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1.  Superiority of preventive antibiotic treatment compared with standard treatment of poststroke pneumonia in experimental stroke: a bed to bench approach 
Stroke patients are prone to life-threatening bacterial pneumonia. Previous experimental stroke studies have demonstrated that preventive antibiotic treatment (PAT) improves outcome compared with placebo treatment, which however does not model the clinical setting properly. Here we investigate whether PAT is superior to the current clinical ‘gold standard' for treating poststroke infections. Therefore, we modeled stroke care according to the current stroke guidelines recommending early antibiotic treatment after diagnosing infections. To reliably diagnose pneumonia in living mice, we established a general health score and a magnetic resonance imaging protocol for radiologic confirmation. Compared with standard treatment after diagnosis by these methods, PAT not only abolished pneumonia successfully but also improved general medical outcome. Both, preventive and standard antibiotic treatment using enrofloxacin improved survival in a similar way compared with placebo treatment. However, in contrast to standard treatment, only PAT improved functional outcome assessed by gait analysis. In conclusion, standard and preventive treatment approach reduced poststroke mortality, however at the cost of a worse neurologic outcome compared with preventive approach. These data support the concept of PAT for treating patients at risk for poststroke infections and warrant phase III trials to prove this concept in clinical setting.
PMCID: PMC3677122  PMID: 23361393
antibiotic prophylaxis; brain ischemia; gait analysis; lung MRI; mouse model of stroke; pneumonia; stroke-induced immunodepression
2.  Fading Signatures of Critical Brain Dynamics during Sustained Wakefulness in Humans 
The Journal of Neuroscience  2013;33(44):17363-17372.
Sleep encompasses approximately a third of our lifetime, yet its purpose and biological function are not well understood. Without sleep optimal brain functioning such as responsiveness to stimuli, information processing, or learning may be impaired. Such observations suggest that sleep plays a crucial role in organizing or reorganizing neuronal networks of the brain toward states where information processing is optimized.
Increasing evidence suggests that cortical neuronal networks operate near a critical state characterized by balanced activity patterns, which supports optimal information processing. However, it remains unknown whether critical dynamics is affected in the course of wake and sleep, which would also impact information processing. Here, we show that signatures of criticality are progressively disturbed during wake and restored by sleep. We demonstrate that the precise power-laws governing the cascading activity of neuronal avalanches and the distribution of phase-lock intervals in human electroencephalographic recordings are increasingly disarranged during sustained wakefulness. These changes are accompanied by a decrease in variability of synchronization. Interpreted in the context of a critical branching process, these seemingly different findings indicate a decline of balanced activity and progressive distance from criticality toward states characterized by an imbalance toward excitation where larger events prevail dynamics. Conversely, sleep restores the critical state resulting in recovered power-law characteristics in activity and variability of synchronization. These findings support the intriguing hypothesis that sleep may be important to reorganize cortical network dynamics to a critical state thereby assuring optimal computational capabilities for the following time awake.
PMCID: PMC3858643  PMID: 24174669
3.  Deficient EBV-Specific B- and T-Cell Response in Patients with Chronic Fatigue Syndrome 
PLoS ONE  2014;9(1):e85387.
Epstein-Barr virus (EBV) has long been discussed as a possible cause or trigger of Chronic Fatigue Syndrome (CFS). In a subset of patients the disease starts with infectious mononucleosis and both enhanced and diminished EBV-specific antibody titers have been reported. In this study, we comprehensively analyzed the EBV-specific memory B- and T-cell response in patients with CFS. While we observed no difference in viral capsid antigen (VCA)-IgG antibodies, EBV nuclear antigen (EBNA)-IgG titers were low or absent in 10% of CFS patients. Remarkably, when analyzing the EBV-specific memory B-cell reservoir in vitro a diminished or absent number of EBNA-1- and VCA-antibody secreting cells was found in up to 76% of patients. Moreover, the ex vivo EBV-induced secretion of TNF-α and IFN-γ was significantly lower in patients. Multicolor flow cytometry revealed that the frequencies of EBNA-1-specific triple TNF-α/IFN-γ/IL-2 producing CD4+ and CD8+ T-cell subsets were significantly diminished whereas no difference could be detected for HCMV-specific T-cell responses. When comparing EBV load in blood immune cells, we found more frequently EBER-DNA but not BZLF-1 RNA in CFS patients compared to healthy controls suggesting more frequent latent replication. Taken together, our findings give evidence for a deficient EBV-specific B- and T-cell memory response in CFS patients and suggest an impaired ability to control early steps of EBV reactivation. In addition the diminished EBV response might be suitable to develop diagnostic marker in CFS.
PMCID: PMC3893202  PMID: 24454857
5.  Regulatory T cells accumulate and proliferate in the ischemic hemisphere for up to 30 days after MCAO 
Local and peripheral immune responses are activated after ischemic stroke. In our present study, we investigated the temporal distribution, location, induction, and function of regulatory T cells (Tregs) and the possible involvement of microglia, macrophages, and dendritic cells after middle cerebral artery occlusion (MCAO). C57BL/6J and Foxp3EGFP transgenic mice were subjected to 30 minutes MCAO. On days 7, 14, and 30 after MCAO, Tregs and antigen presenting cells were analyzed using fluorescence activated cell sorting multicolor staining and immunohistochemistry. A strong accumulation of Tregs was observed on days 14 and 30 in the ischemic hemisphere accompanied by the elevated presence and activation of microglia. Dendritic cells and macrophages were found on each analyzed day. About 60% of Foxp3+ Tregs in ischemic hemispheres were positive for the proliferation marker Ki-67 on days 7 and 14 after MCAO. The transfer of naive CD4+ cells depleted of Foxp3+ Tregs into RAG1−/− mice 1 day before MCAO did not lead to a de novo generation of Tregs 14 days after surgery. After depletion of CD25+ Tregs, no changes regarding neurologic outcome were detected. The sustained presence of Tregs in the brain after MCAO indicates a long-lasting immunological alteration and involvement of brain cells in immunoregulatory mechanisms.
PMCID: PMC3597367  PMID: 22968321
focal ischemia; immunology; leukocytes; macrophages; microglia; neuroprotection; T cells
6.  Dysfunction of alveolar macrophages after cardiac surgery and postoperative pneumonia? - an observational study 
Critical Care  2013;17(6):R285.
Patients undergoing cardiac surgery have an increased risk of postoperative pneumonia. Pulmonary immune dysfunction might be a contributing factor. We therefore determined changes of the surface molecules on alveolar macrophages (AMs). To characterize modulation in patients with pneumonia we correlated these changes to the development of postoperative pneumonia.
After ethical approval and written informed consent, 33 patients undergoing elective coronary bypass grafting surgery were included in this observational study. Peripheral blood cells and alveolar lavage fluid were collected directly after induction of anesthesia and two hours after separation from cardiopulmonary bypass (CPB). Human leukocyte antigen-DR (HLA-DR) and toll-like receptors (TLR) 2/4 expression on monocytes and AM were assessed by flow cytometry. A total of three patients developed postoperative pneumonia determined according to the criteria of the Center of Disease Control. Statistical analysis was performed with the Mann–Whitney-U test and Wilcoxon test.
We found significant changes of phenotypic and functional immune markers on AMs after cardiac surgery. HLA-DR expression on peripheral blood monocytes and AMs was significantly reduced compared to baseline in all patients (each approximately 30%). After surgery patients who developed postoperative pneumonia revealed a trend of stronger reduction of HLA-DR expression (83.7% versus 27.1%) and TLR4 expression on AMs (46.1% versus 9.9%) compared to patients without pneumonia. Already before surgery, the baseline of TLR2 expression on AM was significantly lower (27.7%) in patients who developed postoperative pneumonia.
As far as we know this is the first study that shows an early impairment of lung cellular immune response after cardiac surgery. These findings can help to understand the role of cell-mediated immunosuppression and its association to the development of postoperative pneumonia.
PMCID: PMC4056566  PMID: 24321282
7.  The SCIentinel study - prospective multicenter study to define the spinal cord injury-induced immune depression syndrome (SCI-IDS) - study protocol and interim feasibility data 
BMC Neurology  2013;13:168.
Infections are the leading cause of death in the acute phase following spinal cord injury and qualify as independent risk factor for poor neurological outcome (“disease modifying factor”). The enhanced susceptibility for infections is not stringently explained by the increased risk of aspiration in tetraplegic patients, neurogenic bladder dysfunction, or by high-dose methylprednisolone treatment. Experimental and clinical pilot data suggest that spinal cord injury disrupts the balanced interplay between the central nervous system and the immune system. The primary hypothesis is that the Spinal Cord Injury-induced Immune Depression Syndrome (SCI-IDS) is 'neurogenic’ including deactivation of adaptive and innate immunity with decreased HLA-DR expression on monocytes as a key surrogate parameter. Secondary hypotheses are that the Immune Depression Syndrome is i) injury level- and ii) severity-dependent, iii) triggers transient lymphopenia, and iv) causes qualitative functional leukocyte deficits, which may endure the post-acute phase after spinal cord injury.
SCIentinel is a prospective, international, multicenter study aiming to recruit about 118 patients with acute spinal cord injury or control patients with acute vertebral fracture without neurological deficits scheduled for spinal surgery. The assessment points are: i) <31 hours, ii) 31–55 hours, iii) 7 days, iv) 14 days, and v) 10 weeks post-trauma. Assessment includes infections, concomitant injury, medication and neurological classification using American Spinal Injury Association impairment scale (AIS) and neurological level. Laboratory analyses comprise haematological profiling, immunophenotyping, including HLA-DR expression on monocytes, cytokines and gene expression of immune modulators. We provide an administrative interim analysis of the recruitment schedule of the trial.
The objectives are to characterize the dysfunction of the innate and adaptive immune system after spinal cord injury and to explore its proposed 'neurogenic’ origin by analyzing its correlation with lesion height and severity. The trial protocol considers difficulties of enrolment in an acute setting, and loss to follow up. The administrative interim analysis confirmed the feasibility of the protocol. Better understanding of the SCI-IDS is crucial to reduce co-morbidities and thereby to attenuate the impact of disease modifying factors to protect neurological “outcome at risk”. This putatively results in improved spinal cord injury medical care.
Trial registration
DRKS-ID: DRKS00000122 (German Clinical Trials Registry)
PMCID: PMC3827331  PMID: 24206943
Spinal cord injury; Immune paralysis; Lesion height dependency; High-dose methylprednisolone treatment; Infections
8.  Standardization of whole blood immune phenotype monitoring for clinical trials: panels and methods from the ONE study 
Immune monitoring by flow cytometry is a fast and highly informative way of studying the effects of novel therapeutics aimed at reducing transplant rejection or treating autoimmune diseases. The ONE Study consortium has recently initiated a series of clinical trials aimed at using different cell therapies to promote tolerance to renal allografts. To compare the effectiveness of different cell therapies, the consortium developed a robust immune monitoring strategy, including procedures for whole blood (WB) leukocyte subset profiling by flow cytometry.
Six leukocyte profiling panels computing 7- to 9-surface marker antigens for monitoring the major leukocyte subsets as well as characteristics of T cell, B cell, and dendritic cell (DC) subsets were designed. The precision and variability of these panels were estimated. The assay was standardized within eight international laboratories using Flow-Set Pro beads for mean fluorescence intensity target definition and the flow cytometer setup procedure. Standardization was demonstrated by performing inter-site comparisons.
Optimized methods for sample collection, storage, preparation, and analysis were established, including protocols for gating target subsets. WB specimen age testing demonstrated that staining must be performed within 4 hours of sample collection to keep variability low, meaning less than or equal to 10% for the majority of defined leukocyte subsets. Inter-site comparisons between all participating centers testing shipped normal WB revealed good precision, with a variability of 0.05% to 30% between sites. Intra-assay analyses revealed a variability of 0.05% to 20% for the majority of subpopulations. This was dependent on the frequency of the particular subset, with smaller subsets showing higher variability. The intra-assay variability performance defined limits of quantitation (LoQ) for subsets, which will be the basis for assessing statistically significant differences achieved by the different cell therapies.
Local performance and central analysis of the ONE Study flow cytometry panel yields acceptable variability in a standardized assay at multiple international sites. These panels and procedures with WB allow unmanipulated analysis of changes in absolute cell numbers of leukocyte subsets in single- or multicenter clinical trials. Accordingly, we propose the ONE Study panel may be adopted as a standardized method for monitoring patients in clinical trials enrolling transplant patients, particularly trials of novel tolerance promoting therapies, to facilitate fair and meaningful comparisons between trials.
PMCID: PMC3827923  PMID: 24160259
Immune monitoring; Organ transplantation; Cell therapy; Tolerance; Kidney; Flow cytometry
9.  Postoperative Immunosuppression After Open and Laparoscopic Liver Resection: Assessment of Cellular Immune Function and Monocytic HLA-DR Expression 
In an experimental setting, the authors demonstrated a trend toward improved preservation of the immune system after laparoscopic hepatic resection compared with open surgery.
Background and Objectives:
Major abdominal procedures are strongly associated with postoperative immunosuppression and subsequent increased patient morbidity. It is believed that laparoscopic surgery causes less depletion of the systemic immune function because of the reduced tissue trauma. Various cytokines and monocytic HLA-DR expression have been successfully implemented to assess postoperative immune function. The aim of our study was to show the difference in immunologic profiles after minimally invasive versus conventional liver resection.
Ten animals underwent either laparoscopic or conventional open left lateral liver resection. Flow cytometric characteristics of HLA-DR expression on monocytes and lipopolysaccharide-stimulated cellular secretion of tumor necrosis factor α, interferon γ, interleukin 6, and interleukin 8 were measured and analyzed in ex vivo whole blood samples. Intraoperative and postoperative clinical outcome parameters were also documented and evaluated.
All animals survived the procedures. Postoperative complications were fever (n = 3), wound infections (n = 2), and biloma (n = 1). Open surgery showed a morbidity rate of 80% compared with 40% after laparoscopic surgery. Laparoscopic liver resection showed no postoperative immunoparalysis. Major histocompatibility complex class II expression in this group was elevated, whereas the open surgery group showed decreased major histocompatibility complex class II expression on postoperative day 1. Postoperative secretion of tumor necrosis factor α, interleukin 6, and interferon γ was lower in the open surgery group. Elevated transaminase levels after laparoscopy might have resulted from an ischemia/reperfusion injury caused by the capnoperitoneum.
Major immunoparalysis depression was not observed in either group. Laparoscopic surgery shows a tendency to improve immunologic recovery after liver resection.
PMCID: PMC3866067  PMID: 24398205
Liver resection; Laparoscopy; HLA-DR expression; Immunology; Stimulated cellular secretion; Postoperative immunosuppression
10.  MLL Becomes Functional through Intra-Molecular Interaction Not by Proteolytic Processing 
PLoS ONE  2013;8(9):e73649.
The mixed lineage leukemia (MLL) protein is an epigenetic transcriptional regulator that controls proliferative expansion of immature hematopoietic progenitors, whose aberrant activation triggers leukemogenesis. A mature MLL protein is produced by formation of an intra-molecular complex and proteolytic cleavage. However the biological significance of these two post-transcriptional events remains unclear. To address their in vivo roles, mouse mutant alleles were created that exclusively express either a variant protein incapable of intra-molecular interaction (designated de) or an uncleavable mutant protein (designated uc). The de homozygous mice died during midgestation and manifested devastating failure in embryonic development and reduced numbers of hematopoietic progenitors, whereas uc homozygous mice displayed no apparent defects. Expression of MLL target genes was severely impaired in de homozygous fibroblasts but unaffected in uc homozygous fibroblasts. These results unequivocally demonstrate that intra-molecular complex formation is a crucial maturation step whereas proteolytic cleavage is dispensable for MLL-dependent gene activation and proliferation in vivo.
PMCID: PMC3769346  PMID: 24040009
11.  Scaling Effects and Spatio-Temporal Multilevel Dynamics in Epileptic Seizures 
PLoS ONE  2012;7(2):e30371.
Epileptic seizures are one of the most well-known dysfunctions of the nervous system. During a seizure, a highly synchronized behavior of neural activity is observed that can cause symptoms ranging from mild sensual malfunctions to the complete loss of body control. In this paper, we aim to contribute towards a better understanding of the dynamical systems phenomena that cause seizures. Based on data analysis and modelling, seizure dynamics can be identified to possess multiple spatial scales and on each spatial scale also multiple time scales. At each scale, we reach several novel insights. On the smallest spatial scale we consider single model neurons and investigate early-warning signs of spiking. This introduces the theory of critical transitions to excitable systems. For clusters of neurons (or neuronal regions) we use patient data and find oscillatory behavior and new scaling laws near the seizure onset. These scalings lead to substantiate the conjecture obtained from mean-field models that a Hopf bifurcation could be involved near seizure onset. On the largest spatial scale we introduce a measure based on phase-locking intervals and wavelets into seizure modelling. It is used to resolve synchronization between different regions in the brain and identifies time-shifted scaling laws at different wavelet scales. We also compare our wavelet-based multiscale approach with maximum linear cross-correlation and mean-phase coherence measures.
PMCID: PMC3281841  PMID: 22363431
12.  Failure of Adaptive Self-Organized Criticality during Epileptic Seizure Attacks 
PLoS Computational Biology  2012;8(1):e1002312.
Critical dynamics are assumed to be an attractive mode for normal brain functioning as information processing and computational capabilities are found to be optimal in the critical state. Recent experimental observations of neuronal activity patterns following power-law distributions, a hallmark of systems at a critical state, have led to the hypothesis that human brain dynamics could be poised at a phase transition between ordered and disordered activity. A so far unresolved question concerns the medical significance of critical brain activity and how it relates to pathological conditions. Using data from invasive electroencephalogram recordings from humans we show that during epileptic seizure attacks neuronal activity patterns deviate from the normally observed power-law distribution characterizing critical dynamics. The comparison of these observations to results from a computational model exhibiting self-organized criticality (SOC) based on adaptive networks allows further insights into the underlying dynamics. Together these results suggest that brain dynamics deviates from criticality during seizures caused by the failure of adaptive SOC.
Author Summary
Over the recent years it has become apparent that the concept of phase transitions is not only applicable to the systems classically considered in physics. It applies to a much wider class of complex systems exhibiting phases, characterized by qualitatively different types of long-term behavior. In the critical states, which are located directly at the transition, small changes can have a large effect on the system. This and other properties of critical states prove to be advantageous for computation and memory. It is therefore suspected that also cerebral neural networks operate close to criticality. This is supported by the in vitro and in vivo measurements of power-laws of certain scaling relationships that are the hallmarks of phase transitions. While critical dynamics is arguably an attractive mode of normal brain functioning, its relation to pathological brain conditions is still unresolved. Here we show that brain dynamics deviates from a critical state during epileptic seizure attacks in vivo. Furthermore, insights from a computational model suggest seizures to be caused by the failure of adaptive self-organized criticality, a mechanism of self-organization to criticality based on the interplay between network dynamics and topology.
PMCID: PMC3252275  PMID: 22241971
14.  No Evidence for XMRV in German CFS and MS Patients with Fatigue Despite the Ability of the Virus to Infect Human Blood Cells In Vitro 
PLoS ONE  2010;5(12):e15632.
Xenotropic murine leukemia virus-related virus (XMRV), a novel human retrovirus originally identified in prostate cancer tissues, has recently been associated with chronic fatigue syndrome (CFS), a disabling disease of unknown etiology affecting millions of people worldwide. However, several subsequent studies failed to detect the virus in patients suffering from these illnesses or in healthy subjects. Here we report the results of efforts to detect antibody responses and viral sequences in samples from a cohort of German CFS and relapsing remitting multiple sclerosis (MS) patients with fatigue symptoms.
Blood samples were taken from a cohort of 39 patients fulfilling the Fukuda/CDC criteria (CFS), from 112 patients with an established MS diagnosis and from 40 healthy donors. Fatigue severity in MS patients was assessed using the Fatigue Severity Scale (FSS). Validated Gag- and Env-ELISA assays were used to screen sera for XMRV antibodies. PHA-activated PBMC were cultured for seven days in the presence of IL-2 and DNA isolated from these cultures as well as from co-cultures of PBMC and highly permissive LNCaP cells was analyzed by nested PCR for the presence of the XMRV gag gene. In addition, PBMC cultures were exposed to 22Rv1-derived XMRV to assess infectivity and virus production.
None of the screened sera from CFS and MS patients or healthy blood donors tested positive for XMRV specific antibodies and all PBMC (and PBMC plus LNCaP) cultures remained negative for XMRV sequences by nested PCR. These results argue against an association between XMRV infection and CFS and MS in Germany. However, we could confirm that PBMC cultures from healthy donors and from CFS patients can be experimentally infected by XMRV, resulting in the release of low levels of transmittable virus.
PMCID: PMC3008728  PMID: 21203514
15.  High-Mobility Group Box-1 Protein Serum Levels Do Not Reflect Monocytic Function in Patients with Sepsis-Induced Immunosuppression 
Mediators of Inflammation  2010;2010:745724.
Background. High-mobility group box-1 (HMGB-1) protein is released during “late sepsis” by activated monocytes. We investigated whether systemic HMGB-1 levels are associated with indices of monocytic activation/function in patients with sepsis-induced immunosuppression. Methodology. 36 patients (31 male, 64 ± 14 years) with severe sepsis/septic shock and monocytic deactivation (reduced mHLA-DR expression and TNF-α release) were assessed in a subanalysis of a placebo-controlled immunostimulatory trial using GM-CSF. HMGB-1 levels were assessed over a 9-day treatment interval. Data were compared to standardized biomarkers of monocytic immunity (mHLA-DR expression, TNF-α release). Principle findings. HMGB-1 levels were enhanced in sepsis but did not differ between treatment and placebo groups at baseline (14.6 ± 13.5 versus 12.5 ± 11.5 ng/ml, P = .62). When compared to controls, HMGB-1 level increased transiently in treated patients at day 5 (27.8 ± 21.7 versus 11.0 ± 14.9, P = .01). Between group differences were not noted at any other point of assessment. HMGB-1 levels were not associated with markers of monocytic function or clinical disease severity. Conclusions. GM-CSF treatment for sepsis-induced immunosuppression induces a moderate but only transient increase in systemic HMGB-1 levels. HMGB-1 levels should not be used for monitoring of monocytic function in immunostimulatory trials as they do not adequately portray contemporary changes in monocytic immunity.
PMCID: PMC2905954  PMID: 20652004
16.  Bronchoalveoloar lavage fluid cytokines and chemokines as markers and predictors for the outcome of interstitial lung disease in systemic sclerosis patients 
Arthritis Research & Therapy  2009;11(4):R111.
Interstitial lung disease (ILD) is a frequent manifestation of systemic sclerosis (SSc), and cytokines can contribute to the disease pathology. The aim of the current study was to identify specific changes in cytokine levels that may serve as disease markers and possible targets for therapy.
Cytokines were measured with bioplex analysis in 38 bronchoalveolar fluids (BALFs) from 32 SSc patients (27 with alveolitis and 11 without alveolitis) and 26 control patients. In the case of SSc patients, cytokines were correlated with the respective bronchoalveolar lavage (BAL) cell differentiation, lung function, and thoracic HR-CT score. For 35 BALF samples derived from 29 SSc patients, follow-up investigations of clinical data, lung-function parameter, or thoracic HR-CT scans were available to evaluate the predictive capacity of BALF cytokines and chemokines.
High IL-7 levels were characteristic of SSc-associated interstitial lung disease (ILD) and, in addition, when compared with ILD-negative SSc patients, ILD-positive SSc patients revealed higher IL-4, IL-6, IL-8, and CCL2 (MCP-1) BALF levels. High CCL2 and IL-8 BALF concentrations were associated with neutrophilic and mixed alveolitis. Cytokine levels of IL-4, IL-8, and CCL2 correlated negatively with lung-function parameters; CCL2 concentrations also correlated with HR-CT scores. High concentrations of several cytokines were associated with the progress of ILD and end-stage ILD. Univariate analyses revealed high IL-2 and tumor necrosis factor-alpha (TNF-α) levels as the best predictors for progressive disease, together with lung-function parameters, young age, and neutrophilic alveolitis. Multivariate analyses partially confirmed these results but did not sufficiently converge because of the limited number of patients.
The association of BALF cytokines with lung fibrosis and its progress suggests that cytokines contribute to the pathogenesis of ILD and hence could be regarded as potential therapeutic targets.
PMCID: PMC2745793  PMID: 19615053
17.  Phenotype changes and impaired function of dendritic cell subsets in patients with sepsis: a prospective observational analysis 
Critical Care  2009;13(4):R119.
Patients with sepsis often demonstrate severely impaired immune responses. The hallmark of this state of immunoparalysis is monocytic deactivation characterized by decreased human leukocyte antigen (HLA)-DR expression and reduced production of proinflammatory cytokines. Recently, diminished numbers of dendritic cells (DCs) were reported in patients with sepsis. However, little is known about DC phenotype and function in human sepsis. We therefore compared phenotypic and functional changes in monocyte and DC subsets in patients with sepsis and immunoparalysis.
In a prospective observational analysis, 16 consecutive patients with severe sepsis and septic shock (age 59.2 ± 9.7 years, 13 male, Sequential Organ Failure Assessment score 6.1 ± 2.7) and immunoparalysis (monocytic HLA-DR expression < 5,000 antibodies/cell) and 16 healthy volunteers were included. Peripheral blood DC counts, HLA-DR expression and ex vivo cytokine production were evaluated in comparison with monocyte subsets over time.
At baseline, a profound reduction in the numbers of myeloid DCs (MDCs), plasmacytoid DCs (PDCs), and CD14dimCD16positive monocytes was observed in sepsis whereas CD14brightCD16negative and CD14brightCD16positive monocyte numbers were increased. HLA-DR expression was reduced on all monocyte and DC subsets. Production of proinflammatory cytokines and intracellular cytokine staining in response to lipopolysaccharide and lipoteichoic acid was impaired in monocyte subsets and MDCs, whereas IL-10 secretion was increased. IFNα response by stimulated PDCs was significantly decreased compared with controls. At day 28, HLA-DR expression and cytokine production of DC and monocyte subsets remained lower in septic patients compared with controls.
In sepsis, long-lasting functional deactivation is common to all circulating monocyte and DC subsets. In addition to decreased peripheral blood DC counts, functional impairment of antigen-presenting cells may contribute to an impaired antimicrobial defense in sepsis.
PMCID: PMC2750167  PMID: 19604380
18.  Preventive Antibacterial Therapy in Acute Ischemic Stroke: A Randomized Controlled Trial 
PLoS ONE  2008;3(5):e2158.
Pneumonia is a major risk factor of death after acute stroke. In a mouse model, preventive antibacterial therapy with moxifloxacin not only prevents the development of post-stroke infections, it also reduces mortality, and improves neurological outcome significantly. In this study we investigate whether this approach is effective in stroke patients.
Preventive ANtibacterial THERapy in acute Ischemic Stroke (PANTHERIS) is a randomized, double-blind, placebo-controlled trial in 80 patients with severe, non-lacunar, ischemic stroke (NIHSS>11) in the middle cerebral artery (MCA) territory. Patients received either intravenous moxifloxacin (400 mg daily) or placebo for 5 days starting within 36 hours after stroke onset. Primary endpoint was infection within 11 days. Secondary endpoints included neurological outcome, survival, development of stroke-induced immunodepression, and induction of bacterial resistance.
On intention-to treat analysis (79 patients), the infection rate at day 11 in the moxifloxacin treated group was 15.4% compared to 32.5% in the placebo treated group (p = 0.114). On per protocol analysis (n = 66), moxifloxacin significantly reduced infection rate from 41.9% to 17.1% (p = 0.032). Stroke associated infections were associated with a lower survival rate. In this study, neurological outcome and survival were not significantly influenced by treatment with moxifloxacin. Frequency of fluoroquinolone resistance in both treatment groups did not differ. On logistic regression analysis, treatment arm as well as the interaction between treatment arm and monocytic HLA-DR expression (a marker for immunodepression) at day 1 after stroke onset was independently and highly predictive for post-stroke infections.
PANTHERIS suggests that preventive administration of moxifloxacin is superior in reducing infections after severe non-lacunar ischemic stroke compared to placebo. In addition, the results emphasize the pivotal role of immunodepression in developing post-stroke infections.
Trial Registration ISRCTN74386719
PMCID: PMC2373885  PMID: 18478129
19.  Stroke-induced Immunodeficiency Promotes Spontaneous Bacterial Infections and Is Mediated by Sympathetic Activation Reversal by Poststroke T Helper Cell Type 1–like Immunostimulation 
Infections are a leading cause of death in stroke patients. In a mouse model of focal cerebral ischemia, we tested the hypothesis that a stroke-induced immunodeficiency increases the susceptibility to bacterial infections. 3 d after ischemia, all animals developed spontaneous septicemia and pneumonia. Stroke induced an extensive apoptotic loss of lymphocytes and a shift from T helper cell (Th)1 to Th2 cytokine production. Adoptive transfer of T and natural killer cells from wild-type mice, but not from interferon (IFN)-γ–deficient mice, or administration of IFN-γ at day 1 after stroke greatly decreased the bacterial burden. Importantly, the defective IFN-γ response and the occurrence of bacterial infections were prevented by blocking the sympathetic nervous system but not the hypothalamo-pituitary-adrenal axis. Furthermore, administration of the β-adrenoreceptor blocker propranolol drastically reduced mortality after stroke. These data suggest that a catecholamine-mediated defect in early lymphocyte activation is the key factor in the impaired antibacterial immune response after stroke.
PMCID: PMC2194193  PMID: 12939340
T lymphocytes; natural killer cells; interferon γ; pneumonia; brain ischemia
20.  Crucial Role of the Interleukin 1 Receptor Family Member T1/St2 in T Helper Cell Type 2–Mediated Lung Mucosal Immune Responses 
T1/ST2 is an orphan receptor of unknown function that is expressed on the surface of murine T helper cell type 2 (Th2), but not Th1 effector cells. In vitro blockade of T1/ST2 signaling with an immunoglobulin (Ig) fusion protein suppresses both differentiation to and activation of Th2, but not Th1 effector populations. In a nascent Th2-dominated response, anti-T1/ST2 monoclonal antibody (mAb) inhibited eosinophil infiltration, interleukin 5 secretion, and IgE production. To determine if these effects were mediated by a direct effect on Th2 cells, we next used a murine adoptive transfer model of Th1- and Th2-mediated lung mucosal immune responses. Administration of either T1/ST2 mAb or T1/ST2-Ig abrogated Th2 cytokine production in vivo and the induction of an eosinophilic inflammatory response, but failed to modify Th1-mediated inflammation. Taken together, our data demonstrate an important role of T1/ST2 in Th2-mediated inflammatory responses and suggest that T1/ST2 may prove to be a novel target for the selective suppression of Th2 immune responses.
PMCID: PMC2195643  PMID: 10510079
inflammation; eosinophil; asthma; cytokines; immunoglobulin superfamily

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