Comparisons of resistance of CF and Non-CF pathogens to Hydrogen Peroxide and Hypochlorous Acid Oxidants In Vitro

<p>Abstract</p> <p>Background</p> <p>Cystic fibrosis (CF) lung disease has a unique profile of pathogens predominated by <it>Pseudomonas aeruginosa </it>(PsA) and <it>Staphylococcus aureus </it>(SA). These microorganisms must overcome host immune defense to colonize the CF lungs. Polymorphonuclear neutrophils are a major component of the host defense against bacterial infection. A crucial microbicidal mechanism is the production of oxidants including hydrogen peroxide (H₂O₂) and hypochlorous acid (HOCl) by neutrophils to achieve efficient bacterial killing. To determine to what degrees various CF pathogens resist the oxidants relative to non-CF pathogens, we compared the susceptibility of PsA, SA, <it>Burkholderia cepacia </it>(BC), <it>Klebsiella pneumoniae </it>(KP), and <it>Escherichia coli </it>(EC) to various concentrations of H₂O₂or HOCl, <it>in vitro</it>. The comparative oxidant-resistant profiles were established. Oxidant-induced damage to ATP production and cell membrane integrity of the microbes were quantitatively assessed. Correlation of membrane permeability and ATP levels with bacterial viability was statistically evaluated.</p> <p>Results</p> <p>PsA was relatively resistant to both H₂O₂(LD₅₀= 1.5 mM) and HOCl (LD₅₀= 0.035 mM). SA was susceptible to H₂O₂(LD₅₀= 0.1 mM) but resistant to HOCl (LD₅₀= 0.035 mM). Interestingly, KP was extremely resistant to high doses of H₂O₂(LD₅₀= 2.5-5.0 mM) but was very sensitive to low doses of HOCl (LD₅₀= 0.015 mM). BC was intermediate to resist both oxidants: H₂O₂(LD₅₀= 0.3-0.4 mM) and HOCl (LD₅₀= 0.025 mM). EC displayed the least resistance to H₂O₂(LD₅₀= 0.2-0.3 mM) and HOCl (LD₅₀= 0.015 mM). The identified profile of H₂O₂-resistance was KP > PsA > BC > EC > SA and the profile of HOCl-resistance PsA > SA > BC > EC > KP. Moreover, both oxidants affected ATP production and membrane integrity of the cells. However, the effects varied among the tested organisms and, the oxidant-mediated damage correlated differentially with the bacterial viability.</p> <p>Conclusions</p> <p>The order of HOCl-resistance identified herein best fits the clinical profile of CF infections. Even though oxidants are able to disrupt ATP production and cell membrane integrity, the degrees of damage vary among the organisms and correlate differentially with their viability.</p

Comparison of the Therapeutic Effects of Human and Mouse Adipose-Derived Stem Cells in a Murine Model of Lipopolysaccharide-Induced Acute Lung Injury

Introduction. Adipose-derived stem cells (ASCs) have emerged as important regulators of inflammatory/immune responses in vitro and in vivo and represent attractive candidates for cell-based therapies for diseases that involve excessive inflammation. Acute lung injury (ALI) is an inflammatory condition for which treatment is mainly supportive due to lack of effective therapies. In this study, the therapeutic effects of ASC-based therapy were assessed in vivo by comparison of the anti-inflammatory properties of both human and murine ASCs in a mouse model of lipopolysaccharide (LPS)-induced ALI. Methods. Human ASCs (hASCs) or mouse ASCs (mASCs) were delivered to C57Bl/6 mice (7.5 x 105 total cells/mouse) by oropharyngeal aspiration (OA) four hours after the animals were challenged with lipopolysaccharide (15 mg/kg). Mice were sacrificed 24 and 72 hours after LPS exposure, and lung histology examined for evaluation of inflammation and injury. Bronchoalveolar lavage fluid (BALF) was analyzed to determine total and differential cell counts, total protein and albumin concentrations, and myeloperoxidase (MPO) activity. Cytokine expression in the injured lungs was measured at the steady-state mRNA levels and protein levels for assessment of the degree of lung inflammation. Results: Both human and mouse ASC treatments provided protective anti-inflammatory responses. There were decreased levels of leukocyte (for example neutrophil) migration into the alveoli, total protein and albumin concentrations in BALF, and MPO activity after the induction of ALI following both therapies. Additionally, cell therapy with both cell types effectively suppressed the expression of proinflammatory cytokines and increased the anti-inflammatory cytokine interleukin 10 (IL-10). Overall, the syngeneic mASC therapy had a more potent therapeutic effect than the xenogeneic hASC therapy in this model. Conclusions: Treatment with hASCs or mASCs significantly attenuated LPS-induced acute lung injury in mice. These results suggest a potential benefit for using an ASC-based therapy to treat clinical ALI and may possibly prevent the development of acute respiratory distress syndrome (ARDS)

Interleukin 6 Mediates the Therapeutic Effects of Adipose-Derived Stromal/Stem Cells in Lipopolysaccharide-Induced Acute Lung Injury

Adipose-derived stromal/stem cells (ASCs) have anti-inflammatory as well as immunosuppressive activities and are currently the focus of clinical trials for a number of inflammatory diseases. Acute lung injury (ALI) is an inflammatory condition of the lung for which standard treatment is mainly supportive due to lack of effective therapies. Our recent studies have demonstrated the ability of both human ASCs (hASCs) and mouse ASCs (mASCs) to attenuate lung damage and inflammation in a rodent model of lipopolysaccharide-induced ALI, suggesting that ASCs may also be beneficial in treating ALI. To better understand how ASCs may act in ALI and to elucidate the mechanism(s) involved in ASC modulation of lung inflammation, gene expression analysis was performed in ASC-treated (hASCs or mASCs) and control sham-treated lungs. The results revealed a dramatic difference between the expression of anti-inflammatory molecules by hASCs and mASCs. These data show that the beneficial effects of hASCs and mASCs in ALI may result from the production of different paracrine factors. Interleukin 6 (IL-6) expression in the mASC-treated lungs was significantly elevated as compared to sham-treated controls 20 hours after delivery of the cells by oropharyngeal aspiration. Knockdown of IL-6 expression in mASCs by RNA interference abrogated most of their therapeutic effects, suggesting that the anti-inflammatory properties of mASCs in ALI are explained, at least in part, by activation of IL-6 secretion

Multipotent Stromal Cells Alleviate Inflammation, Neuropathology, and Symptoms Associated with Globoid Cell Leukodystrophy in the Twitcher Mouse

Globoid cell leukodystrophy (GLD) is a common neurodegenerative lysosomal storage disorder caused by a deficiency in galactocerebrosidase (GALC), an enzyme that cleaves galactocerebroside during myelination. Bone marrow transplantation has shown promise when administered to late-onset GLD patients. However, the side effects (e.g., graft vs. host disease), harsh conditioning regimens (e.g., myelosuppression), and variable therapeutic effects make this an unsuitable option for infantile GLD patients. We previously reported modest improvements in the twitcher mouse model of GLD after intracerebroventricular (ICV) injections of a low-dose of multipotent stromal cells (MSCs). Goals of this study were to improve bone marrow-derived MSC (BMSC) therapy for GLD by increasing the cell dosage and comparing cell type (e.g., transduced vs. native), treatment timing (e.g., single vs. weekly), and administration route (e.g., ICV vs. intraperitoneal [IP]). Neonatal twitcher mice received (a) 2 x 105 BMSCs by ICV injection, (b) 1 x 106 BMSCs by IP injection, (c) weekly IP injections of 1 x 106 BMSCs, or (d) 1 x 106 lentiviral-transduced BMSCs overexpressing GALC (GALC-BMSC) by IP injection. All treated mice lived longer than untreated mice. However, the mice receiving peripheral MSC therapy had improved motor function (e.g., hind limb strength and rearing ability), twitching symptoms, and weight compared to both the untreated and ICV-treated mice. Inflammatory cell, globoid cell, and apoptotic cell levels in the sciatic nerves were significantly decreased as a result of the GALC-BMSC or weekly IP injections. The results of this study indicate a promising future for peripheral MSC therapy as a noninvasive, adjunct therapy for patients affected with GLD

A Nonhuman Primate Model of Lung Regeneration: Detergent-Mediated Decellularization and Initial in vitro Recellularization with Mesenchymal Stem Cells

Currently, patients with end-stage lung disease are limited to lung transplantation as their only treatment option. Unfortunately, the lungs available for transplantation are few. Moreover, transplant recipients require life-long immune suppression to tolerate the transplanted lung. A promising alternative therapeutic strategy is decellularization of whole lungs, which permits the isolation of an intact scaffold comprised of innate extracellular matrix (ECM) that can theoretically be recellularized with autologous stem or progenitor cells to yield a functional lung. Nonhuman primates (NHP) provide a highly relevant preclinical model with which to assess the feasibility of recellularized lung scaffolds for human lung transplantation. Our laboratory has successfully accomplished lung decellularization and initial stem cell inoculation of the resulting ECM scaffold in an NHP model. Decellularization of normal adult rhesus macaque lungs as well as the biology of the resulting acellular matrix have been extensively characterized. Acellular NHP matrices retained the anatomical and ultrastructural properties of native lungs with minimal effect on the content, organization, and appearance of ECM components, including collagen types I and IV, laminin, fibronectin, and sulfated glycosaminoglycans (GAG), due to decellularization. Proteomics analysis showed enrichment of ECM proteins in total tissue extracts due to the removal of cells and cellular proteins by decellularization. Cellular DNA was effectively removed after decellularization (~92% reduction), and the remaining nuclear material was found to be highly disorganized, very-low-molecular-weight fragments. Both bone marrow- and adipose-derived mesenchymal stem cells (MSC) attach to the decellularized lung matrix and can be maintained within this environment in vitro, suggesting that these cells may be promising candidates and useful tools for lung regeneration. Analysis of decellularized lung slice cultures to which MSC were seeded showed that the cells attached to the decellularized matrix, elongated, and proliferated in culture. Future investigations will focus on optimizing the recellularization of NHP lung scaffolds toward the goal of regenerating pulmonary tissue. Bringing this technology to eventual human clinical application will provide patients with an alternative therapeutic strategy as well as significantly reduce the demand for transplantable organs and patient wait-list time