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1.  Pulmonary Administration of a Water-Soluble Curcumin Complex Reduces Severity of Acute Lung Injury 
Local or systemic inflammation can result in acute lung injury (ALI), and is associated with capillary leakage, reduced lung compliance, and hypoxemia. Curcumin, a plant-derived polyphenolic compound, exhibits potent anti-inflammatory properties, but its poor solubility and limited oral bioavailability reduce its therapeutic potential. A novel curcumin formulation (CDC) was developed by complexing the compound with hydroxypropyl-γ-cyclodextrin (CD). This results in greatly enhanced water solubility and stability that facilitate direct pulmonary delivery. In vitro studies demonstrated that CDC increased curcumin’s association with and transport across Calu-3 human airway epithelial cell monolayers, compared with uncomplexed curcumin solubilized using DMSO or ethanol. Importantly, Calu-3 cell monolayer integrity was preserved after CDC exposure, whereas it was disrupted by equivalent uncomplexed curcumin solutions. We then tested whether direct delivery of CDC to the lung would reduce severity of ALI in a murine model. Fluorescence microscopic examination revealed an association of curcumin with cells throughout the lung. The administration of CDC after LPS attenuated multiple markers of inflammation and injury, including pulmonary edema and neutrophils in bronchoalveolar lavage fluid and lung tissue. CDC also reduced oxidant stress in the lungs and activation of the proinflammatory transcription factor NF-κB. These results demonstrate the efficacy of CDC in a murine model of lung inflammation and injury, and support the feasibility of developing a lung-targeted, curcumin-based therapy for the treatment of patients with ALI.
PMCID: PMC3488693  PMID: 22312018
cyclodextrin; LPS; turmeric; Calu-3; oxidative stress; TEER
2.  PPARs: Regulators and Translational Targets in the Lung 
PPAR Research  2012;2012:342924.
PMCID: PMC3503434  PMID: 23213318
3.  The Nitrated Fatty Acid 10-Nitro-oleate Diminishes Severity of LPS-Induced Acute Lung Injury in Mice 
PPAR Research  2012;2012:617063.
Acute lung injury (ALI) is an inflammatory condition culminating in respiratory failure. There is currently no effective pharmacological treatment. Nitrated fatty acids (NFAs) have been shown to exert anti-inflammatory effects. We therefore hypothesized that delivery of NFAs directly to the site of inflammation would reduce the severity of ALI. Pulmonary delivery of 10-nitro-oleate following endotoxin-induced ALI in mice reduced markers of lung inflammation and injury, including capillary leakage, lung edema, infiltration of neutrophils into the lung, and oxidant stress, as well as plasma levels of proinflammatory cytokines. Nitro-oleate delivery likewise downregulated expression of proinflammatory genes by alveolar macrophages, key cells in regulation of lung inflammation. These effects may be accounted for by the observed increases in the activity of PPAR-γ and the PPAR-γ-induced antioxidant transcription factor Nrf2, together with the decreased activity of NF-κB. Our results demonstrate that pulmonary delivery of NFAs reduces severity of acute lung injury and suggest potential utility of these molecules in other inflammatory lung diseases.
PMCID: PMC3423963  PMID: 22919366
4.  Curcumin-Loaded Apotransferrin Nanoparticles Provide Efficient Cellular Uptake and Effectively Inhibit HIV-1 Replication In Vitro 
PLoS ONE  2011;6(8):e23388.
Curcumin (diferuloylmethane) shows significant activity across a wide spectrum of conditions, but its usefulness is rather limited because of its low bioavailability. Use of nanoparticle formulations to enhance curcumin bioavailability is an emerging area of research.
Methodology/Principal Findings
In the present study, curcumin-loaded apotransferrin nanoparticles (nano-curcumin) prepared by sol-oil chemistry and were characterized by electron and atomic force microscopy. Confocal studies and fluorimetric analysis revealed that these particles enter T cells through transferrin-mediated endocytosis. Nano-curcumin releases significant quantities of drug gradually over a fairly long period, ∼50% of curcumin still remaining at 6 h of time. In contrast, intracellular soluble curcumin (sol-curcumin) reaches a maximum at 2 h followed by its complete elimination by 4 h. While sol-curcumin (GI50 = 15.6 µM) is twice more toxic than nano-curcumin (GI50 = 32.5 µM), nano-curcumin (IC50<1.75 µM) shows a higher anti-HIV activity compared to sol-curcumin (IC50 = 5.1 µM). Studies in vitro showed that nano-curcumin prominently inhibited the HIV-1 induced expression of Topo II α, IL-1β and COX-2, an effect not seen with sol-curcumin. Nano-curcumin did not affect the expression of Topoisomerase II β and TNF α. This point out that nano-curcumin affects the HIV-1 induced inflammatory responses through pathways downstream or independent of TNF α. Furthermore, nano-curcumin completely blocks the synthesis of viral cDNA in the gag region suggesting that the nano-curcumin mediated inhibition of HIV-1 replication is targeted to viral cDNA synthesis.
Curcumin-loaded apotransferrin nanoparticles are highly efficacious inhibitors of HIV-1 replication in vitro and promise a high potential for clinical usefulness.
PMCID: PMC3161739  PMID: 21887247
5.  A Pediatric Dyspnea Scale for use in hospitalized patients with asthma 
Asthma is a leading cause of pediatric hospitalizations across the country yet no clinical instrument exists that incorporates the child’s perception of dyspnea in determining discharge readiness.
We sought to develop a Pediatric Dyspnea Scale (PDS) to support discharge decision-making in hospitalized asthmatic patients, and to compare the performance of PDS with traditional markers of asthma control in predicting outcomes after discharge.
Asthmatic children aged 6 to 18 years hospitalized for an exacerbation were included in the study. The PDS score, demographics, asthma severity, spirometry, peak expiratory flow rate (PEFR) and exhaled nitric oxide (FENO) were assessed at the time of discharge. A telephone-call 14 days after discharge determined relapse, activity limitation, asthma control and asthma-related quality of life outcomes.
Eighty-nine subjects were enrolled of whom 70 completed the phone follow-up. Eight patients had a relapse and 29 complained of limited activity. A PDS score above 2 on the 7-point scale was a significant predictor of these poor outcomes, with each additional point of the PDS doubling the risk. A higher score on PDS also correlated with worse asthma control and poor asthma-specific quality of life. The PDS performed better than FEV1, PEFR or FENO in predicting the outcomes of interest.
The PDS, which is easy to use in children as young as 6 years of age, may be able to predict adverse outcomes after hospitalization for an asthma exacerbation, and should be used as a tool to help guide inpatient discharge decisions.
PMCID: PMC2747623  PMID: 19181371
asthma; discharge; dyspnea; hospitalized; outcome; pediatric; scale; spirometry; symptoms; exhaled nitric oxide
6.  Curcumin is not a ligand for peroxisome proliferator-activated receptor-γ 
Curcumin, a compound found in the spice turmeric, has been shown to possess a number of beneficial biological activities exerted through a variety of different mechanisms. Some curcumin effects have been reported to involve activation of the nuclear transcription factor peroxisome proliferator-activated receptor-γ (PPAR-γ), but the concept that curcumin might be a PPAR-γ ligand remains controversial. Results reported here demonstrate that, in contrast to the PPAR-γ ligands ciglitazone and rosiglitazone, curcumin is inactive in five different reporter or DNA-binding assays, does not displace [3H]rosiglitazone from the PPAR-γ ligand-binding site, and does not induce PPAR-γ–dependent differentiation of preadipocytes, while its ability to inhibit fibroblast-to-myofibroblast differentiation is not affected by any of four PPAR-γ antagonists. These multiple lines of evidence conclusively demonstrate that curcumin is not a PPAR-γ ligand and indicate the need for further investigation of the mechanisms through which the compound acts.
PMCID: PMC2717748  PMID: 19644570
PPAR-γ; TGF-β; rosiglitazone; ciglitazone; PPRE; preadipocyte; fibroblast; turmeric; peroxisome; curcumin
7.  Chemotherapeutic Drugs Induce PPAR-γ Expression and Show Sequence-Specific Synergy with PPAR-γ Ligands in Inhibition of Non-Small Cell Lung Cancer1 
Neoplasia (New York, N.Y.)  2008;10(6):597-603.
Preclinical studies have shown that peroxisome proliferator-activated receptor γ (PPAR-γ) ligands can exert antitumor effects against non-small cell lung cancer (NSCLC) and a variety of other cancers. In this study, we investigate the potential use of a PPAR-γ ligand, troglitazone (Tro), in combination with either of two chemotherapeutic agents, cisplatin (Cis) or paclitaxel (Pac), for the treatment of NSCLC. In vitro, treatment of NSCLC cell lines with Tro potentiated Cis- or Pac-induced growth inhibition. The potentiation of growth inhibition was observed only when Cis or Pac treatment was followed by Tro and not vice versa, demonstrating a sequence-specific effect. Median effect analysis revealed a synergistic interaction between Tro and Cis in the inhibition of NSCLC cell growth and confirmed the sequence-specific effect. We also found that Cis or Pac up-regulated the expression of PPAR-γ protein, accounting for the observed sequence-specific synergy. Similarly, experiments performed using a NSCLC xenograft model demonstrated enhanced effectiveness of combined treatment with Cis and PPAR-γ ligands, Tro or pioglitazone. Tumors from Cis-treated mice also demonstrated enhanced PPAR-γ expression. Together, our data demonstrate a novel sequence-specific synergy between PPAR-γ ligands and chemotherapeutic agents for lung cancer treatment.
PMCID: PMC2386544  PMID: 18516296
8.  Constitutive activation of prosurvival signaling in alveolar mesenchymal cells isolated from patients with nonresolving acute respiratory distress syndrome 
Acute respiratory distress syndrome (ARDS) is a clinical syndrome characterized by stereotypic host inflammatory and repair cellular responses; however, mechanisms regulating the resolution of ARDS are poorly understood. Here, we report the isolation and characterization of a novel population of mesenchymal cells from the alveolar space of ARDS patients via fiber-optic bronchoscopy with bronchoalveolar lavage (BAL). BAL was performed on 17 patients during the course of ARDS. Immunofluorescence staining and multiparameter flow cytometric analysis defined a population of alveolar mesenchymal cells (AMCs) that are CD45−/prolyl-4-hydroxylase+/α-smooth muscle actin+/−. AMCs proliferated in ex vivo cell culture for multiple passages; early passage (3–5) cells were subsequently analyzed in 13 patients. AMCs isolated from patients with persistent or nonresolving ARDS (ARDS-NR, n = 4) demonstrate enhanced constitutive activation of prosurvival signaling pathways involving PKB/Akt, FKHR, and BCL-2 family proteins compared with AMCs from patients with resolving ARDS (ARDS-R, n = 9). Exogenous transforming growth factor-β1 markedly induces PKB/Akt activation in AMCs from ARDS-R. ARDS-NR cells are more resistant to serum deprivation-induced apoptosis compared with ARDS-R. This study identifies a novel population of mesenchymal cells that can be isolated from the alveolar spaces of ARDS patients. AMCs in patients with ARDS-NR acquire an activational profile characterized by enhanced prosurvival signaling and an antiapoptotic phenotype. These findings support the concept that apoptosis of mesenchymal cells may be an essential component of normal repair and resolution of ARDS and suggest that dysregulation of this process may contribute to persistent ARDS.
PMCID: PMC1382273  PMID: 16214815
acute lung injury; fibrosis; BCL-2 genes; fibroblast; protein kinase B; apoptosis; tissue repair
9.  Pioglitazone is as effective as dexamethasone in a cockroach allergen-induced murine model of asthma 
Respiratory Research  2007;8(1):90.
While glucocorticoids are currently the most effective therapy for asthma, associated side effects limit enthusiasm for their use. Peroxisome proliferator-activated receptor-γ (PPAR-γ) activators include the synthetic thiazolidinediones (TZDs) which exhibit anti-inflammatory effects that suggest usefulness in diseases such as asthma. How the ability of TZDs to modulate the asthmatic response compares to that of glucocorticoids remains unclear, however, because these two nuclear receptor agonists have never been studied concurrently. Additionally, effects of PPAR-γ agonists have never been examined in a model involving an allergen commonly associated with human asthma.
We compared the effectiveness of the PPAR-γ agonist pioglitazone (PIO) to the established effectiveness of a glucocorticoid receptor agonist, dexamethasone (DEX), in a murine model of asthma induced by cockroach allergen (CRA). After sensitization to CRA and airway localization by intranasal instillation of the allergen, Balb/c mice were challenged twice at 48-h intervals with intratracheal CRA. Either PIO (25 mg/kg/d), DEX (1 mg/kg/d), or vehicle was administered throughout the period of airway CRA exposure.
PIO and DEX demonstrated similar abilities to reduce airway hyperresponsiveness, pulmonary recruitment of inflammatory cells, serum IgE, and lung levels of IL-4, IL-5, TNF-α, TGF-β, RANTES, eotaxin, MIP3-α, Gob-5, and Muc5-ac. Likewise, intratracheal administration of an adenovirus containing a constitutively active PPAR-γ expression construct blocked CRA induction of Gob-5 and Muc5-ac.
Given the potent effectiveness shown by PIO, we conclude that PPAR-γ agonists deserve investigation as potential therapies for human asthma.
PMCID: PMC2231357  PMID: 18053220
10.  Increased Yield of High Purity Recombinant Human Interferon-γ Utilizing Reversed Phase Column Chromatography 
Increasing therapeutic applications for recombinant human interferon-γ (rhIFN-γ), an antiviral proinflammatory cytokine, has broadened interest in optimizing methods for its production and purification. We describe a reversed phase chromatography (RPC) procedure using Source-30™ matrix in the purification of rhIFN-γ from Escherichia coli that results in a higher yield than previously reported. The purified rhIFN-γ monomer from the RPC column is refolded in Tris buffer. Optimal refolding occurs at protein concentrations between 50–100 μg/ml. This method yields greater than 90% of the dimer form with a yield of 40 mg g−1 cell mass. Greater than 99% purity is achieved with further purification over a Superdex G-75 column to obtain specific activities of from 2 to 4 × 107 IU/mg protein as determined via cytopathic antiviral assay. The improved yield of rhIFN-γ in a simple chromatographic purification procedure promises to enhance the development and therapeutic application of this biologically potent molecule.
PMCID: PMC2015061  PMID: 17049266
recombinant human interferon-gamma; protein expression; reversed phase chromatography; protein purification; antiviral assay
11.  Stimulatory Effects of Peroxisome Proliferator-Activated Receptor-γ on Fcγ Receptor-Mediated Phagocytosis by Alveolar Macrophages 
PPAR Research  2007;2007:52546.
Alveolar macrophages abundantly express PPAR-γ, with both natural and synthetic agonists maintaining the cell in a quiescent state hyporesponsive to antigen stimulation. Conversely, agonists upregulate expression and function of the cell-surface receptor CD36, which mediates phagocytosis of lipids, apoptotic neutrophils, and other unopsonized materials. These effects led us to investigate the actions of PPAR-γ agonists on the Fcγ receptor, which mediates phagocytosis of particles opsonized by binding of immunoglobulin G antibodies. We found that troglitazone, rosiglitazone, and 15-deoxy-Δ12,14-prostaglandin J2 increase the ability of alveolar, but not peritoneal, macrophages to carry out phagocytosis mediated by the Fcγ receptor. Receptor expression was not altered but activation of the downstream signaling proteins Syk, ERK-1, and ERK-2 was observed. Although it was previously known that PPAR-γ ligands stimulate phagocytosis of unopsonized materials, this is the first demonstration that they stimulate phagocytosis of opsonized materials as well.
PMCID: PMC2220046  PMID: 18253476
12.  PPARs in Alveolar Macrophage Biology 
PPAR Research  2007;2007:23812.
PPARs, most notably PPAR-γ, play a crucial role in regulating the activation of alveolar macrophages, which in turn occupy a pivotal place in the immune response to pathogens and particulates drawn in with inspired air. In this review, we describe the dual role of the alveolar macrophage as both a first-line defender through its phagocytotic activity and a regulator of the immune response. Depending on its state of activation, the alveolar macrophage may either enhance or suppress different aspects of immune function in the lung. We then review the role of PPAR-γ and its ligands in deactivating alveolar macrophages—thus limiting the inflammatory response that, if unchecked, could threaten the essential respiratory function of the alveolus—while upregulating the cell's phagocytotic activity. Finally, we examine the role that inadequate or inappropriate PPAR-γ responses play in specific lung diseases.
PMCID: PMC2066181  PMID: 18000531
13.  PPAR-γ Activation Inhibits Angiogenesis by Blocking ELR+CXC Chemokine Production in Non-small Cell Lung Cancer1 
Neoplasia (New York, N.Y.)  2005;7(3):294-301.
Activation of peroxisome proliferator-activated receptor-γ (PPAR-γ) results in inhibition of tumor growth in various types of cancers, but the mechanism( s) by which PPAR-γ induces growth arrest has not been completely defined. In a recent study, we demonstrated that treatment of A549 (human non small cell lung cancer cell line) tumor-bearing SCID mice with PPAR-γ ligands troglitazone (Tro) and pioglitazone significantly inhibits primary tumor growth. In this study, immunohistochemical analysis of Tro-treated and Pio-treated tumors with factor VIII antibody revealed a significant reduction in blood vessel density compared to tumors in control animals, suggesting inhibition of angiogenesis. Further analysis showed that treatment of A549 cells in vitro with Tro or transient transfection of A549 cells with constitutively active PPAR-γ (VP16-PPAR-γ) construct blocked the production of the angiogenic ELR +CXC chemokines IL-8 (CXCL8), ENA-78 (CXCL5), and Gro-α (CXCL1). Similarly, an inhibitor of NF-κB activation (PDTC) also blocked CXCL8, CXCL5, and CXCL1 production, consistent with their NF-κB-dependent regulation. Conditioned media from A549 cells induce human microvascular endothelial cell (HMVEC) chemotaxis. However, conditioned media from Tro-treated A549 cells induced significantly less HMVEC chemotaxis compared to untreated A549 cells. Furthermore, PPAR-γ activation inhibited NF-κB transcriptional activity, as assessed by TransAM reporter gene assay. Collectively, our data suggest that PPAR-γ ligands can inhibit tumor-associated angiogenesis by blocking the production of ELR+CXC chemokines, which is mediated through antagonizing NF-κB activation. These antiangiogenic effects likely contribute to the inhibition of primary tumor growth by PPAR-γ ligands.
PMCID: PMC1501135  PMID: 15799829
PPAR-γ; NSCLC; angiogenesis; CXC chemokines; NF-κB
14.  Chemokine-Dependent Neutrophil Recruitment in a Murine Model of Legionella Pneumonia: Potential Role of Neutrophils as Immunoregulatory Cells 
Infection and Immunity  2001;69(4):2017-2024.
The roles of CXC chemokine-mediated host responses were examined with an A/J mouse model of Legionella pneumophila pneumonia. After intratracheal inoculation of 106 CFU of L. pneumophila, the bacterial numbers in the lungs increased 10-fold by day 2; this increase was accompanied by the massive accumulation of neutrophils. Reverse transcription-PCR data demonstrated the up-regulation of CXC chemokines, such as keratinocyte-derived chemokine, macrophage inflammatory protein 2 (MIP-2), and lipopolysaccharide-induced CXC chemokine (LIX). Consistent with these data, increased levels of KC, MIP-2, and LIX proteins were observed in the lungs and peaked at days 1, 2, and 2, respectively. Although the administration of anti-KC or anti–MIP-2 antibody resulted in an approximately 20% decrease in neutrophil recruitment on day 2, no increase in mortality was observed. In contrast, the blockade of CXC chemokine receptor 2 (CXCR2), a receptor for CXC chemokines, including KC and MIP-2, strikingly enhanced mortality; this effect coincided with a 67% decrease in neutrophil recruitment. Interestingly, anti-CXCR2 antibody did not affect bacterial burden by day 2, even in the presence of a lethal challenge of bacteria. Moreover, a significant decrease in interleukin-12 (IL-12) levels, in contrast to the increases in KC, MIP-2, and LIX levels, was demonstrated for CXCR2-blocked mice. These data indicated that CXCR2-mediated neutrophil accumulation may play a crucial role in host defense against L. pneumophila pneumonia in mice. The increase in lethality without a change in early bacterial clearance suggested that neutrophils may exert their protective effect not through direct killing but through more immunomodulatory actions in L. pneumophila pneumonia. We speculate that a decrease in the levels of the protective cytokine IL-12 may explain, at least in part, the high mortality in the setting of reduced neutrophil recruitment.
PMCID: PMC98125  PMID: 11254553
15.  Alveolar Macrophage Deactivation in Murine Septic Peritonitis: Role of Interleukin 10 
Infection and Immunity  2001;69(3):1394-1401.
Sepsis predisposes the host to a number of infectious sequelae, particularly the development of nosocomial pneumonia. Mechanisms by which sepsis results in impairment of lung antibacterial host defense have not been well defined. Alveolar macrophages (AM) represent important immune effector cells of the lung airspace. In this study, we examined the effects of cecal ligation and puncture (CLP) on murine AM function ex vivo, including the expression of proinflammatory cytokines and AM phagocytic activity. AM were harvested from mice subjected to a sham operation and CLP 24 h after laparotomy, adherence purified, and challenged with lipopolysaccharide (LPS) or left unstimulated. Both unstimulated and LPS-stimulated AM from mice subjected to CLP (CLP mice) produced significantly smaller amounts of proinflammatory cytokines tumor necrosis factor alpha and interleukin (IL-12) and C-X-C chemokines KC and macrophage inflammatory protein 2 than similarly treated AM from animals subjected to a sham operation. Furthermore, AM isolated from CLP mice displayed a marked impairment in phagocytic activity, as determined by flow cytometry, with this defect persisting to 48 h post-CLP. Induction of peritoneal sepsis syndrome resulted in a time-dependent increase in IL-10 in plasma and peritoneal fluid. Interestingly, the impairment in AM proinflammatory-cytokine production and phagocytic activity observed in AM from CLP mice was partially reversed by the in vivo neutralization of IL-10 prior to AM harvest. These observations suggest that abdominal sepsis syndrome results in significant impairment in AM effector cell function, which is mediated, in part, by sepsis-induced expression of IL-10.
PMCID: PMC98033  PMID: 11179304

Results 1-15 (15)