Brent W. Winston

Safety of Cell Therapy with Mesenchymal Stromal Cells (SafeCell): A Systematic Review and Meta-Analysis of Clinical Trials

Background Mesenchymal stromal cells (MSCs, “adult stem cells”) have been widely used experimentally in a variety of clinical contexts. There is interest in using these cells in critical illness, however, the safety profile of these cells is not well known. We thus conducted a systematic review of clinical trials that examined the use MSCs to evaluate their safety. Methods and Findings MEDLINE, EMBASE, and the Cochrane Central Register of Controlled Trials (to June 2011), were searched. Prospective clinical trials that used intravascular delivery of MSCs (intravenously or intra-arterially) in adult populations or mixed adult and pediatric populations were identified. Studies using differentiated MSCs or additional cell types were excluded. The primary outcome adverse events were grouped according to immediate events (acute infusional toxicity, fever), organ system complications, infection, and longer term adverse events (death, malignancy). 2347 citations were reviewed and 36 studies met inclusion criteria. A total of 1012 participants with clinical conditions of ischemic stroke, Crohns disease, cardiomyopathy, myocardial infarction, graft versus host disease, and healthy volunteers were included. Eight studies were randomized control trials (RCTs) and enrolled 321 participants. Meta-analysis of the RCTs did not detect an association between acute infusional toxicity, organ system complications, infection, death or malignancy. There was a significant association between MSCs and transient fever. Conclusions Based on the current clinical trials, MSC therapy appears safe. However, further larger scale controlled clinical trials with rigorous reporting of adverse events are required to further define the safety profile of MSCs.

TNF-α-induced Regulation and Signalling in Macrophages

Tumor necrosis factor alpha (TNF-alpha) is a pleiotropic cytokine produced predominantly by macrophages. In addition, macrophages respond to TNF-alpha by differentiating to express different groups of gene products. Our laboratory recently showed that the context in which TNF-alpha is recognized by macrophages dramatically impacts the pattern of gene expression and hence investigating the mechanism of TNF-alpha signal transduction will be important in understanding how this molecule regulates macrophage differentiation. TNF-alpha is recognized by two cell surface receptors, CD120a (p55) and CD120b (p75) that belong to the TNF/NGF receptor family. Signalling is initiated by receptor multimerization in the plane of the plasma membrane. The initial signalling events activated by receptor cross-linking are unknown although activation of the mitogen-activated protein kinase (MAPK) cascade occurs shortly after ligand binding to CD120a (p55). We have investigated the upstream kinases that mediate the activation of p42mapk/erk2 following cross-linking of CD120a (p55) in mouse macrophages. Exposure of mouse macrophages to TNF-alpha stimulated a time-dependent increase in the activity of MEK1, that temporally preceded peak activation of p42mapk/erk2. MEKs, dual specificity T/Y kinases, act as a convergence point for several signalling pathways including Ras/Raf, MEKK and Mos. Incubation of macrophages with TNF-alpha was found to transiently stimulate an MEKK that peaked in activity within 30 sec of exposure and progressively declined towards basal levels by 5 min. By contrast, under these conditions, activation of either c-Raf-1 or Raf-B was not detected. These data suggest that the activation of the MAPK cascade in response to TNF-alpha is mediated by the sequential activation of an MEKK and MEK1 in a c-Raf-1 and Raf-B-independent fashion. The implications of these findings will be discussed in the context of the regulation of macrophage gene expression.

Complement Factor B Gene Regulation: Synergistic Effects of TNF-α and IFN-γ in Macrophages

Complement factor B (Bf) plays an important role in activating the alternative complement pathway. The inflammatory cytokines, in particular TNF-α and IFN-γ, are critical in the regulation of Bf gene expression in macrophages. In this study, we investigated the mechanisms of Bf gene regulation by TNF-α and IFN-γ in murine macrophages. Northern analysis revealed that Bf mRNA expression was synergistically up-regulated by TNF-α and IFN-γ in MH-S cells. Truncations of the 5′ Bf promoter identified a region between −556 and −282 bp that mediated TNF-α responsiveness as well as the synergistic effect of TNF-α and IFN-γ on Bf expression. Site-directed mutagenesis of a NF-κB-binding element in this region (−433 to −423 bp) abrogated TNF-α responsiveness and decreased the synergistic effect of TNF-α and IFN-γ on Bf expression. EMSAs revealed nuclear protein binding to this NF-κB cis-binding element on TNF-α stimulation. Supershift analysis revealed that both p50 and p65 proteins contribute to induction of Bf by TNF-α. An I-κB dominant negative mutant blocked Bf induction by TNF-α and reduced the synergistic induction by TNF-α and IFN-γ. In addition, the proteasome inhibitor MG132, which blocks NF-κB induction, blocked TNF-α-induced Bf promoter activity and the synergistic induction of Bf promoter activity by TNF-α and IFN-γ. LPS was found to induce Bf promoter activity through the same NF-κB cis-binding site. These findings suggest that a NF-κB cis-binding site between −433 and −423 bp is required for TNF-α responsiveness and for TNF-α- and IFN-γ-stimulated synergistic responsiveness of the Bf gene.

Metabolic profiling of serum samples by 1H nuclear magnetic resonance spectroscopy as a potential diagnostic approach for septic shock.

Objectives:To determine whether a nuclear magnetic resonance–based metabolomics approach can be useful for the early diagnosis and prognosis of septic shock in ICUs. Design:Laboratory-based study. Setting:University research laboratory. Subjects:Serum samples from septic shock patients and ICU controls (ICU patients with systemic inflammatory response syndrome but not suspected of having an infection) were collected within 24 hours of admittance to the ICU. Interventions:None. Measurements and Main Results:1H nuclear magnetic resonance spectra of septic shock and ICU control samples were analyzed and quantified using a targeted profiling approach. By applying multivariate statistical analysis (e.g., orthogonal partial least squares discriminant analysis), we were able to distinguish the patient groups and detect specific metabolic patterns. Some of the metabolites were found to have a significant impact on the separation between septic shock and control samples. These metabolites could be interpreted in terms of a biological human response to septic shock and they might serve as a biomarker pattern for septic shock in ICUs. Additionally, nuclear magnetic resonance–based metabolomics was evaluated in order to detect metabolic variation between septic shock survivors and nonsurvivors and to predict patient outcome. The area under the receiver operating characteristic curve indicated an excellent predictive ability for the constructed orthogonal partial least squares discriminant analysis models (septic shock vs ICU controls: area under the receiver operating characteristic curve = 0.98; nonsurvivors vs survivors: area under the receiver operating characteristic curve = 1). Conclusions:Our results indicate that nuclear magnetic resonance–based metabolic profiling could be used for diagnosis and mortality prediction of septic shock in the ICU.

Integration of metabolic and inflammatory mediator profiles as a potential prognostic approach for septic shock in the intensive care unit

IntroductionSeptic shock is a major life-threatening condition in critically ill patients and it is well known that early recognition of septic shock and expedient initiation of appropriate treatment improves patient outcome. Unfortunately, to date no single compound has shown sufficient sensitivity and specificity to be used as a routine biomarker for early diagnosis and prognosis of septic shock in the intensive care unit (ICU). Therefore, the identification of new diagnostic tools remains a priority for increasing the survival rate of ICU patients. In this study, we have evaluated whether a combined nuclear magnetic resonance spectroscopy-based metabolomics and a multiplex cytokine/chemokine profiling approach could be used for diagnosis and prognostic evaluation of septic shock patients in the ICU.MethodsSerum and plasma samples were collected from septic shock patients and ICU controls (ICU patients with the systemic inflammatory response syndrome but not suspected of having an infection). 1H Nuclear magnetic resonance spectra were analyzed and quantified using the targeted profiling methodology. The analysis of the inflammatory mediators was performed using human cytokine and chemokine assay kits.ResultsBy using multivariate statistical analysis we were able to distinguish patient groups and detect specific metabolic and cytokine/chemokine patterns associated with septic shock and its mortality. These metabolites and cytokines/chemokines represent candidate biomarkers of the human response to septic shock and have the potential to improve early diagnosis and prognosis of septic shock.ConclusionsOur findings show that integration of quantitative metabolic and inflammatory mediator data can be utilized for the diagnosis and prognosis of septic shock in the ICU.

Mechanisms of human complement factor B induction in sepsis and inhibition by activated protein C

To investigate the potential role of the local expression of alternative complement factor B (hBf) in human sepsis, we examined the induction of Bf gene expression in human peripheral blood monocytes (PBMCs) from patients with septic shock and the mechanisms of hBf gene regulation by tumor necrosis factor (TNF)-alpha, interferon (IFN)-gamma, and lipopolysaccharide (LPS) in human monocytes. PBMCs from septic shock patients showed increased hBf mRNA expression when compared with control patients. Costimulation with TNF-alpha and IFN-gamma or stimulation with LPS demonstrated a time- and dose-dependent induction of hBf mRNA expression in human PBMCs. A region of the hBf promoter between -735 and +128 bp was found to mediate IFN-gamma, TNF-alpha, and LPS responsiveness as well as the synergistic effect of IFN-gamma/TNF-alpha on hBf promoter activity. Site-directed mutagenesis of a IFN-gamma-activation site (GAS) cis element (-90 to -82 bp) abrogated IFN-gamma responsiveness. Mutagenesis of a nuclear factor (NF)-kappaB cis element at -466 to -456 bp abrogated TNF-alpha and LPS responsiveness of the Bf promoter. Thus hBf gene expression is induced in PBMCs from septic shock patients, and the induction of hBf by IFN-gamma, TNF-alpha, and LPS is through GAS and NF-kappaB cis-binding sites on the hBf promoter. Furthermore, activated protein C (APC) inhibited LPS-stimulated hBf promoter activity and protein expression in human monocytes suggesting that the beneficial effect of APC therapy in sepsis may in part be due to inhibition of complement induction and/or activation via the alternative pathway.

Characterization of IFN‐γ regulation of the complement factor B gene in macrophages

Complement factor B (Bf) is involved in the activation of the alternative complement cascade. Bf is induced by IFN‐γ; however, the mechanisms of Bf gene regulation have not been well characterized in general, and not in macrophages specifically. Northern analysis reveals that IFN‐γ induces a dose‐ and time‐dependent increase in Bf mRNA expression in primary macrophages and macrophage cell lines. MH‐S cells transfected with reporter constructs containing truncated regions of the Bf promoter reveal that IFN‐γ responsiveness lies between –154 and –53 bp on the Bf promoter.This region of the Bf promoter contains both an IFN‐γ‐activation site (GAS) and an interferon‐stimulated response element (ISRE). Site‐directed mutagenesis of the GAS binding site or the ISRE binding site in this region of the Bf promoter partially inhibits IFN‐γ responsiveness. Mutagenesis of both the GAS and ISRE cis elements totally abrogates IFN‐γ responsiveness of the Bf promoter. Electrophoretic mobility shift assays reveal nuclear binding complexes involving both Bf‐GAS and Bf‐ISRE oligonucleotide sequences upon IFN‐γ stimulation. In competition assays, both Bf‐GAS and Bf‐ISRE oligonucleotides, but not mutant Bf‐GAS nor mutant Bf‐ISRE oligonucleotides, compete for the DNA binding. Supershift analysis reveals that Stat1‐GAS and IRF‐1‐ISRE nuclear binding complexes contribute to induction of Bf by IFN‐γ. Western analysis confirms an IFN‐γ‐stimulated increase in tyrosine phosphorylation of Stat1. These findings suggest that both GAS and ISRE cis bindingsites have an additive effect on IFN‐γ‐stimulated Bf gene expression and that both are required for full expression of Bf by IFN‐γ. Stat1 and IRF‐1 take part in IFN‐γ‐stimulated Bf geneinduction in macrophages through their respective cis binding elements.

Akt/Protein Kinase B Activation by Adenovirus Vectors Contributes to NFκB-Dependent CXCL10 Expression

ABSTRACT In gene therapy, the innate immune system is a significant barrier to the effective application of adenovirus (Ad) vectors. In kidney epithelium-derived (REC) cells, serotype 5 Ad vectors induce the expression of the chemokine CXCL10 (IP-10), a response that is dependent on NFκB. Compared to the parental vector AdLuc, transduction with the RGD-deleted vector AdL.PB resulted in reduced CXCL10 activation despite increasing titers, implying that RGD-αV integrin interactions contribute to adenovirus induction of inflammatory genes. Akt, a downstream effector of integrin signaling, was activated within 10 min of transduction with Ad vectors in a dose-dependent manner. Akt activation was not present following transduction with AdL.PB, confirming the importance of capsid-αV integrin interactions in Ad vector Akt activation. Inhibition of the phosphoinositide-3-OH kinase/Akt pathway by Wortmannin or Ly294002 compounds decreased Ad vector induction of CXCL10 mRNA. Similarly, adenovirus-mediated overexpression of the dominant negative AktAAA decreased CXCL10 mRNA expression compared to the reporter vector AdLacZ alone. The effect of Akt on CXCL10 mRNA expression occurred via NFκB-dependent transcriptional activation, since AktAAA overexpression and Ly294002 both inhibited CXCL10 and NFκB promoter activation in luciferase reporter experiments. These results show that Akt plays a role in the Ad vector activation of NFκB and CXCL10 expression. Understanding the mechanism underlying the regulation of host immunomodulatory genes by adenovirus vectors will lead to strategies that will improve the efficacy and safety of these agents for clinical use.

Development of metabolic and inflammatory mediator biomarker phenotyping for early diagnosis and triage of pediatric sepsis.

IntroductionThe first steps in goal-directed therapy for sepsis are early diagnosis followed by appropriate triage. These steps are usually left to the physician’s judgment, as there is no accepted biomarker available. We aimed to determine biomarker phenotypes that differentiate children with sepsis who require intensive care from those who do not.MethodsWe conducted a prospective, observational nested cohort study at two pediatric intensive care units (PICUs) and one pediatric emergency department (ED). Children ages 2–17 years presenting to the PICU or ED with sepsis or presenting for procedural sedation to the ED were enrolled. We used the judgment of regional pediatric ED and PICU attending physicians as the standard to determine triage location (PICU or ED). We performed metabolic and inflammatory protein mediator profiling with serum and plasma samples, respectively, collected upon presentation, followed by multivariate statistical analysis.ResultsNinety-four PICU sepsis, 81 ED sepsis, and 63 ED control patients were included. Metabolomic profiling revealed clear separation of groups, differentiating PICU sepsis from ED sepsis with accuracy of 0.89, area under the receiver operating characteristic curve (AUROC) of 0.96 (standard deviation [SD] 0.01), and predictive ability (Q2) of 0.60. Protein mediator profiling also showed clear separation of the groups, differentiating PICU sepsis from ED sepsis with accuracy of 0.78 and AUROC of 0.88 (SD 0.03). Combining metabolomic and protein mediator profiling improved the model (Q2 =0.62), differentiating PICU sepsis from ED sepsis with accuracy of 0.87 and AUROC of 0.95 (SD 0.01). Separation of PICU sepsis or ED sepsis from ED controls was even more accurate. Prespecified age subgroups (2–5 years old and 6–17 years old) improved model accuracy minimally. Seventeen metabolites or protein mediators accounted for separation of PICU sepsis and ED sepsis with 95 % confidence.ConclusionsIn children ages 2–17 years, combining metabolomic and inflammatory protein mediator profiling early after presentation may differentiate children with sepsis requiring care in a PICU from children with or without sepsis safely cared for outside a PICU. This may aid in making triage decisions, particularly in an ED without pediatric expertise. This finding requires validation in an independent cohort.

Growth factor regulation and manipulation in wound repair: to scar or not to scar, that is the question

The process of tissue repair following injury is in the large part mediated by secreted growth factors which, in an autocrine or paracrine fashion, stimulate immune and mesenchymal cells at the site of injury. The complex process of replacing damaged tissue with newly formed tissue involves components of the blood, coagulation, immune and mesenchymal systems as well as cytokines, chemokines, metalloproteinases and growth factors. This review will focus on growth factors as the controllers of this process and includes members of the transforming growth factor (TGF), platelet derived growth factor (PDGF), fibroblast growth factor (FGF), connective tissue derived growth factor (CTGF) and insulin-like growth factor-I (IGF-I) families of growth factors. These growth factors stimulate re-epithelialisation, angiogenesis, extracellular matrix (ECM) formation and cell proliferation each of which plays a role in tissue replacement and restoration of tissue function following injury. Normal wound healing frequently involves the formation of scar tissue, including increased mesenchymal cell proliferation and excessive production of ECM proteins. While scar tissue rapidly and effectively closes wounds, it leaves visually apparent tissue structure changes and may reduce the function of the tissue leading to compromised organ function. Growth factors, the conductors of these processes, are targets for therapeutic manipulation of wound healing and scar formation. Recent patents involving growth factors may be implicated in the treatment of wound healing following tissue injury. Enhanced growth factor activity may be beneficial to increase the rate of wound healing in chronic non-healing wounds, whereas reduction of growth factor presence or activity may reduce scar formation in the skin and internal organs, which may be particularly relevant where scar formation is associated with pathologic loss of life sustaining organ function.