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Background: Accumulating evidence indicates that neuropathic pain is a neuro-immune disorder with enhanced activation of the immune system. Recent data provided proof that neuropathic pain patients exhibit increased numbers of immunosuppressive regulatory T cells (Tregs), which may represent an endogenous attempt to limit inflammation and to reduce pain levels. We here investigate the molecular mechanisms underlying these alterations., Methods: Our experimental approach includes functional analyses of primary human T cells, 3'-UTR reporter assays, and expression analyses of neuropathic pain patients' samples., Results: We demonstrate that microRNAs (miRNAs) are involved in the differentiation of Tregs in neuropathic pain. We identify miR-124a and miR-155 as direct repressors of the histone deacetylase sirtuin1 (SIRT1) in primary human CD4(+) cells. Targeting of SIRT1 by either specific siRNA or by these two miRNAs results in an increase of Foxp3 expression and, consecutively, of anti-inflammatory Tregs (siRNA: 1.7 ± 0.4; miR-124a: 1.5 ± 0.4; miR-155: 1.6 ± 0.4; p < 0.01). As compared to healthy volunteers, neuropathic pain patients exhibited an increased expression of miR-124a (2.5 ± 0.7, p < 0.05) and miR-155 (1.3 ± 0.3; p < 0.05) as well as a reduced expression of SIRT1 (0.5 ± 0.2; p < 0.01). Moreover, the expression of these two miRNAs was inversely correlated with SIRT1 transcript levels., Conclusions: Our findings suggest that in neuropathic pain, enhanced targeting of SIRT1 by miR-124a and miR-155 induces a bias of CD4(+) T cell differentiation towards Tregs, thereby limiting pain-evoking inflammation. Deciphering miRNA-target interactions that influence inflammatory pathways in neuropathic pain may contribute to the discovery of new roads towards pain amelioration., Trial Registration: German Clinical Trial Register DRKS00005954.

Background: Despite substantial progress, pathogenesis and therapy of chronic pain are still the focus of many investigations. The ATP-gated P2X7 receptor (P2X7R) has previously been shown to play a central role in animal models of nociceptive inflammatory and neuropathic pain. Recently, we found that the adaptive immune system is involved in the pathophysiology of chronic nociceptive and neuropathic pain in humans. So far, data regarding P2X7R expression patterns on cells of the adaptive immune system of pain patients are scarce. We therefore analyzed the P2X7R expression on peripheral blood lymphocytes and monocytes, as well as serum levels of IL-1β in patients suffering from chronic nociceptive and neuropathic pain in comparison to healthy volunteers in order to identify individuals who might benefit from a P2X7R modulating therapy., Methods: P2X7R messenger RNA (mRNA) and protein expression were determined in patients with either chronic nociceptive low back pain (CLBP) or neuropathic pain (NeP), and in healthy volunteers by quantitative real-time PCR (qPCR) and by fluorescence-assisted cell-sorting (FACS), respectively. IL-1β serum levels were measured with a multiplex cytokine assay., Results: Compared to healthy volunteers, P2X7R mRNA (1.6-fold, p = 0.038) and protein levels were significantly increased on monocytes (NeP: 24.6 ± 6.2, healthy volunteers: 17.0 ± 5.4; p = 0.002) and lymphocytes (NeP: 21.8 ± 6.5, healthy volunteers: 15.6 ± 5.2; p = 0.009) of patients with NeP, but not in patients with CLBP. Similarly, IL-1β serum concentrations were significantly elevated only in NeP patients (1.4-fold, p = 0.04)., Conclusions: A significant upregulation of P2X7R and increased IL-1β release seems to be a particular phenomenon in patients with NeP. P2X7R inhibitors may therefore represent a potential option for the treatment of this frequently intractable type of pain. German Clinical Trial Register (DRKS): Registration Trial DRKS00005954.

About half of the known miRNA genes are located within protein-coding host genes, and are thus subject to co-transcription. Accumulating data indicate that this coupling may be an intrinsic mechanism to directly regulate the host gene's expression, constituting a negative feedback loop. Inevitably, the cell requires a yet largely unknown repertoire of methods to regulate this control mechanism. We propose APA as one possible mechanism by which negative feedback of intronic miRNA on their host genes might be regulated. Using in-silico analyses, we found that host genes that contain seed matching sites for their intronic miRNAs yield longer 32UTRs with more polyadenylation sites. Additionally, the distribution of polyadenylation signals differed significantly between these host genes and host genes of miRNAs that do not contain potential miRNA binding sites. We then transferred these in-silico results to a biological example and investigated the relationship between ZFR and its intronic miRNA miR-579 in a U87 cell line model. We found that ZFR is targeted by its intronic miRNA miR-579 and that alternative polyadenylation allows differential targeting. We additionally used bioinformatics analyses and RNA-Seq to evaluate a potential cross-talk between intronic miRNAs and alternative polyadenylation. CPSF2, a gene previously associated with alternative polyadenylation signal recognition, might be linked to intronic miRNA negative feedback by altering polyadenylation signal utilization.

Background: The classification of pain into nociceptive and neuropathic pain is based on characteristic symptoms and different pathophysiological mechanisms. In a recent investigation, we found a disrupted TH17/Treg balance in patients suffering from chronic unspecific low back pain (CLBP). These patients did not show any signs of neuropathy. There is evidence for a considerable impact of the immune system also in neuropathic pain. However, the role of the adaptive immune system is still unclear. In the present study, we investigated systemic T-cell subset responses and T-cell related cytokine profiles in patients with chronic neuropathic pain., Methods: We analyzed T-cell subsets, mRNA expression and T-cell-related cytokine profiles in 26 patients suffering from neuropathic pain in comparison to 26 healthy controls. Using multicolor flow cytometry (FACS), we quantified the number of T helper cells 1 (TH1), TH2, TH17 and regulatory T-cells (Tregs). Forkhead-Box-Protein 3 (FoxP3), Transforming growth factor-β (TGF-β) and RAR-related orphan receptor-γT (ROR-γT) mRNA expression was determined by quantitative real-time PCR (qPCR) and levels of pain-related cytokines were measured by Human Cytokine Multiplex Immunoassay (Macrophage inflammatory protein-1α (MIP-1α), Tumor necrosis factor-α (TNF-α), Interferon-γ (IFN-γ), Interleukin (IL) -4, IL-6, IL-10, IL-17, and IL-23)., Results: We found a TH17/Treg imbalance with significantly increased anti-inflammatory Tregs and decreased pro-inflammatory TH17 cells in patients with neuropathic pain as compared to healthy controls. These results were confirmed on mRNA level: Treg-related FoxP3 and TGF-β mRNA expression was elevated, whereas expression of TH17-related RORγT was reduced. Cytokine analyses revealed only marginal changes., Conclusions: Our investigation revealed a clear shift of T-cell subsets towards anti-inflammation in patients with neuropathic pain. Interestingly, this is quite similar to our previous findings in CLBP patients, but even more pronounced. Therefore, it remains to be elucidated in future investigations whether the immune changes represent an underlying pathophysiological mechanism or an epiphenomenon induced by ongoing pain and stress., German Clinical Trial Register (drks): Trial registration number: DRKS00005954.

T-cell functions must be tightly controlled to keep the balance between vital proinflammatory activity and detrimental overactivation. MicroRNA-146a (miR-146a) has been identified as a key negative regulator of T-cell responses in mice. Its role in human T cells and its relevance to human inflammatory disease, however, remains poorly defined. In this study, we have characterized miR-146a-driven pathways in primary human T cells. Our results identify miR-146a as a critical gatekeeper of Th1-cell differentiation processes acting via molecular mechanisms not uncovered so far. MiR-146a targets protein kinase C epsilon (PRKCε), which is part of a functional complex consisting of PRKCε and signal transducer and activator of transcription 4 (STAT4). Within this complex, PRKCε phosphorylates STAT4, which in turn is capable of promoting Th1-cell differentiation processes in human CD4(+) T lymphocytes. In addition, we observed that T cells of sepsis patients had reduced levels of miR-146a and an increased PRKCε expression in the initial hyperinflammatory phase of the disease. Collectively, our results identify miR-146a as a potent inhibitor of Th1-cell differentiation in human T cells and suggest that dysregulation of miR-146a contributes to the pathogenesis of sepsis., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

MicroRNAs (miRNAs) are a class of small (∼22 nucleotides) non-coding RNAs that post-transcriptionally regulate gene expression by interacting with target mRNAs. A majority of miRNAs is located within intronic or exonic regions of protein-coding genes (host genes), and increasing evidence suggests a functional relationship between these miRNAs and their host genes. Here, we introduce miRIAD, a web-service to facilitate the analysis of genomic and structural features of intragenic miRNAs and their host genes for five species (human, rhesus monkey, mouse, chicken and opossum). miRIAD contains the genomic classification of all miRNAs (inter- and intragenic), as well as classification of all protein-coding genes into host or non-host genes (depending on whether they contain an intragenic miRNA or not). We collected and processed public data from several sources to provide a clear visualization of relevant knowledge related to intragenic miRNAs, such as host gene function, genomic context, names of and references to intragenic miRNAs, miRNA binding sites, clusters of intragenic miRNAs, miRNA and host gene expression across different tissues and expression correlation for intragenic miRNAs and their host genes. Protein-protein interaction data are also presented for functional network analysis of host genes. In summary, miRIAD was designed to help the research community to explore, in a user-friendly environment, intragenic miRNAs, their host genes and functional annotations with minimal effort, facilitating hypothesis generation and in-silico validations. Database URL: http://www.miriad-database.org., (© The Author(s) 2014. Published by Oxford University Press.)

Background: Despite well-designed concepts of perioperative pain management, recent studies have revealed that a large number of patients still suffer from unacceptable pain after surgery. The purpose of this prospective evaluation was to critically analyze postoperative pain treatment provided by a routinely established, DIN certified acute pain service (APS) at the University Hospital Großhadern in Munich., Materials and Methods: A total of 1,000 consecutive patients received one of the following analgesic procedures: continuous epidural analgesia (EA, n = 401), continuous and patient-controlled epidural analgesia (PCEA, n = 305), intravenous patient-controlled analgesia with opioids (PCA, n = 169) or continuous peripheral nerve block (CPNB, n = 125). For EA and PCEA, ropivacaine 0.2 % and sufentanil 0.24 µg/ml were administered while peripheral regional analgesia was performed with infusion of ropivacaine 0.2 % only. Patients with PCEA were allowed a 3 mg bolus once per hour on demand. Standardized intravenous PCA was performed with piritramide 2.5 mg/ml, a bolus of 2.5 mg, a lock-out time of 15 min, a maximum of 25 mg/4 h and no background infusion. During the daily visits the APS assessed pain intensity at rest and during movement on a numerical rating scale from 0 (no pain) to 10 (maximum pain), acceptance of pain, satisfaction with the analgesic procedure, demand of additional non-opioid analgesics, the need for optimization including bolus applications and changes of the infusion rate or retraction of the epidural catheter. The duration of the procedures, side effects and complications were documented. The catheter insertion sites were inspected daily for redness and tenderness on palpation., Results: In general, epidural and peripheral regional analgesic techniques were superior in terms of postoperative analgesia to intravenous opioid PCA and were associated with fewer side effects, such as sedation, nausea, vomiting, obstipation and sensorimotor deficits. A subgroup analysis revealed that in major upper abdominal surgery, EA provided significantly better analgesia at rest and during movement than PCA. In lower abdominal surgery PCEA induced significantly better analgesia than both PCA and EA, especially during movement. Patient satisfaction was generally high and was best with PCEA (95 %) followed by CPNB (94 %), EA (91 %) and PCA (88 %). On the first postoperative day analgesic procedures had to be optimized (e.g. by bolus administration, retraction of catheters or changes to standardized PCA) in 23 % of EA patients, 10 % of PCEA patients, 6 % of PCA patients and 12 % of CPNB patients. Major complications, such as neuraxial hematoma, infections or respiratory depression were not observed., Conclusions: As described in many prospective studies, this evaluation revealed that for postoperative pain control, regional anesthesia is superior to intravenous patient-controlled analgesia with strong opioids in terms of analgesia and side effects. In the setting of a well-organized acute pain service with frequent education and training of all members involved, postoperative pain management is safe and effective. However, regular re-evaluation of the defined and certified procedures is necessary.

Favorable outcome after chemotherapy of glioblastomas cannot unequivocally be linked to promoter hypermethylation of the O6-methylguanine-DNA methyltransferase (MGMT) gene encoding a DNA repair enzyme associated with resistance to alkylating agents. This indicates that molecular mechanisms determining MGMT expression have not yet been fully elucidated. We here show that glioblastomas are capable to downregulate MGMT expression independently of promoter methylation by elongation of the 3'-UTR of the mRNA, rendering the alternatively polyadenylated transcript susceptible to miRNA-mediated suppression. While the elongated transcript is poorly expressed in normal brain, its abundance in human glioblastoma specimens is inversely correlated with MGMT mRNA expression. Using a bioinformatically guided experimental approach, we identified miR-181d, miR-767-3p, and miR-648 as significant post-transcriptional regulators of MGMT in glioblastomas; the first two miRNAs induce MGMT mRNA degradation, the latter affects MGMT protein translation. A regression model including the two miRNAs influencing MGMT mRNA expression and the MGMT methylation status reliably predicts The Cancer Genome Atlas MGMT expression data. Responsivity of MGMT expressing T98G glioma cells to temozolomide was significantly enhanced after transfection of miR-181d, miR-767-3p, and miR-648. Taken together, our results uncovered alternative polyadenylation of the MGMT 3'-UTR and miRNA targeting as new mechanisms of MGMT silencing.

In the recent past, microRNAs (miRNAs) have gained significant attention as potent regulators of gene expression. These small noncoding RNA molecules are currently of major interest when investigating regulatory circuits of the cell. After identification of potential miRNA-target gene interactions (e.g., using computational algorithms), biomolecular validation is necessary. In the current chapter, we present a protocol for validation of an miRNA target interaction implying cloning of a dual-luciferase miRNA target expression vector, transfection of cells with this vector and a precursor miRNA (pre-miRNA), and the subsequent luciferase assay.

In the recent past, intragenic microRNAs (miRNAs) have gained significant attention. Due to the unique linkage to their host gene's transcription, these miRNAs offer more information than intergenic miRNAs as they associate with some of their hosts' properties. However, genome wide analysis of intronic miRNA data can be very challenging, especially if it relies on Web-based tools only. We therefore describe in this chapter how to set a database and how to link the different publicly available information resources on miRNAs and host genes. We also provide an example of a simple, but useful analysis technique. The basic structures and ideas suggested in this chapter can easily be extended to integrating other data and be applied to different analysis techniques.

Fungal infections are becoming an increasing menace in the hospital care setting. Among them, non-albicans Candida species have gained significant attention. Especially in the ICU setting, therapeutic options are limited in many cases by the side-effects of conventional antifungal therapy. Echinocandins are a relatively new class of antifungal agents that promise good effectiveness against Candida and Aspergillus species. Due to their underlying mechanisms of action, they yield good tolerability and few limitations of usage. In the current manuscript we describe the patents of two recently approved echinocandins, micafungin (US approved 2005) and anidulafungin (2006) and provide an overview of the mechanisms, clinical effectiveness and safety of antifungal therapy with these agents.

Immunosuppressive signaling via the adenosine A2A receptor (A2AR) is an important pathway to control inflammation. In immune cells, expression levels of A2ARs influence responsiveness to inflammatory stimuli. However, mechanisms driving expressional changes of A2ARs are still largely elusive. In the current study, we have investigated the impact of microRNAs (miRNAs) on A2AR expression in human polymorphonuclear leukocytes (PMNs) and T cells. Bioinformatic analyses and reporter gene assays revealed that A2AR expression is controlled by miRNA-214, miRNA-15, and miRNA-16. We detected all three miRNAs in both human PMNs and T cells. However, in PMNs, up to 10-fold higher levels of miRNA-16 and miRNA-214 were detected as compared with T cells. Upon in vitro stimulation, no significant expressional changes occurred. Expression levels of all three miRNAs strongly differed between individuals. A2AR expression also exhibited significant differences between PMNs and T cells: In PMNs, more than a 60-fold increase was seen upon LPS stimulation, whereas in T cells only a 2-fold increase was observed upon anti-CD3/CD28 activation. The extent of A2AR upregulation in PMNs strongly differed between individuals (from less than 10-fold to more than 100-fold). In PMNs, the increase in A2AR mRNA expression upon stimulation was inversely correlated with the expression levels of miRNA-214, miRNA-15, and miRNA-16 (R = -0.87, P < 0.0001); no correlation was found in human T cells. These results indicate that individual miRNA profiles gain important influence on A2AR expression regulation in PMNs upon stimulation. Determination of miRNA expression levels may help to identify patients with an increased risk for severe inflammation.

Introduction: Combined aneurysms of aortic and iliac arteries are rare with a prevalence of less than 0.1%. However, these combined aneurysms are associated with a high incidence of thrombosis, distal embolism, or rupture. Endovascular repair is a therapeutic option and includes embolization of the internal iliac artery in numerous cases. Embolization of the internal iliac artery may cause severe ischemia with hip and buttock complications in 2-5%. Therefore, preservation of internal iliac arteries is essential to reduce complications., Patient and Method: We describe in detail an endovascular procedure for combined abdominal aortic (diameter of 8.6 cm) and bilateral common and internal iliac aneurysm (diameter of the left side: 6.4 cm; diameter of the right side: 4.3 cm) in a 44-year-old patient caused by media necrosis Erdheim-Gsell. The blood flow of both internal arteries was preserved in this patient., Result: Operation was done without any complications. Conversion to an open procedure was not necessary. During follow up (3, 6, and 12 months after operation) the patient did not develop any signs of severe hip and buttock complications. Furthermore, sonography and CT angiography revealed a good blood flow via the internal and external iliac arteries., Conclusion: Endovascular repair of abdominal aortic aneurysms combined with bilateral common and internal iliac aneurysms offers a promising minimal invasive procedure. Most importantly, this technique is less invasive than open operations and reduces complications by preserving the pelvic perfusion., (Copyright © 2011 Wiley-Liss, Inc.)

Background: We analyzed prospectively whether MGMT (O(6)-methylguanine-DNA methyltransferase) mRNA expression gains prognostic/predictive impact independent of MGMT promoter methylation in malignant glioma patients undergoing radiotherapy with concomitant and adjuvant temozolomide or temozolomide alone. As DNA-methyltransferases (DNMTs) are the enzymes responsible for setting up and maintaining DNA methylation patterns in eukaryotic cells, we analyzed further, whether MGMT promoter methylation is associated with upregulation of DNMT expression., Methodology/principal Findings: ADULT PATIENTS WITH A HISTOLOGICALLY PROVEN MALIGNANT ASTROCYTOMA (GLIOBLASTOMA: N = 53, anaplastic astrocytoma: N = 10) were included. MGMT promoter methylation was determined by methylation-specific PCR (MSP) and sequencing analysis. Expression of MGMT and DNMTs mRNA were analysed by real-time qPCR. Prognostic factors were obtained from proportional hazards models. Correlation between MGMT mRNA expression and MGMT methylation status was validated using data from the Cancer Genome Atlas (TCGA) database (N = 229 glioblastomas). Low MGMT mRNA expression was strongly predictive for prolonged time to progression, treatment response, and length of survival in univariate and multivariate models (p<0.0001); the degree of MGMT mRNA expression was highly correlated with the MGMT promoter methylation status (p<0.0001); however, discordant findings were seen in 12 glioblastoma patients: Patients with methylated tumors with high MGMT mRNA expression (N = 6) did significantly worse than those with low transcriptional activity (p<0.01). Conversely, unmethylated tumors with low MGMT mRNA expression (N = 6) did better than their counterparts. A nearly identical frequency of concordant and discordant findings was obtained by analyzing the TCGA database (p<0.0001). Expression of DNMT1 and DNMT3b was strongly upregulated in tumor tissue, but not correlated with MGMT promoter methylation and MGMT mRNA expression., Conclusions/significance: MGMT mRNA expression plays a direct role for mediating tumor sensitivity to alkylating agents. Discordant findings indicate methylation-independent pathways of MGMT expression regulation. DNMT1 and DNMT3b are likely to be involved in CGI methylation. However, their exact role yet has to be defined.

Background: miRNAs are small, non-coding RNA molecules that mainly act as negative regulators of target gene messages. Due to their regulatory functions, they have lately been implicated in several diseases, including malignancies. Roughly half of known miRNA genes are located within previously annotated protein-coding regions ("intragenic miRNAs"). Although a role of intragenic miRNAs as negative feedback regulators has been speculated, to the best of our knowledge there have been no conclusive large-scale studies investigating the relationship between intragenic miRNAs and host genes and their pathways., Results: miRNA-containing host genes were three times longer, contained more introns and had longer 5' introns compared to a randomly sampled gene cohort. These results are consistent with the observation that more than 60% of intronic miRNAs are found within the first five 5' introns. Host gene 3'-untranslated regions (3'-UTRs) were 40% longer and contained significantly more adenylate/uridylate-rich elements (AREs) compared to a randomly sampled gene cohort. Coincidentally, recent literature suggests that several components of the miRNA biogenesis pathway are required for the rapid decay of mRNAs containing AREs. A high-confidence set of predicted mRNA targets of intragenic miRNAs also shared many of these features with the host genes. Approximately 20% of intragenic miRNAs were predicted to target their host mRNA transcript. Further, KEGG pathway analysis demonstrated that 22 of the 74 pathways in which host genes were associated showed significant overrepresentation of proteins encoded by the mRNA targets of associated intragenic miRNAs., Conclusions: Our findings suggest that both host genes and intragenic miRNA targets may potentially be subject to multiple layers of regulation. Tight regulatory control of these genes is likely critical for cellular homeostasis and absence of disease. To this end, we examined the potential for negative feedback loops between intragenic miRNAs, host genes, and miRNA target genes. We describe, how higher-order miRNA feedback on hosts' interactomes may at least in part explain correlation patterns observed between expression of host genes and intragenic miRNA targets in healthy and tumor tissue.

Medical errors rank high amongst leading causes of death. Especially in emergency care, when there is limited time to think, the "human factor", the interface between human action and the environmental system, has been recognized to be a critical part that determines the outcome. Recent models of human error are based on the principle that critical incidents are of multifactorial origin and reflect insufficiencies of the underlying system itself. The Human Simulation Center (HSC) was built specifically to train interaction between medical teams and to investigate the human factor in medical emergencies. In the following article we present "MevidIO", a live-monitoring and debriefing application framework. Developed for a full-scale simulation center designed to model error transduction in medical emergency care process chains, the framework integrates educational and scientific aspects.

Introduction: The goal of personalised medicine in the intensive care unit (ICU) is to predict which diagnostic tests, monitoring interventions and treatments translate to improved outcomes given the variation between patients. Unfortunately, processes such as gene transcription and drug metabolism are dynamic in the critically ill; that is, information obtained during static non-diseased conditions may have limited applicability. We propose an alternative way of personalising medicine in the ICU on a real-time basis using information derived from the application of artificial intelligence on a high-resolution database. Calculation of maintenance fluid requirement at the height of systemic inflammatory response was selected to investigate the feasibility of this approach., Methods: The Multi-parameter Intelligent Monitoring for Intensive Care II (MIMIC II) is a database of patients admitted to the Beth Israel Deaconess Medical Center ICU in Boston. Patients who were on vasopressors for more than six hours during the first 24 hours of admission were identified from the database. Demographic and physiological variables that might affect fluid requirement or reflect the intravascular volume during the first 24 hours in the ICU were extracted from the database. The outcome to be predicted is the total amount of fluid given during the second 24 hours in the ICU, including all the fluid boluses administered., Results: We represented the variables by learning a Bayesian network from the underlying data. Using 10-fold cross-validation repeated 100 times, the accuracy of the model in predicting the outcome is 77.8%. The network generated has a threshold Bayes factor of seven representing the posterior probability of the model given the observed data. This Bayes factor translates into p < 0.05 assuming a Gaussian distribution of the variables., Conclusions: Based on the model, the probability that a patient would require a certain range of fluid on day two can be predicted. In the presence of a larger database, analysis may be limited to patients with identical clinical presentation, demographic factors, co-morbidities, current physiological data and those who did not develop complications as a result of fluid administration. By better predicting maintenance fluid requirements based on the previous day's physiological variables, one might be able to prevent hypotensive episodes requiring fluid boluses during the course of the following day.