Inhibition of Neutrophil Recruitment Partially Explained by Impaired Mobilization from Bone Marrow and Reduced Chemokine Levels

Rapid activation of the innate immune system is critical for an efficient host response to invading pathogens. However, the inflammatory reaction has to be strictly controlled to minimize harmful immunopathology. A number of mediators including the cytokine interleukin-27 (IL-27) appear to be responsible for limitation and resolution of inflammation. Despite increasing knowledge of its suppressive effects on T cells, the influence on neutrophils and macrophages is poorly understood. To determine the role of IL-27 in innate immune responses we analysed the effect of IL-27 in a T cell independent model of zymosan-induced peritonitis. Early administration of recombinant IL-27 strongly reduced the number of neutrophils recruited to the peritoneal cavity after zymosan application as well as the neutrophil frequency in the blood. Simultaneously, IL-27 reduced the release of neutrophils from the bone marrow upon inflammation. Although cytokine levels were not affected by IL-27 treatment, the levels of the chemokines KC, MCP-1 and MIP-1α in the peritoneal fluid were strongly decreased. These findings demonstrate that IL-27 is able to control mobilisation and recruitment of neutrophils into the peritoneal cavity and identify a novel mechanism to limit inflammation caused by innate immune cells

a clinical study protocol

Introduction The approved analgesic and anti-inflammatory drugs ibuprofen and indometacin block the small GTPase RhoA, a key enzyme that impedes axonal sprouting after axonal damage. Inhibition of the Rho pathway in a central nervous system-effective manner requires higher dosages compared with orthodox cyclooxygenase-blocking effects. Preclinical studies on spinal cord injury (SCI) imply improved motor recovery after ibuprofen/indometacin-mediated Rho inhibition. This has been reassessed by a meta-analysis of the underlying experimental evidence, which indicates an overall effect size of 20.2% regarding motor outcome achieved after ibuprofen/indometacin treatment compared with vehicle controls. In addition, ibuprofen/indometacin may also limit sickness behaviour, non-neurogenic systemic inflammatory response syndrome (SIRS), neuropathic pain and heterotopic ossifications after SCI. Consequently, ‘small molecule’-mediated Rho inhibition after acute SCI warrants clinical investigation. Methods and analysis Protocol of an investigator-initiated clinical open-label pilot trial on high-dose ibuprofen treatment after acute traumatic, motor-complete SCI. A sample of n=12 patients will be enrolled in two cohorts treated with 2400 mg/day ibuprofen for 4 or 12 weeks, respectively. The primary safety end point is an occurrence of serious adverse events, primarily gastroduodenal bleedings. Secondary end points are pharmacokinetics, feasibility and preliminary effects on neurological recovery, neuropathic pain and heterotopic ossifications. The primary safety analysis is based on the incidence of severe gastrointestinal bleedings. Additional analyses will be mainly descriptive and casuistic. Ethics and dissemination The clinical trial protocol was approved by the responsible German state Ethics Board, and the Federal Institute for Drugs and Medical Devices. The study complies with the Declaration of Helsinki, the principles of Good Clinical Practice and all further applicable regulations. This safety and pharmacokinetics trial informs the planning of a subsequent randomised controlled trial. Regardless of the result of the primary and secondary outcome assessments, the clinical trial will be reported as a publication in a peer-reviewed journal. Trial registration number NCT02096913; Pre-results

protocol of a prospective, longitudinal study

Background Natural killer (NK) cells comprise the main components of lymphocyte-mediated nonspecific immunity. Through their effector function they play a crucial role combating bacterial and viral challenges. They are also thought to be key contributors to the systemic spinal cord injury-induced immune-deficiency syndrome (SCI-IDS). SCI-IDS increases susceptibility to infection and extends to the post-acute and chronic phases after SCI. Methods and design The prospective study of NK cell function after traumatic SCI was carried out in two centers in Berlin, Germany. SCI patients and control patients with neurologically silent vertebral fracture also undergoing surgical stabilization were enrolled. Furthermore healthy controls were included to provide reference data. The NK cell function was assessed at 7 (5–9) days, 14 days (11–28) days, and 10 (8–12) weeks post-trauma. Clinical documentation included the American Spinal Injury Association (ASIA) impairment scale (AIS), neurological level of injury, infection status, concomitant injury, and medications. The primary endpoint of the study is CD107a expression by NK cells (cytotoxicity marker) 8–12 weeks following SCI. Secondary endpoints are the NK cell’s TNF-α and IFN-γ production by the NK cells 8–12 weeks following SCI. Discussion The protocol of this study was developed to investigate the hypotheses whether i) SCI impairs NK cell function throughout the post-acute and sub-acute phases after SCI and ii) the degree of impairment relates to lesion height and severity. A deeper understanding of the SCI-IDS is crucial to enable strategies for prevention of infections, which are associated with poor neurological outcome and elevated mortality. Trial registration DRKS00009855

The spinal cord injury-induced immune deficiency syndrome: results of the SCIentinel study

Infections are prevalent after spinal cord injury (SCI), constitute the main cause of death and are a rehabilitation confounder associated with impaired recovery. We hypothesize that SCI causes an acquired lesion-dependent (neurogenic) immune suppression as an underlying mechanism to facilitate infections. The international prospective multicentre cohort study (SCIentinel; protocol registration DRKS00000122; n = 111 patients) was designed to distinguish neurogenic from general trauma-related effects on the immune system. Therefore, SCI patient groups differing by neurological level, i.e. high SCI [thoracic (Th)4 or higher]; low SCI (Th5 or lower) and severity (complete SCI; incomplete SCI), were compared with a reference group of vertebral fracture (VF) patients without SCI. The primary outcome was quantitative monocytic Human Leukocyte Antigen-DR expression (mHLA-DR, synonym MHC II), a validated marker for immune suppression in critically ill patients associated with infection susceptibility. mHLA-DR was assessed from Day 1 to 10 weeks after injury by applying standardized flow cytometry procedures. Secondary outcomes were leucocyte subpopulation counts, serum immunoglobulin levels and clinically defined infections. Linear mixed models with multiple imputation were applied to evaluate group differences of logarithmic-transformed parameters. Mean quantitative mHLA-DR [ln (antibodies/cell)] levels at the primary end point 84 h after injury indicated an immune suppressive state below the normative values of 9.62 in all groups, which further differed in its dimension by neurological level: high SCI [8.95 (98.3% confidence interval, CI: 8.63; 9.26), n = 41], low SCI [9.05 (98.3% CI: 8.73; 9.36), n = 29], and VF without SCI [9.25 (98.3% CI: 8.97; 9.53), n = 41, P = 0.003]. Post hoc analysis accounting for SCI severity revealed the strongest mHLA-DR decrease [8.79 (95% CI: 8.50; 9.08)] in the complete, high SCI group, further demonstrating delayed mHLA-DR recovery [9.08 (95% CI: 8.82; 9.38)] and showing a difference from the VF controls of -0.43 (95% CI: -0.66; -0.20) at 14 days. Complete, high SCI patients also revealed constantly lower serum immunoglobulin G [-0.27 (95% CI: -0.45; -0.10)] and immunoglobulin A [-0.25 (95% CI: -0.49; -0.01)] levels [ln (g/l × 1000)] up to 10 weeks after injury. Low mHLA-DR levels in the range of borderline immunoparalysis (below 9.21) were positively associated with the occurrence and earlier onset of infections, which is consistent with results from studies on stroke or major surgery. Spinal cord injured patients can acquire a secondary, neurogenic immune deficiency syndrome characterized by reduced mHLA-DR expression and relative hypogammaglobulinaemia (combined cellular and humoral immune deficiency). mHLA-DR expression provides a basis to stratify infection-risk in patients with SCI

Enhanced axonal response of mitochondria to demyelination offers neuroprotection:implications for multiple sclerosis

Axonal loss is the key pathological substrate of neurological disability in demyelinating disorders, including multiple sclerosis (MS). However, the consequences of demyelination on neuronal and axonal biology are poorly understood. The abundance of mitochondria in demyelinated axons in MS raises the possibility that increased mitochondrial content serves as a compensatory response to demyelination. Here, we show that upon demyelination mitochondria move from the neuronal cell body to the demyelinated axon, increasing axonal mitochondrial content, which we term the axonal response of mitochondria to demyelination (ARMD). However, following demyelination axons degenerate before the homeostatic ARMD reaches its peak. Enhancement of ARMD, by targeting mitochondrial biogenesis and mitochondrial transport from the cell body to axon, protects acutely demyelinated axons from degeneration. To determine the relevance of ARMD to disease state, we examined MS autopsy tissue and found a positive correlation between mitochondrial content in demyelinated dorsal column axons and cytochrome c oxidase (complex IV) deficiency in dorsal root ganglia (DRG) neuronal cell bodies. We experimentally demyelinated DRG neuron-specific complex IV deficient mice, as established disease models do not recapitulate complex IV deficiency in neurons, and found that these mice are able to demonstrate ARMD, despite the mitochondrial perturbation. Enhancement of mitochondrial dynamics in complex IV deficient neurons protects the axon upon demyelination. Consequently, increased mobilisation of mitochondria from the neuronal cell body to the axon is a novel neuroprotective strategy for the vulnerable, acutely demyelinated axon. We propose that promoting ARMD is likely to be a crucial preceding step for implementing potential regenerative strategies for demyelinating disorders.</p

Enhanced axonal response of mitochondria to demyelination offers neuroprotection:implications for multiple sclerosis

Axonal loss is the key pathological substrate of neurological disability in demyelinating disorders, including multiple sclerosis (MS). However, the consequences of demyelination on neuronal and axonal biology are poorly understood. The abundance of mitochondria in demyelinated axons in MS raises the possibility that increased mitochondrial content serves as a compensatory response to demyelination. Here, we show that upon demyelination mitochondria move from the neuronal cell body to the demyelinated axon, increasing axonal mitochondrial content, which we term the axonal response of mitochondria to demyelination (ARMD). However, following demyelination axons degenerate before the homeostatic ARMD reaches its peak. Enhancement of ARMD, by targeting mitochondrial biogenesis and mitochondrial transport from the cell body to axon, protects acutely demyelinated axons from degeneration. To determine the relevance of ARMD to disease state, we examined MS autopsy tissue and found a positive correlation between mitochondrial content in demyelinated dorsal column axons and cytochromecoxidase (complex IV) deficiency in dorsal root ganglia (DRG) neuronal cell bodies. We experimentally demyelinated DRG neuron-specific complex IV deficient mice, as established disease models do not recapitulate complex IV deficiency in neurons,and found that these mice are able to demonstrate ARMD, despite the mitochondrial perturbation.Enhancement of mitochondrial dynamics in complex IV deficient neurons protects the axon upon demyelination. Consequently, increased mobilisation of mitochondria from the neuronal cell body to the axon is a novel neuroprotective strategy for the vulnerable, acutely demyelinated axon. We propose that promoting ARMD is likely to be a crucial preceding step for implementing potential regenerative strategies for demyelinating disorders.</p

Olfactory Ensheathing Cell Transplantation in Experimental Spinal Cord Injury:Effect size and Reporting Bias of 62 Experimental Treatments: A Systematic Review and Meta-Analysis

Olfactory ensheathing cell (OEC) transplantation is a candidate cellular treatment approach for human spinal cord injury (SCI) due to their unique regenerative potential and autologous origin. The objective of this study was, through a meta-epidemiologic approach, (i) to assess the efficacy of OEC transplantation on locomotor recovery after traumatic experimental SCI and (ii) to estimate the likelihood of reporting bias and/or missing data. A study protocol was finalized before data collection. Embedded into a systematic review and meta-analysis, we conducted a literature research of databases including PubMed, EMBASE, and ISI Web of Science from 1949/01 to 2014/10 with no language restrictions, screened by two independent investigators. Studies were included if they assessed neurobehavioral improvement after traumatic experimental SCI, administrated no combined interventions, and reported the number of animals in the treatment and control group. Individual effect sizes were pooled using a random effects model. Details regarding the study design were extracted and impact of these on locomotor outcome was assessed by meta-regression. Missing data (reporting bias) was determined by Egger regression and Funnel-plotting. The primary study outcome assessed was improvement in locomotor function at the final time point of measurement. We included 49 studies (62 experiments, 1,164 animals) in the final analysis. The overall improvement in locomotor function after OEC transplantation, measured using the Basso, Beattie, and Bresnahan (BBB) score, was 20.3% (95% CI 17.8-29.5). One missing study was imputed by trim and fill analysis, suggesting only slight publication bias and reducing the overall effect to a 19.2% improvement of locomotor activity. Dose-response ratio supports neurobiological plausibility. Studies were assessed using a 9-point item quality score, resulting in a median score of 5 (interquartile range [IQR] 3-5). In conclusion, OEC transplantation exerts considerable beneficial effects on neurobehavioral recovery after traumatic experimental SCI. Publication bias was minimal and affirms the translational potential of efficacy, but safety cannot be adequately assessed. The data justify OECs as a cellular substrate to develop and optimize minimally invasive and safe cellular transplantation paradigms for the lesioned spinal cord embedded into state-of-the-art Phase I/II clinical trial design studies for human SCI

Post-Rückenmarkquerschnittsverletzung auftretendes Immundefizienz-Syndrom und therapeutische Wirksamkeit der pharmakologischen Inhibitoren des RhoA/ROCK- Signaltransduktionsweges - ein translationales Vorhaben

Introduction: Secondary complications such as infections are the leading cause of death after acute spinal cord injury (SCI). Impaired motor function and poor clinical condition do not sufficiently explain the increased infection susceptibility after SCI suggesting the presence of a neurogenic immune deficiency (SCI-induced immune deficiency syndrome, SCI-IDS). The publications summarized here investigate the functional relevance of the SCI-IDS, aim at further characterizing it with immunophenotyping and haematological profiling and provide a systematic identification of possible therapeutic interventions for SCI-IDS triggered consequences such as increased secondary axonal damage or impaired functional recovery. Methods: Neurogenic implications of the SCI- IDS were investigated in a mouse model of experimental pneumonia related to SCI at different thoracic lesion levels. Additionally, the relevance of an intact sympathetic innervation of secondary immune organs was investigated by selective spleen denervation before SCI induction. A level dependency of pneumonia was then evaluated in an observational multicenter human study using multiple logistic regression models. Moreover, SCI-IDS was characterized in a longitudinal study (SCIentinel) including an interim-analysis to evaluate the feasibility. To systematically identify experimental interventions for SCI and SCI-IDS related consequences, electronic databases were searched for studies reporting neurobehavioral outcome after treatment with RhoA/ROCK-inhibitors. An overall effect size was calculated and study characteristics significantly associated with different observed effect sizes were identified. Publication bias was addressed using funnel plots, Egger regression and the trim and fill method. Results: Susceptibility for experimental pneumonia was is increased for T3 spinal cord lesions compared to T9 lesions indicated by elevated bacterial load in the lungs and pronounced spleen atrophy. The transected spinal cord below T3 SCI was then identified to propagate SCI-IDS through decentralized sympathetic innervation of the spleen as a main immune organ. The lesion dependent susceptibility for pneumonia was confirmed in human SCI by multiple regression analysis incorporating 1221 patients. The administrative interim-analysis of the SCIentinel study confirmed its feasibility after inclusion of 60 patients (51% of calculated sample size). Systematic review and meta-analysis of Rho/ROCK-inhibition calculated an overall neurobehavioral efficacy of 21% including data from 725 animals. This was reduced by 5.6% after trim and fill analysis indicating underlying publication bias. Conclusions: SCI-IDS was identified to increase the susceptibility for pneumonia in a lesion level dependent manner. Early detection of SCI-IDS and development of prognostic markers could improve clinical care and reduce mortality. Evaluation of preclinical candidates targeting SCI-IDS supports a translational approach to the clinics.Einleitung: Sekundäre Komplikationen wie Infektionen sind die häufigste Todesursache nach traumatischer Rückenmarkverletzung. Die erhöhte Infektanfälligkeit nach einer Querschnittlähmung erklärt sich nicht vollständig durch verschlechterte motorische Funktion und den klinischen Zustand, was auf ein vorliegendes neurogenes Immundefizienz-Syndrom (SCI-IDS) hinweist. Die hier zusammengefassten Publikationen untersuchen die funktionelle Relevanz des SCI-IDS, zielen auf eine weitere immunphänotypische und hämatologische Charakterisierung ab und verschaffen einen systematischen Überblick über potentielle therapeutische Interventionen für durch das SCI-IDS verstärkte Komplikationen, wie ein gesteigerter axonaler Sekundärschaden oder eine verschlechterte funktionelle Erholung. Methodik: Neurogene Auswirkungen des SCI-IDS wurden im Mausmodell einer experimentellen Pneumonie in Abhängigkeit von Rückenmarkverletzungen unterschiedlicher thorakaler Läsionshöhen untersucht. Ferner wurde nach einer solchen Verletzung durch selektive Milzdenervierung die Relevanz einer intakten sympathischen Innervation sekundärer Immunorgane geprüft. Eine Läsionshöhenabhängigkeit wurde mittels multipler Regressionsmodelle zusätzlich in einer multizentrischen humanen Observationsstudie beurteilt. Eine weitere Charakterisierung des SCI-IDS fand in einer longitudinalen Patientenstudie statt (SCIentinel). Die Interimsanalyse zur Machbarkeit ist Teil dieser Dissertation. Um systematisch experimentelle Studien zu identifizieren, die auf die Behandlung von SCI oder SCI-IDS bedingten Konsequenzen abzielen, wurden elektronische Datenbanken nach Studien durchsucht, die den Wiedergewinn neurologischer Funktionen nach Behandlung mit RhoA/ROCK-Inhibitoren untersuchen. Für diese wurde eine Gesamteffektgröße berechnet und diejenigen Studiendetails bestimmt, die signifikant mit unterschiedlichen Effektgrößen assoziiert waren. Folgende Methoden wurden benutzt, um Publikations-Bias (Verzerrung durch nichtveröffentlichte Studien) zu bestimmen: Funnel-Plots, Egger-Regression und Trim-and-Fill. Ergebnisse: Die Infektionsanfälligkeit nach experimenteller Pneumonie zeigte sich im Rahmen von T3 Rückenmarkquerschnittverletzungen erhöht, was sich im Vergleich zu T9 Verletzungen durch eine vermehrte Bakterienanzahl in der Lunge und eine ausgeprägtere Milzatrophie darstellte. Das vom Gehirn isolierte Rückenmark unterhalb der Transektionsverletzung auf Höhe T3 wurde als eine Ursache für das SCI-IDS identifiziert, bedingt durch eine sympathische Fehlinnvervation der Milz als immunologisches Effektororgan. Die Läsionshöhenabhängigkeit wurde mittels multipler logistischer Regression bei 1221 Patienten nach Rückenmarkverletzung bestätigt. Die administrative Zwischenanalyse der SCIentinel-Studie bestätigte die Machbarkeit nach Einschluss von 60 Patienten, was 51% gemäß Fallzahlberechnung darstellt. Die Meta-Analyse ergab für 725 eingeschlossene Tiere eine Gesamteffektgröße von 21% und die Adjustierung mittels Trim-and-Fill reduzierte diese um 5.6%, was einen vorliegenden Publikations-Bias impliziert. Schlussfolgerung: Das SCI-IDS in Abhängigkeit von der Läsionshöhe wurde als ursächlich für eine erhöhte Anfälligkeit für Pneumonien identifiziert. Die frühe Erkennung eines SCI-IDS und die Bestimmung von prognostischen Markern könnten die klinische Behandlung verbessern und die Mortalität senken. Die Evaluation von präklinischen Therapeutika zur Behandlung einer Rückenmarkverletzung und derer Konsequenzen unterstützen Bemühungen, die auf eine Translation in die klinische Anwendung abzielen

Gene Cloning, Protein Characterization, and Alteration of Product Selectivity for the Trehalulose Hydrolase and Trehalulose Synthase from “Pseudomonas mesoacidophila” MX-45▿ †

The naturally occurring structural isomer of sucrose, trehalulose, is produced by sucrose isomerase (SI). Screening of chromosomal DNA from “Pseudomonas mesoacidophila” MX-45 with an SI-specific probe facilitated the cloning of two adjacent gene homologs, mutA and mutB. Both genes were expressed separately in Escherichia coli, and their enzyme products were characterized. MutA hydrolyzed the substrates trehalulose, isomaltulose, and sucrose into glucose and fructose. Due to its highest activity on trehalulose, MutA was referred to as trehalulase. mutB encodes the SI (trehalulose synthase) and catalyzes the isomerization of sucrose to mainly trehalulose. From Northern blot analysis it is apparent that the mutB gene is not transcribed as part of an operon and was transcriptionally upregulated when P. mesoacidophila MX-45 cells were grown in sucrose medium, whereas under investigated conditions no transcript for mutA was detected. Mutants of mutB were created by a random mutagenesis approach in order to alter the product specificity of MutB. Two types of mutants have emerged, one type that prefers the hydrolytic reaction on sucrose and another type that still acts as an SI but with a significant shift in the product from trehalulose to isomaltulose. The hydrolytic character of MutB R311C was demonstrated through its higher catalytic efficiency for glucose production over trehalulose production. MutB D442N favored the transfer reaction, with an isomer preference for isomaltulose