causes chronic pulmonary infections in patients with cystic fibrosis (CF).
mucoid conversion, defined by overproduction of the exopolysaccharide alginate, correlates with accelerated decline in CF ...patient lung function. Recalcitrance of the mucoid phenotype to clearance by antibiotics and the immune response is well documented. However, despite advantages conferred by mucoidy, mucoid variants often revert to a nonmucoid phenotype both
and
Mixed populations of mucoid isolates and nonmucoid revertants are recovered from CF lungs, suggesting a selective benefit for coexistence of these variants. In this study, cocultures of mucoid and nonmucoid variants exhibited enhanced resistance to two host antimicrobials: LL-37, a cationic antimicrobial peptide, and hydrogen peroxide (H
O
). Alginate production by mucoid isolates protected nonmucoid variants in consortia from LL-37, as addition of alginate exogenously to nonmucoid variants abrogated LL-37 killing. Conversely, nonmucoid revertants shielded mucoid variants from H
O
stress via catalase (KatA) production, which was transcriptionally repressed by AlgT and AlgR, central regulators of alginate biosynthesis. Furthermore, extracellular release of KatA by nonmucoid revertants was dependent on
, encoding an endolysin implicated in autolysis and extracellular DNA (eDNA) release. Overall, these data provide a rationale to study interactions of
mucoid and nonmucoid variants as contributors to evasion of innate immunity and persistence within the CF lung.
mucoid conversion within lungs of cystic fibrosis (CF) patients is a hallmark of chronic infection and predictive of poor prognosis. The selective benefit of mixed populations of mucoid and nonmucoid variants, often isolated from chronically infected CF patients, has not been explored. Here, we show that mixed-variant communities of
demonstrate advantages in evasion of innate antimicrobials via production of shared goods: alginate and catalase. These data argue for therapeutically targeting multiple constituents (both mucoid and nonmucoid variants) within diversified
communities
, as these variants can differentially shield one another from components of the host response.
Cell‐based therapy for the treatment of inflammatory disorders has focused on the application of mesenchymal stromal cells (MSCs) and multipotent adult progenitor cells (MAPCs). Despite the recent ...positive findings in industry‐sponsored clinical trials of MSCs and MAPCs for graft vs host disease (GvHD), cell therapy is efficacious in some but not all patients, highlighting the need to identify strategies to enhance cell‐based therapeutic efficacy. Here, we demonstrate the capacity for interferon (IFN)‐γ licensing to enhance human MAPC efficacy and retention following early administration in a humanized mouse model of acute GvHD (aGvHD). Activation of the nuclear receptor peroxisome proliferator‐activated receptor delta (PPARδ) negatively influenced the retention and efficacy of human MAPCs as well as IFN‐γ‐licensed MAPCs in the aGvHD model. PPARδ antagonism significantly enhanced the efficacy of human MAPCs when administered early in the humanized aGvHD model. COX‐2 expression in human MAPC was significantly decreased in IFN‐γ licensed MAPCs exposed to a PPARδ agonist. Importantly, MAPC exposure to the PPARδ antagonist in the presence of a COX‐2 inhibitor indomethacin before administration significantly reduced the efficacy of PPARδ antagonized MAPCs in the aGvHD humanized mouse model. This is the first study to demonstrate the importance of PPARδ in human MAPC efficacy in vivo and highlights the importance of understanding the disease microenvironment in which cell‐based therapies are to be administered. In particular, the presence of PPARδ ligands may negatively influence MAPC or MSC therapeutic efficacy.
Interferon‐γ (IFN‐γ) licensing enhances the therapeutic efficacy of human multipotent adult progenitor cells (MAPCs) in a mouse model of acute graft vs host disease (aGvHD). Activation of peroxisome proliferator‐activated receptor (PPAR) δ reduces, whereas antagonism of PPARδ(−) enhances MAPC efficacy in aGvHD. PPARδ agonism reduces COX‐2 expression in IFN‐γ licensed MAPC. Figure created with BioRender.com.
Transferrin receptor (TfR) has been shown to be significantly overexpressed in different types of cancers. We discovered TfR as a target for gambogic acid (GA), used in traditional Chinese medicine ...and a previously undiscovered link between TfR and the rapid activation of apoptosis. The binding site of GA on TfR is independent of the transferrin binding site, and it appears that GA potentially inhibits TfR internalization. Down-regulation of TfR by RNA interference decreases sensitivity to GA-induced apoptosis, further supporting TfR as the primary GA receptor. In summary, GA binding to TfR induces a unique signal leading to rapid apoptosis of tumor cells. These results suggest that GA may provide an additional approach for targeting the TfR and its use in cancer therapy.
T-cell depletion therapy is used to prevent acute allograft rejection, treat autoimmunity and create space for bone marrow or hematopoietic cell transplantation. The evolved response to T-cell loss ...is a transient increase in IL-7 that drives compensatory homeostatic proliferation (HP) of mature T cells. Paradoxically, the exaggerated form of this process that occurs following lymphodepletion expands effector T-cells, often causing loss of immunological tolerance that results in rapid graft rejection, autoimmunity, and exacerbated graft-versus-host disease (GVHD). While standard immune suppression is unable to treat these pathologies, growing evidence suggests that manipulating the incipient process of HP increases allograft survival, prevents autoimmunity, and markedly reduces GVHD. Multipotent adult progenitor cells (MAPC) are a clinical grade immunomodulatory cell therapy known to alter γ-chain cytokine responses in T-cells. Herein, we demonstrate that MAPC regulate HP of human T-cells, prevent the expansion of Th1, Th17, and Th22 effectors, and block the development of pathogenic allograft responses. This occurs via IL-1β-primed secretion of PGE2 and activates T-cell intrinsic regulatory mechanisms (SOCS2, GADD45A). These data provide proof-of-principle that HP of human T-cells can be targeted by cellular and molecular therapies and lays a basis for the development of novel strategies to prevent immunopathology in lymphodepleted patients.
Lymphodepletion strategies are used in the setting of transplantation (including bone marrow, hematopoietic cell, and solid organ) to create space or to prevent allograft rejection and graft versus ...host disease. Following lymphodepletion, there is an excess of IL-7 available, and T cells that escape depletion respond to this cytokine undergoing accelerated proliferation. Moreover, this environment promotes the skew of T cells to a Th1 pro-inflammatory phenotype. Existing immunosuppressive regimens fail to control this homeostatic proliferative (HP) response, and thus the development of strategies to successfully control HP while sparing T cell reconstitution (providing a functioning immune system) represents a significant unmet need in patients requiring lymphodepletion. Multipotent adult progenitor cells (MAPC
) have the capacity to control T cell proliferation and Th1 cytokine production. Herein, this study shows that MAPC cells suppressed anti-thymocyte globulin-induced cytokine production but spared T cell reconstitution in a pre-clinical model of lymphodepletion. Importantly, MAPC cells administered intraperitoneally were efficacious in suppressing interferon-γ production and in promoting the expansion of regulatory T cells in the lymph nodes. MAPC cells administered intraperitoneally accumulated in the omentum but were not present in the spleen suggesting a role for soluble factors. MAPC cells suppressed lymphopenia-induced cytokine production in a prostaglandin E2-dependent manner. This study suggests that MAPC cell therapy may be useful as a novel strategy to target lymphopenia-induced pathogenic T cell responses in lymphodepleted patients.
Nonsteroidal anti-inflammatory drugs (NSAIDs) are known to prevent colorectal tumorigenesis. Although antitumor effects of
NSAIDs are mainly due to inhibition of cyclooxygenase activity, there is ...increasing evidence that cyclooxygenase-independent
mechanisms may also play an important role. The early growth response-1 ( EGR-1 ) gene is a member of the immediate-early gene family and has been identified as a tumor suppressor gene. Tolfenamic acid
is a NSAID that exhibits anticancer activity in a pancreatic cancer model. In the present study, we investigated the anticancer
activity of tolfenamic acid in human colorectal cancer cells. Tolfenamic acid treatment inhibited cell growth and induced
apoptosis as measured by caspase activity and bioelectric impedance. Tolfenamic acid induced EGR-1 expression at the transcription
level, and analysis of the EGR-1 promoter showed that a putative ETS-binding site, located at −400 and −394 bp, was required for activation by tolfenamic
acid. The electrophoretic mobility shift assay and chromatin immunoprecipitation assay confirmed that this sequence specifically
bound to the ETS family protein epithelial-specific ETS-1 (ESE-1) transcription factor. Tolfenamic acid also facilitated translocation
of endogenous and exogenous ESE-1 to the nucleus in colorectal cancer cells, and gene silencing using ESE-1 small interfering RNA attenuated tolfenamic acid-induced EGR-1 expression and apoptosis. Overexpression of EGR-1 increased
apoptosis and decreased bioelectrical impedance, and silencing of endogenous EGR-1 prevented tolfenamic acid-induced apoptosis.
These results show that activation of ESE-1 via enhanced nuclear translocation mediates tolfenamic acid-induced EGR-1 expression,
which plays a critical role in the activation of apoptosis. Mol Cancer Ther 2008;7(12):3739–50
Indium tin oxide (ITO) biosensors are used to perform simultaneous optical and electrical measurements in order to examine the dynamic cellular attachment, spreading, and proliferation of endothelial ...cells (ECs) as well as cytotoxic effects when exposed to cytochalasin D. A detailed description of the fabrication of these sensors is provided and their superior optical characteristics are qualitatively shown using four different microscopic images. Differential interference contrast microscopy (DICM) images were acquired simultaneously with micro-impedance measurements as a function of frequency and time. A digital image processing algorithm quantified the cell-covered electrode area as a function of time. In addition, cytotoxicity effects, produced by the toxic agent cytochalasin D, were examined using micro-impedance measurements, confocal microscopy images of stained actin-filaments, and interference reflection contrast microscopy (IRCM) capable of examining the bottom morphology of a cell. The results of this study show (1) the dynamic optical and electrical cellular characteristics using optically thin ITO biosensors; (2) qualitative agreement between cell-covered electrode area and electrical impedance during cellular attachment; (3) in vitro cytotoxicity detection of ECs due to 3 mM cytochalasin D. The present opto-electric biosensor system is unique in that a simultaneous and integrated cellular analysis is possible for a variety of living cells.
The surface micromechanical properties of 2-hydroxyethyl methacrylate (HEMA) and 2-methacryloxyethyl trimethyl ammonium chloride (MAETAC) copolymer hydrogels are probed using atomic force microscopy. ...HEMA–MAETAC polyelectrolyte hydrogels with increasing positive charge concentrations ranging from 0 to 400
mM in increments of 40
mM, are fabricated using different proportions of HEMA and MAETAC monomers. Increasing proportions of positively charged MAETAC monomers produce hydrogels with increasingly swollen states and correspondingly decreasing measures of stiffness, or Young’s modulus. Increasing the relative proportion of charged monomers also increases the hysteresis in the approaching and retracting components of the force spectroscopy curves. When these hydrogels are equilibrated in cell-culture media without fetal bovine serum and a pH-controlled CO
2 environment, precipitation reactions increase the variability of the Young’s modulus estimates. Adding a buffer, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, maintains physiological pH without the use of a CO
2 environment, and thus reduces salt precipitation reactions and the variability of the Young’s modulus. The attachment of porcine pulmonary artery endothelial cells increases with increasing prepared hydrogel charge concentration and decreasing elasticity.
Cellular transformation is the first step in cancer development. Two features of cellular transformation are proliferation in reduced serum and loss of contact inhibition. Electronic Cell-Substrate ...Impedance Sensing (ECIS) measurements have been used to measure cellular proliferation, cytotoxicity, apoptosis, and attachment. We have used impedance measurements to distinguish normal cells from cells transformed with a constitutively active chemokine receptor, CXCR2. CXCR2, a member of the G-protein coupled receptor (GPCR) family, is normally involved in cellular activation and migration, but a single amino acid substitution leads to constitutive activity. NIH3T3 cells were transformed with a constitutively active CXCR2 (D143V_CXCR2) and growth in reduced serum and foci formation were measured using established biological assays and compared to data from ECIS. The results of this study show that impedance measurements provide a quick and reliable way of measuring cellular transformation and provide real time assessment of transformed cellular parameters. Use of the ECIS system could allow a rapid screening of anti-cancer drugs that alter cellular transformation.
This study describes the fabrication and performance of an endothelial cell compatible, optically thin, indium tin oxide (ITO) microimpedance biosensor. The biosensor was constructed by sputtering a ...thin insulating layer of silicon nitride (Si
3N
4) onto a 100
nm thick ITO layer. Indium tin oxide electrodes were formed by chemically etching 250 or 500
μm diameter holes through the Si
3N
4 insulating layer. The exposed ITO electrode was electrically connected to an ITO counter electrode, approximately 2
cm
2 in area, via a 400
μL well containing cell culture media. A lock-in amplifier circuit monitored the impedance of porcine pulmonary artery endothelial cells (PPAECs) cultivated on the electrodes as a function of frequency, between 10 and 100
kHz, and as a function of time, at 5.62
kHz. The ITO–Si
3N
4 microelectrodes provided consistent and repeatable impedance measurements to the attachment and spreading of PPAECs. In addition, the ITO–Si
3N
4 electrodes were recyclable, robust, resistant to ethanol sterilization, and had a high optical transmittance. Most importantly, the ITO–Si
3N
4 electrodes allowed optical access for dynamic cellular attachment imaging. The 5.62
kHz time dependent cellular impedance response to the drug Cytochalasin D further demonstrated the feasibility of using this electrode configuration for dynamic cellular impedance studies.
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