadunandam Anandam ◽

Omar A. Alwan ◽

Veedamali S. Subramanian ◽

Padmanabhan Srinivasan ◽

Rubina Kapadia ◽

...

Keyword(s):

Ex Vivo ◽

Significant Inhibition ◽

Mrna Levels ◽

In Vivo Models ◽

Uptake Inhibition ◽

Tnf Α ◽

Vitro Model ◽

Factor Α

Riboflavin (RF), is essential for normal cellular metabolism/function. Intestinal RF absorption occurs via a specific carrier-mediated process that involves the apical transporter RFVT-3 ( SLC52A3) and the basolateral RFVT-1 (SLC52A1). Previously, we characterized different cellular/molecular aspects of the intestinal RF uptake process, but nothing is known about the effect of proinflammatory cytokines on the uptake event. We addressed this issue using in vitro, ex vivo, and in vivo models. First, we determined the level of mRNA expression of the human (h)RFVT-3 and hRFVT-1 in intestinal tissue of patients with inflammatory bowel disease (IBD) and observed a markedly lower level compared with controls. In the in vitro model, exposing Caco-2 cells to tumor necrosis factor-α (TNF-α) led to a significant inhibition in RF uptake, an effect that was abrogated upon knocking down TNF receptor 1 (TNFR1). The inhibition in RF uptake was associated with a significant reduction in the expression of hRFVT-3 and -1 protein and mRNA levels, as well as in the activity of the SLC52A3 and SLC52A1 promoters. The latter effects appear to involve Sp1 and NF-κB sites in these promoters. Similarly, exposure of mouse small intestinal enteroids and wild-type mice to TNF-α led to a significant inhibition in physiological and molecular parameters of intestinal RF uptake. Collectively, these findings demonstrate that exposure of intestinal epithelial cells to TNF-α leads to inhibition in RF uptake and that this effect is mediated, at least in part, via transcriptional mechanism(s). These findings may explain the significantly low RF levels observed in patients with IBD.

Download Full-text

Modelling Bovine Granuloma Formation In Vitro upon Infection with Mycobacterium Avium Subspecies Paratuberculosis

Veterinary Sciences ◽

10.3390/vetsci6040080 ◽

2019 ◽

Vol 6 (4) ◽

pp. 80 ◽

Cited By ~ 1

Author(s):

J. Hunter Rice ◽

Margaret M. McDaniel ◽

Alyson Holland ◽

Shigetoshi Eda

Keyword(s):

Mycobacterium Avium ◽

Mycobacterium Avium Subspecies Paratuberculosis ◽

Clinical Signs ◽

Host Cells ◽

Economic Losses ◽

Multiple Parameters ◽

Cell Mediated Immune Response ◽

A Cell ◽

Vitro Model

Mycobacterium avium subspecies paratuberculosis (Map) causes chronic granulomatous disease in cattle and ruminant livestock, causing substantial economic losses. Current vaccines delay clinical signs but cannot train the immune system to fully eradicate latent Map. During latency, Map uses host defenses, cage-like macrophage clusters called granuloma, as incubators for months or years. We used an in vitro model to investigate the early coordination of macrophages into granuloma upon Map infection over ten days. We found that at multiplicities of infection (MOI; Map:macrophages) of 1:2 and below, the macrophages readily form clusters and evolve pro-inflammatory cytokines in keeping with a cell-mediated immune response. At higher MOIs, viability of host macrophages is negatively impacted. At 1:4 MOI, we quantified viable Map in our model and confirmed that intracellular Map reproduced over the first five days of infection. Host cells expressed Type 1-specific cytokines, and Map-infected macrophages displayed reduced motility compared to Map-exposed, uninfected macrophages, suggesting an important role for uninfected macrophages in the early aggregative response. Reported is the first in vitro JD granuloma model capturing Map and macrophage viability, size distribution of resulting clusters, motility of monocyte-derived macrophages, and cytokine response during clustering, allowing quantitative analysis of multiple parameters of the Map-specific granulomatous response.

Download Full-text

Differential expression and regulation of ADAM17 and TIMP3 in acute inflamed intestinal epithelia

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.90641.2008 ◽

2009 ◽

Vol 296 (6) ◽

pp. G1332-G1343 ◽

Cited By ~ 37

Author(s):

Annabelle Cesaro ◽

Abakar Abakar-Mahamat ◽

Patrick Brest ◽

Sandra Lassalle ◽

Eric Selva ◽

...

Keyword(s):

High Activity ◽

Early Phase ◽

Active Form ◽

Tissue Inhibitor Of Metalloproteinase ◽

Intestinal Epithelial ◽

T84 Cells ◽

Intestinal Epithelia ◽

Tnf Α ◽

Vitro Model

The acute phase of Crohn's disease (CD) is characterized by a large afflux of polymorphonuclear leukocytes (PMNL) into the mucosa and by the release of TNF-α. Conversion of inactive TNF-α into an active form requires the cleavage of a transmembrane TNF-α precursor by the TNF-α-converting enzyme (ADAM17), a protease mainly regulated by the tissue inhibitor of metalloproteinase 3 (TIMP3). The aim of the present study was to investigate in an in vitro model of PMNL transepithelial migration and in the intestinal mucosa of patients with CD the expression and regulation of ADAM17 and TIMP3 in intestinal epithelial cells (IEC). ADAM17 and TIMP3 expression was analyzed by Western blotting, RT-PCR, confocal microscopy, and immunohistochemistry by using the T84 model and digestive biopsies. ADAM17 expression in IEC was increased at a posttranscriptional level during the early phase (from 2 to 4 h) of PMNL transepithelial migration whereas TIMP3 was only increased 24 h later. TNF-α induced an early upregulation of ADAM17 in T84 cells, whereas PMNL adhesion, H2O2, or epithelial tight junction opening alone did not affect the amount of ADAM17. Immunohistochemistry of intestinal biopsies revealed that strong expression of ADAM17 was associated with a high activity of CD. In contrast, TIMP3 was very poorly expressed in these biopsies. ADAM17 and TIMP3 profiling did not correlated with the NOD2/CARD15 status. The ADAM17 activity was higher both in the early phase of PMNL transepithelial migration and in active CD. These results showed early posttranscriptional upregulation of ADAM17 in IEC linked to PMNL transepithelial migration and a high activity of CD.

Download Full-text

Pharmacokinetic/Pharmacodynamic Investigation of Colistin against Pseudomonas aeruginosa Using an In Vitro Model

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00903-09 ◽

2010 ◽

Vol 54 (9) ◽

pp. 3783-3789 ◽

Cited By ~ 107

Author(s):

Phillip J. Bergen ◽

Jurgen B. Bulitta ◽

Alan Forrest ◽

Brian T. Tsuji ◽

Jian Li ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Growth Control ◽

Multidrug Resistant ◽

Type Model ◽

Time Curve ◽

Killing Effect ◽

Optimal Dosing ◽

Dosing Strategies ◽

Vitro Model

ABSTRACT Colistin plays a key role in treatment of serious infections by Pseudomonas aeruginosa. The aims of this study were to (i) identify the pharmacokinetic/pharmacodynamic (PK/PD) index (i.e., the area under the unbound concentration-time curve to MIC ratio [ƒAUC/MIC], the unbound maximal concentration to MIC ratio [ƒC max/MIC], or the cumulative percentage of a 24-h period that unbound concentrations exceed the MIC [ƒT >MIC]) that best predicts colistin efficacy and (ii) determine the values for the predictive PK/PD index required to achieve various magnitudes of killing effect. Studies were conducted in a one-compartment in vitro PK/PD model for 24 h using P. aeruginosa ATCC 27853, PAO1, and the multidrug-resistant mucoid clinical isolate 19056 muc. Six intermittent dosing intervals, with a range of ƒC max colistin concentrations, and two continuous infusion regimens were examined. PK/PD indices varied from 0.06 to 18 for targeted ƒC max/MIC, 0.36 to 312 for ƒAUC/MIC, and 0 to 100% for ƒT >MIC. A Hill-type model was fit to killing effect data, which were expressed as the log10 ratio of the area under the CFU/ml curve for treated regimens versus control. With ƒC max values equal to or above the MIC, rapid killing was observed following the first dose; substantial regrowth occurred by 24 h with most regimens. The overall killing effect was best correlated with ƒAUC/MIC (R 2 = 0.931) compared to ƒC max/MIC (R 2 = 0.868) and ƒT >MIC (R 2 = 0.785). The magnitudes of ƒAUC/MIC required for 1- and 2-log10 reductions in the area under the CFU/ml curve relative to growth control were 22.6 and 30.4, 27.1 and 35.7, and 5.04 and 6.81 for ATCC 27853, PAO1, and 19056 muc, respectively. The PK/PD targets identified will assist in designing optimal dosing strategies for colistin.

Download Full-text

A Novel Lactic Acid Bacteria Mixture: Macrophage-Targeted Prophylactic Intervention in Colorectal Cancer Management

Microorganisms ◽

10.3390/microorganisms8030387 ◽

2020 ◽

Vol 8 (3) ◽

pp. 387

Author(s):

Petra Hradicka ◽

Jane Beal ◽

Monika Kassayova ◽

Andrew Foey ◽

Vlasta Demeckova

Keyword(s):

Colorectal Cancer ◽

Male Rats ◽

Cancer Management ◽

Immune Parameters ◽

Chemically Induced ◽

Tnf Α ◽

Vitro Model ◽

Macrophage Subsets

Colorectal cancer (CRC) is one of the most common forms of cancer. Its onset from chronic inflammation is widely accepted. Moreover, dysbiosis plays an undeniable role, thus the use of probiotics in CRC has been suggested. They exhibit both anti- and pro-inflammatory properties and restore balance in the microbiota. The aim of this study was to investigate the immunomodulatory properties of six lactobacilli with probiotic features in an in vitro model of macrophage-like cells and to test these pooled probiotics for their anti-tumour properties in a chemically induced CRC model using Wistar male rats. Upon co-culture of M1- and M2-like macrophages with lactobacilli, cytokine release (TNF-α, IL-1β, IL-18, IL-23) and phagocytic activity using fluorescent-labelled bacteria were tested. The effects of orally administered probiotics on basic cancer and immune parameters and cytokine concentration (TNF-α, IL-1β, IL-18) in colon tumours were studied. Tested lactobacilli exhibited both pro- and anti-inflammatory properties in in vitro conditions. In vivo study showed that the administration of probiotics was able to decrease multiplicity, volume and total tumour numbers, restore colon length (p < 0.05) and increase IL-18 production (p < 0.05) in tumour tissue. These data indicate both an immunomodulatory effect of probiotics on distinct macrophage subsets and a protective effect against chemically-induced CRC.

Download Full-text

Beneficial effects of nontoxic ozone on H2O2-induced stress and inflammation

Biochemistry and Cell Biology ◽

10.1139/bcb-2016-0033 ◽

2016 ◽

Vol 94 (6) ◽

pp. 577-583 ◽

Cited By ~ 6

Author(s):

Altug Kucukgul ◽

Suat Erdogan ◽

Ramazan Gonenci ◽

Gonca Ozan

Keyword(s):

Cell Loss ◽

Alveolar Cells ◽

Sod Activity ◽

Beneficial Effects ◽

Tnf Α ◽

Ozone Pretreatment ◽

Anti Oxidant ◽

Anti Inflammatory ◽

Vitro Model

In this study, the anti-oxidant and anti-inflammatory efficacy of ozone oxidative preconditioning (OOP) were investigated on hydrogen peroxide (H2O2)-induced human lung alveolar cells. In MTT and trypan blue viability tests, while 100 μmol/L H2O2caused a 17.3% and 21.9% decrease in the number of living cells, respectively, ozone at 20 μmol/L regenerated cell proliferation and prevented 9.6% and 11.0% of cell loss, respectively. In addition, H2O2decreased the transcription levels of catalase (CAT), glutathione peroxidase (GPx), and superoxide dismutase (SOD) 5.43-, 2.89-, and 5.33-fold, respectively, while it increased Bax, NF-κβ, TNF-α, and iNOS expression 1.57-, 1.32-, 1.40-, and 1.41-fold, respectively. Ozone pretreatment, however, increased CAT, GPx, and SOD transcription levels 7.08-, 5.17-, and 6.49-fold and decreased Bax, NF-κβ, TNF-α, and iNOS transcriptions by 1.25-, 0.76-, 3.63-, and 7.91-fold, respectively. Moreover, intracellular glutathione (GSH) level and SOD activity were decreased by 46.2% and 45.0% in the H2O2treatment group, and OOP recovered 58.5% and 20.1% of the decreases caused by H2O2. H2O2also increased nitrite levels 7.84-fold, and OOP reduced this increase by half. Consequently, OOP demonstrated potent anti-oxidant and anti-inflammatory effects on in vitro model of oxidative stress-induced lung injury.

Download Full-text

TNF-α Induces MMP2 Gelatinase Activity and MT1-MMP Expression in an In Vitro Model of Nucleus Pulposus Tissue Degeneration

Spine ◽

10.1097/brs.0b013e3181642a5e ◽

2008 ◽

Vol 33 (4) ◽

pp. 356-365 ◽

Cited By ~ 55

Author(s):

Cheryle A. Séguin ◽

Robert M. Pilliar ◽

Joseph A. Madri ◽

Rita A. Kandel

Keyword(s):

Nucleus Pulposus ◽

In Vitro Model ◽

Gelatinase Activity ◽

Nucleus Pulposus Tissue ◽

Tissue Degeneration ◽

Tnf Α ◽

Vitro Model

Download Full-text

Mycobacterium avium Biofilm Attenuates Mononuclear Phagocyte Function by Triggering Hyperstimulation and Apoptosis during Early Infection

Infection and Immunity ◽

10.1128/iai.00820-13 ◽

2013 ◽

Vol 82 (1) ◽

pp. 405-412 ◽

Cited By ~ 25

Author(s):

Sasha J. Rose ◽

Luiz E. Bermudez

Keyword(s):

Immune System ◽

Mycobacterium Avium ◽

Innate Immune System ◽

Innate Immune ◽

Mononuclear Phagocytes ◽

Bacterial Killing ◽

Content Type ◽

Tnf Α

ABSTRACTMycobacterium aviumsubsp.hominissuisis an opportunistic human pathogen that has been shown to form biofilmin vitroandin vivo. Biofilm formationin vivoappears to be associated with infections in the respiratory tract of the host. The reasoning behind howM. aviumsubsp.hominissuisbiofilm is allowed to establish and persist without being cleared by the innate immune system is currently unknown. To identify the mechanism responsible for this, we developed anin vitromodel using THP-1 human mononuclear phagocytes cocultured with establishedM. aviumsubsp.hominissuisbiofilm and surveyed various aspects of the interaction, including phagocyte stimulation and response, bacterial killing, and apoptosis.M. aviumsubsp.hominissuisbiofilm triggered robust tumor necrosis factor alpha (TNF-α) release from THP-1 cells as well as superoxide and nitric oxide production. Surprisingly, the hyperstimulated phagocytes did not effectively eliminate the cells of the biofilm, even when prestimulated with gamma interferon (IFN-γ) or TNF-α or cocultured with natural killer cells (which have been shown to induce anti-M. aviumsubsp.hominissuisactivity when added to THP-1 cells infected with planktonicM. aviumsubsp.hominissuis). Time-lapse microscopy and the TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) assay determined that contact with theM. aviumsubsp.hominissuisbiofilm led to early, widespread onset of apoptosis, which is not seen until much later in planktonicM. aviumsubsp.hominissuisinfection. Blocking TNF-α or TNF-R1 during interaction with the biofilm significantly reduced THP-1 apoptosis but did not lead to elimination ofM. aviumsubsp.hominissuis. Our data collectively indicate thatM. aviumsubsp.hominissuisbiofilm induces TNF-α-driven hyperstimulation and apoptosis of surveilling phagocytes, which prevents clearance of the biofilm by cells of the innate immune system and allows the biofilm-associated infection to persist.

Download Full-text