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ZusammenfassungMultiresistente Tuberkulosen (MDR-TB) machen zwar einen nur geringen Anteil an der Gesamtzahl aller TB-Erkrankungen aus, durch die erforderliche komplexe Medikation mit potenziell schweren und lebensbedrohlichen Nebenwirkungen, Langzeitfolgen und ungünstigem Verlauf ist jedoch besondere Sorgfalt geboten. Wir berichten über eine 30-jährige Altenpflegerin mit chronischem Husten und angeborenem Alpha-1-Antitrypsin-Mangel (AATM), die an einer MDR-TB erkrankte und erhebliche Medikamenten-Nebenwirkungen erlitt.

Poor solubility of drug candidates is a well-known and thoroughly studied challenge in the development of oral dosage forms. One important approach to tackle this challenge is the formulation as an amorphous solid dispersion (ASD). To reach the desired biopharmaceutical improvement a high supersaturation has to be reached quickly and then be conserved long enough for absorption to take place. In the presented study, various formulations of regorafenib have been produced and characterized in biorelevant in-vitro experiments. Povidone-based formulations, which are equivalent to the marketed product Stivarga®, showed a fast drug release but limited stability and robustness after that. In contrast, HPMCAS-based formulations exhibited excellent stability of the supersaturated solution, but unacceptably slow drug release. The attempt to combine the desired attributes of both formulations by producing a ternary ASD failed. Only co-administration of HPMCAS as an external stabilizer to the rapidly releasing Povidone-based ASDs led to the desired dissolution profile and high robustness. This optimized formulation was tested in a pharmacokinetic animal model using Wistar rats. Despite the promising in-vitro results, the new formulation did not perform better in the animal model. No differences in AUC could be detected when compared to the conventional (marketed) formulation. These data represent to first in-vivo study of the new concept of external stabilization of ASDs. Subsequent in-vitro studies revealed that temporary exposure of the ASD to gastric medium had a significant and long-lasting effect on the dissolution performance and externally administered stabilizer could not prevent this sufficiently. By applying the co-administered HPMCAS as an enteric coating onto Stivarga tablets, a new bi-functional approach was realized. This approach achieved the desired tailoring of the dissolution profile and high robustness against gastric medium as well as against seeding.

Association studies have linked alterations of blood‐derived microRNAs (miRNAs) with colorectal cancer (CRC). Here, we performed a microarray‐based comparison of the profiles of 2549 miRNAs in 80 blood samples from healthy donors and patients with colorectal adenomas, colorectal diverticulitis and CRC at different stages. Confirmation by quantitative real‐time PCR (RT‐PCR) was complemented by validation of identified molecules in another 36 blood samples. No variations in miRNA levels were observed in samples from patients with colorectal adenomas and diverticulitis or from healthy donors. However, there were 179 CRC‐associated miRNAs of differential abundance compared to healthy controls. Only three – miR‐1225‐5p, miR‐1207‐5p and miR‐4459 – exhibited increased levels at all CRC stages. Most deregulated miRNAs (128/179, 71%) specifically predicted metastatic CRC. Pathway analysis found several cancer‐related pathways to which the miRNAs contribute in various ways. In conclusion, miRNA levels in blood vary throughout CRC progression and affect cellular functions relevant to haematogenous CRC progression and dissemination. The identified biomarker and therapeutic candidates require further confirmation of their clinical relevance., Here, we performed a comprehensive study on the abundance variations of 2549 miRNAs in blood from 80 healthy donors and patients with colorectal disease. We further validated the results in 36 independent samples by quantitative real‐time PCR. Patients with colorectal adenomas or diverticulitis and healthy donors exhibited no difference in their miRNA profiles, while patients with colorectal cancer displayed miRNAs with differential abundance in a stage‐specific manner. The miRNAs identified in our study contribute to several cancer‐related pathways relevant to colorectal cancer progression and haematogenous dissemination.

Objective Psychogenic non-epileptic seizures (PNES), a common phenomenon in neurological settings, are regarded as a paroxysmal type of functional neurological disorder (FND). In a substantial proportion, PNES are disabling with poor long-term outcomes and high economic costs. Despite the clinical and financial consequences of PNES, there is still a lack of controlled clinical trials on the treatment of this challenging disorder. The study aims to evaluate the feasibility and collect first evidence of the efficacy of a group based-intervention in PNES-patients. Methods A pilot randomized controlled feasibility study with a parallel-group design was performed in adult outpatients with PNES to evaluate a new body-focused group therapy (CORDIS) versus guided self-help groups. Self-assessment of dissociation (Dissociation Experience Scale—DES-20) and seizure severity (Liverpool Seizure Severity Scale—LSSS) were assessed two weeks before and two weeks after the treatment intervention and also six months after treatment as primary outcome parameters. Results A total of 53 patients were recruited from a specialized outpatient clinic, and out of those, 29 patients completed either the body-focused group therapy program (n = 15) or a guided self-help group (SHG) therapy (n = 14). When analyzing the ITT sample (n = 22 CORDIS group, n = 20 SHG), both groups showed an effect on seizure severity and level of dissociation. In the per protocol sample (n = 13 CORDIS group, n = 12 SHG), CORDIS was superior to the self-help group for reducing seizure severity 6 months after the treatment. Significance CORDIS is a newly developed body-focused group therapy program for adults with PNES. Further studies should include a multicentric design with a higher number of participants.

Neuroactive substances released by activated microglia contribute to hyperexcitability of spinal dorsal horn neurons in many animal models of chronic pain. An important feedback loop mechanism is via release of fractalkine (CX3CL1) from primary afferent terminals and dorsal horn neurons and binding to CX3CR1 receptors on microglial cells. We studied the involvement of fractalkine signaling in latent and manifest spinal sensitization induced by two injections of nerve growth factor (NGF) into the lumbar multifidus muscle as a model for myofascial low back pain. Single dorsal horn neurons were recorded in vivo to study their receptive fields and spontaneous activity. Under intrathecal vehicle application, the two NGF injections led to an increased proportion of neurons responding to stimulation of deep tissues (41%), to receptive field expansion into the hindlimb (15%), and to resting activity (53%). Blocking fractalkine signaling by continuous intrathecal administration of neutralizing antibodies completely prevented these signs of spinal sensitization to a similar extent as in a previous study with the microglia inhibitor minocycline. Reversely, fractalkine itself induced similar sensitization in a dose-dependent manner (for 200 ng/mL: 45% deep tissue responses, 24% receptive field expansion, and 45% resting activity) as repeated nociceptive stimulation by intramuscular NGF injections. A subsequent single NGF injection did not have an additive effect. Our data suggest that neuron-to-microglia signaling via the CX3CL1-CX3CR1 pathway is critically involved in the initiation of nonspecific, myofascial low back pain through repetitive nociceptive stimuli.

Introduction: Chronic obstructive pulmonary disease (COPD) is one of the most common chronic diseases associated with high mortality. Previous studies suggested a prognostic role for peak oxygen uptake (VO2peak) assessed during cardiopulmonary exercise testing (CPET) in patients with COPD. However, most of these studies had small sample sizes or short follow-up periods, and despite their relevance, CPET parameters are not included in the Global Initiative for Chronic Obstructive Lung Disease (GOLD) tool for assessment of severity. Objectives: We therefore aimed to assess the prognostic value of CPET parameters in a large cohort of outpatients with COPD. Methods: In this retrospective, multicentre cohort study, medical records of patients with COPD who underwent CPET during 2004–2017 were reviewed and demographics, smoking habits, GOLD grade and category, exacerbation frequency, dyspnoea score, lung function measurements, and CPET parameters were documented. Relationships with survival were evaluated using Kaplan-Meier analysis, Cox regression, and receiver operating characteristic (ROC) curves. Results: Of a total of 347 patients, 312 patients were included. Five-year and 10-year survival probability was 75% and 57%, respectively. VO2peak significantly predicted survival (hazard ratio: 0.886 [95% confidence interval: 0.830; 0.946]). The optimal VO2peak threshold for discrimination of 5-year survival was 14.6 mL/kg/min (area under ROC curve: 0.713). Five-year survival in patients with VO2peak Conclusions: We confirm that VO2peak is a highly significant predictor of survival in COPD patients and recommend the incorporation of VO2peak into the assessment of COPD severity.

Background: Helicobacter pylori (H. pylori) is a bacterial carcinogen and the leading risk factor for noncardia gastric cancer (NCGC). Detecting antibodies against specific H. pylori proteins in peripheral blood can be applied to characterize infection and determine disease associations. Most studies analyzing the association between H. pylori infection and gastric cancer have focused on previously identified antigens, predominantly the virulence factor cytotoxin-associated gene A (CagA). Selecting antigens in an unbiased approach may, however, allow the identification of novel biomarkers. Methods: Using a combination of multiple spotting technique and cell-free, on-chip protein expression, we displayed the H. pylori genome (strain 26695) on high-density microarrays. Immunogenic proteins were identified by serum pool incubations and henceforth analyzed in individual samples. To test its applicability, we used sera from a multicase–control (MCC)-Spain study. Serologic responses between NCGC cases and controls were assessed by conditional logistic regression estimating ORs and 95% confidence intervals. Results: We successfully expressed 93% of the 1,440 H. pylori open reading frames in situ. Of these, 231 (17%) were found to be immunogenic. By comparing 58 NCGC cases with 58 matched controls, we confirmed a higher seroprevalence of CagA among cases (66%) than controls (31%). We further identified a potential novel marker, the Helicobacter outer membrane protein A (HopA). Conclusions: In this study, we provide evidence that our H. pylori whole-proteome microarray offers a platform for unbiased de novo identification of serologic biomarkers. Impact: Given its versatile workflow, antibody responses against other H. pylori strains and possible associations with diverse H. pylori–related outcomes can be systematically analyzed.

Aberrant DNA methylation is often involved in carcinogenesis. Our initial goal was to identify DNA methylation biomarkers associated with pancreatic cancer. A genomewide methylation study was performed on DNA from pancreatic ductal adenocarcinoma (PDAC) and endocrine pancreas tumors. Validation of DNA methylation patterns and concomitant alterations in expression of gene candidates was performed on patient samples and pancreatic cancer cell lines. Furthermore, validation was done on independent data from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO). Finally, droplet digital PCR was employed to detect DNA methylation marks in cell‐free (cf) DNA isolated from plasma samples of PDAC patients and cancer‐free blood donors. Hypermethylation of the SST gene (encoding somatostatin) and concomitant downregulation of its expression were discovered in PDAC and endocrine tumor tissues while not being present in chronic pancreatitis (inflamed) tissues and normal pancreas. Fittingly, treatment with a somatostatin agonist (octreotide) reduced cell proliferation and migration of pancreatic cancer cells. Diagnostic performance of SST methylation in a receiver operating characteristic curve analysis was 100% and 89% for tissue and plasma samples, respectively. A large body of TCGA and GEO data confirmed SST hypermethylation and downregulation in PDAC and showed a similar effect in a broad spectrum of other tumor entities. SST promoter methylation represents a sensitive and promising molecular, pan‐cancer biomarker detectable in tumor tissue, and liquid biopsy samples., Hypermethylation of the SST gene (encoding somatostatin) and concomitant downregulation of its expression were found to be common in tumor entities, representing a promising pan‐cancer biomarker. Somatostatin agonist octreotide reduced proliferation and migration of pancreatic cancer cells, indicating a tumor‐suppressive function of somatostatin. Variation in SST methylation was detectable in not only tumor tissues but also patients’ plasma samples.

ZusammenfassungPsychogene nicht-epileptische Anfälle (PNES) treten im Kontext verschiedener Erkrankungen auf. Die Ätiologie ist noch weitgehend unverstanden. Bisherige Konzepte gehen von einer erhöhten Rate an Trauma – Folgestörungen als ätiologische Erklärung für PNES aus, was sich durch Studien mehrfach belegen ließ 1 2. Klinisch imponiert das Krankheitsbild bedrohlich, wodurch die Betroffenen häufig intensivmedizinische Maßnahmen erhalten, ohne dass sie davon profitieren 3. v. a. in Epilepsiezentren häufen sich Betroffene, da hier eine diagnostische Abgrenzung zu epileptischen Anfällen möglich ist. Oft gestaltet sich der Übergang von der Diagnosestellung in der Epileptologie zur Weiterbehandlung in die Psychosomatik schwierig, da sowohl Patienten/innen als auch Behandelnde oft in somatischen Krankheitskonzepten verhaftet sind 28. Aufgrund der notwendigen Interdisziplinarität zwischen Neurologie und Psychosomatik wurde an der Charité Universitätsmedizin Berlin eine Spezialambulanz für Menschen mit dissoziativen Anfällen gegründet, welche sich im Übergang von Neurologie zu Psychosomatik ansiedelt und als Kooperationsprojekt funktioniert 27. Aus der Ambulanz heraus wurde zudem ein Gruppenbehandlungsprogramm (Kördis) entwickelt. Dieses modularisierte 10-wöchige Therapieprogramm wird im Folgenden inhaltlich vorgestellt und ist weiterhin Gegenstand einer aktuell noch laufenden randomisierten, kontrollierten Evaluationsstudie. In den hier vorgestellten Pilotdaten aus der laufenden RCT Studie zeigte sich eine signifikante Verbesserung der Anfallsstärke (gemessen mit der Liverpool Seizure Severity Scale LSSS) sowie der Dissoziationsneigung (gemessen mit dem Fragebogen für dissoziative Symptome FDS-20) durch das Therapieprogramm (LSSS-Wert prä-interventionell: 49,2±4,2; LSSS-Wert post-interventionell: 46,8±5,4; t(10)=2,73; p=0,02; FDS-20 -Wert prä-interventionell: 25,3±14,6; FDS-20 -Wert post-interventionell: 19,7±8,8; t(10)=2,18; p=0,05).

BACKGROUND: Insecticide applications in blueberry production systems play a crucial role in the control of Drosophila suzukii populations. Here, quantitative spray deposition patterns were obtained under replicated field experiments in blueberry during two field seasons with three sprayers, i.e. cannon, electrostatic, and air‐blast. Seven insecticides were tested (at 6 hours using a Potter spray tower) to determine the mortality data for adult D. suzukii. Spray deposition and mortality data for adult D. suzukii were used to create model simulations for insect populations. Model simulations included field deposition rates of sprayers and insecticide mortality as factors. Simulations were applied in different combinations with five applications over a 6‐week period. RESULTS: Relative deposition rates for the cannon sprayer were elevated in the upper zones of the canopy, whereas for the air‐blast sprayer, deposition was greater in the bottom zones. Electrostatic spray deposition was relatively uniform within the six canopy zones. Clear trends in D. suzukii laboratory mortality were found with lowest to highest mortality recorded for phosmet, spinetoram, spinosad, malathion, cyantraniliprole, zeta‐cypermethrin, and methomyl respectively. Maximum D. suzukii population impacts, as shown by model outputs, were observed with air‐blast sprayers together with zeta‐cypermethrin. CONCLUSION: The electrostatic sprayer had the least variable canopy deposition among the three types of spray equipment, and the air‐blast sprayer had the highest overall deposition rates. This study provides new hypotheses that can be used for field verification with these spray technologies and insecticides as key factors. © 2019 Society of Chemical Industry

In this study, protein profiling was performed on gastric cancer tissue samples in order to identify proteins that could be utilized for an effective diagnosis of this highly heterogeneous disease and as targets for therapeutic approaches. To this end, 16 pairs of postoperative gastric adenocarcinomas and adjacent non-cancerous control tissues were analyzed on microarrays that contain 813 antibodies targeting 724 proteins. Only 17 proteins were found to be differentially regulated, with much fewer molecules than the numbers usually identified in studies comparing tumor to healthy control tissues. Insulin-like growth factor-binding protein 7 (IGFBP7), S100 calcium binding protein A9 (S100A9), interleukin-10 (IL‐10) and mucin 6 (MUC6) exhibited the most profound variations. For an evaluation of the proteins’ capacity for discriminating gastric cancer, a Receiver Operating Characteristic curve analysis was performed, yielding an accuracy (area under the curve) value of 89.2% for distinguishing tumor from non-tumorous tissue. For confirmation, immunohistological analyses were done on tissue slices prepared from another cohort of patients with gastric cancer. The utility of the 17 marker proteins, and particularly the four molecules with the highest specificity for gastric adenocarcinoma, is discussed for them to act as candidates for diagnosis, even in serum, and targets for therapeutic approaches.

We report the case of a man with intense cough for several months and a few days of severe dyspnea. A massive pleural empyema due to Actinomyces meyeri was diagnosed by radiological, microbiological and thoracoscopic means. Pleural infections caused by this anaerobic bacterium are very rare and should be considered when risk factors like male gender, chronic alcohol abuse, and poor oral hygiene are present. Penicillin-based antibiotic treatment and surgical decortication led to recovery.

Objective To propose a noninvasive diagnostic approach, which allows reliable distinction between low- and high-risk pancreatic intraductal papillary mucinous neoplasms (IPMNs). Background IPMNs are identifiable precursor lesions of pancreatic cancer, of which surgical resection is warranted prior to the development of invasive carcinoma, but low-grade IPMNs should not be unnecessarily resected. However, diagnostic tools that preoperatively enable accurate risk stratification of IPMNs are missing. Methods This single-center, retrospective cohort study included 56 patients who underwent surgical resection for IPMN including 18 low-risk (low-grade) and 38 high-risk (high-grade/invasive carcinoma) IPMNs, from whom clinical features and serum samples were prospectively obtained. An antibody microarray platform was used to analyze the serum proteome. Based on serum markers and selected clinical characteristics support vector machine models were constructed to predict the risk of IPMN malignancy. Results A serum protein signature discriminating low- and high-risk IPMN patients was identified. Combinations of established clinical features and the newly identified serum biomarkers correctly distinguished low- and high-risk IPMNs in 93% on 1000-fold cross-validation. Conclusions This study highlights the synergistic predictive value of combining a novel serum protein signature with conventional clinical characteristics to risk-stratify IPMN patients. If these findings are supported by larger validation studies, they might enable more rational decision-making in clinical management of IPMN patients in conjunction with clinical guidelines.

Background Non-invasive ventilation (NIV) has been shown to improve survival and quality of life in COPD patients with chronic hypercapnic respiratory failure. However, the proportion of COPD patients with chronic hypercapnia is not yet known and clinical data enabling better identification of patients are scarce. The HOmeVent registry was initiated to determine the prevalence of chronic hypercapnia in COPD in an outpatient setting and to evaluate the predictors of hypercapnia. Methods HOmeVent is a multicenter, prospective, observational, non-interventional patient registry that includes COPD patients in GOLD stage 3 or 4. Eligible patients were identified and enrolled in an outpatient setting during routine clinic visits. Assessments included blood gas analyses, pulmonary function testing and quality of life assessment. Results Ten outpatient clinics in Germany enrolled 231 COPD patients in the registry (135 in GOLD stage 3 (58%) and 96 in GOLD stage 4 (42%)). Arterial carbon dioxide pressure (PaCO2) was ≥45 mmHg in 58 patients (25%); of these, 20 (9%) had PaCO2 ≥50 mmHg. The prevalence of hypercapnia at both cut-off values was numerically higher for patients in GOLD stage 4 versus 3. An increased body mass index, a decreased forced vital capacity and an increased bicarbonate level were significant independent predictors of hypercapnia. The proportion of patients who received NIV was 6% overall and 22% of those with hypercapnia. Conclusion A relevant proportion of COPD patients in GOLD stage 3 and 4 exhibits chronic hypercapnia and might, therefore, be candidates for long-term domiciliary NIV treatment.

In orthopaedic medicine, connective tissues are often affected by traumatic or degenerative injuries, and surgical intervention is required. Rotator cuff tears are a common cause of shoulder pain and disability among adults. The development of graft materials for bridging the gap between tendon and bone after chronic rotator cuff tears is essentially required. The limiting factor for the clinical success of a tissue engineering construct is a fast and complete vascularization of the construct. Otherwise, immigrating cells are not able to survive for a longer period of time, resulting in the failure of the graft material. The femur chamber allows the observation of microhaemodynamic parameters inside implants located in close vicinity to the femur in repeated measurements in vivo. We compared a porous polymer patch (a commercially available porous polyurethane-based scaffold from Biomerix™) with electrospun polycaprolactone (PCL) fibre mats and chitosan (CS)-graft-PCL modified electrospun PCL (CS-g-PCL) fibre mats in vivo. By means of intravital fluorescence microscopy, microhaemodynamic parameters were analysed repetitively over 20 days at intervals of 3 to 4 days. CS-g-PCL modified fibre mats showed a significantly increased vascularization at Day 10 compared with Day 6 and at Day 14 compared with the porous polymer patch and the unmodified PCL fibre mats at the same day. These results could be verified by histology. In conclusion, a clear improvement in terms of vascularization and biocompatibility is achieved by graft-copolymer modification compared with the unmodified material. © 2019 The Authors Journal of Tissue Engineering and Regenerative Medicine Published by John Wiley & Sons Ltd

Aim: To identify DNA methylation biomarkers in peripheral blood samples from triple-negative breast cancer (TNBC) patients. Materials & methods: We conducted an epigenome-wide association study (EWAS): the most promising markers were identified in 233 TNBC case–control pairs (discovery set) and subsequently validated in an independent validation set (57 TNBC patients and 124 controls). Results: cg06588802 (LINC00299/ID2) showed a higher methylation in TNBC patients compared with controls (discovery set: 3% increase, p-value = 0.0009; validation set: 2% increase, p-value = 0.01). Consistent results at four neighboring methylation probes and the strong negative correlation (rho = -0.93) with LINC00299 expression add plausibility to this result. Conclusion: Hypermethylation of LINC00299 in peripheral blood may constitute a useful circulating biomarker for TNBC.

Restraint stress followed by weak nociceptive input from multifidus muscle enhances resting activity and sensitization of rat spinal neurons and mimics aspects of back pain., Background: A single injection of nerve growth factor (NGF) into a low back muscle induces a latent sensitization of rat dorsal horn neurons (DHNs) that primes for a manifest sensitization by a subsequent second NGF injection. Repeated restraint stress also causes a latent DHN sensitization. Objective: In this study, we investigated whether repeated restraint stress followed by a single NGF injection causes a manifest sensitization of DHNs. Methods: Rats were stressed repeatedly in a narrow plastic restrainer (1 hour on 12 consecutive days). Control animals were handled but not restrained. Two days after stress paradigm, behavioral tests and electrophysiological in vivo recordings from single DHNs were performed. Mild nociceptive low back input was induced by a single NGF injection into the lumbar multifidus muscle just before the recording started. Results: Restraint stress slightly lowered the low back pressure pain threshold (Cohen d = 0.83). Subsequent NGF injection increased the proportion of neurons responsive to deep low back input (control + NGF: 14%, stress + NGF: 39%; P = 0.041), mostly for neurons with input from outside the low back (7% vs 26%; P = 0.081). There was an increased proportion of neurons with resting activity (28% vs 55%; P = 0.039), especially in neurons having deep input (0% vs 26%; P = 0.004). Conclusions: The results indicate that stress followed by a short-lasting nociceptive input causes manifest sensitization of DHNs to deep input, mainly from tissue outside the low back associated with an increased resting activity. These findings on neuronal mechanisms in our rodent model suggest how stress might predispose to radiating pain in patients.

BACKGROUND: Nociplastic pain has been recently introduced as a third mechanistic descriptor of pain arising primarily from alterations of neural processing, in contrast to pain due to tissue damage leading to nociceptor activation (nociceptive) or due to lesion or disease of the somatosensory nervous system (neuropathic). It is characterized by hyperalgesia and allodynia, inconsistency and reversibility, as well as dynamic cross-system interactions with biological and psychobehavioral factors. Along with this renewed understanding, functional pain disorders, also classified as chronic primary pain, are being reframed as biopsychosocial conditions that benefit from multimodal treatment. OBJECTIVE: To summarize the current understanding of nociplastic pain and functional pain disorders, with a focus on conditions that are common in neurology practice. METHODS: This was a narrative literature review. RESULTS: Chronic back pain, fibromyalgia syndrome and complex regional pain syndrome are best understood within a biopsychosocial framework of pain perception that considers structural factors (predispositions and sequelae) and psychobehavioral mechanisms. Although pain is often the primary complaint, it should not be the only focus of treatment, as accompanying symptoms such as sleep or mood problems can significantly impact quality of life and offer useful leverage points for multimodal treatment. Analgesic pharmacotherapy is rarely helpful on its own, and should always be imbedded in a multidisciplinary setting.

Background and current state of (inter)national research: Due to the German National Cancer Plan, cancer centers have been established. Lung cancer centers are responsible for coordinating the care of lung cancer patients in a region and to diagnose and treat them according to the latest evidence-based[for full text, please go to the a.m. URL], 19. Deutscher Kongress für Versorgungsforschung (DKVF)

BACKGROUND: Preclinical experiments have shown that donor blood cells, modified in vitro by an alkylating agent (modified immune cells [MICs]), induced long-term specific immunosuppression against the allogeneic donor. METHODS: In this phase I trial, patients received either 1.5 × 10(6) MICs per kg BW on day –2 (n = 3, group A), or 1.5 × 10(8) MICs per kg BW on day –2 (n = 3, group B) or day –7 (n = 4, group C) before living donor kidney transplantation in addition to post-transplantation immunosuppression. The primary outcome measure was the frequency of adverse events (AEs) until day 30 (study phase) with follow-up out to day 360. RESULTS: MIC infusions were extremely well tolerated. During the study phase, 10 treated patients experienced a total of 69 AEs that were unlikely to be related or not related to MIC infusion. No donor-specific human leukocyte antigen Abs or rejection episodes were noted, even though the patients received up to 1.3 × 10(10) donor mononuclear cells before transplantation. Group C patients with low immunosuppression during follow-up showed no in vitro reactivity against stimulatory donor blood cells on day 360, whereas reactivity against third-party cells was still preserved. Frequencies of CD19(+)CD24(hi)CD38(hi) transitional B lymphocytes (Bregs) increased from a median of 6% before MIC infusion to 20% on day 180, which was 19- and 68-fold higher, respectively, than in 2 independent cohorts of transplanted controls. The majority of Bregs produced the immunosuppressive cytokine IL-10. MIC-treated patients showed the Immune Tolerance Network operational tolerance signature. CONCLUSION: MIC administration was safe and could be a future tool for the targeted induction of tolerogenic Bregs. TRIAL REGISTRATION: EudraCT number: 2014-002086-30; ClinicalTrials.gov identifier: NCT02560220. FUNDING: Federal Ministry for Economic Affairs and Technology, Berlin, Germany, and TolerogenixX GmbH, Heidelberg, Germany.

Identification of disease-associated autoantibodies is of high importance. Their assessment could complement current diagnostic modalities and assist the clinical management of patients. We aimed at developing and validating high-throughput protein microarrays able to screen patients&rsquo, sera to determine disease-specific autoantibody-signatures for pancreatic cancer (PDAC), chronic pancreatitis (CP), autoimmune pancreatitis and their subtypes (AIP-1 and AIP-2). In-house manufactured microarrays were used for autoantibody-profiling of IgG-enriched preoperative sera from PDAC-, CP-, AIP-1-, AIP-2-, other gastrointestinal disease (GID) patients and healthy controls. As a top-down strategy, three different fluorescence detection-based protein-microarrays were used: large with 6400, intermediate with 345, and small with 36 full-length human recombinant proteins. Large-scale analysis revealed 89 PDAC, 98 CP and 104 AIP immunogenic antigens. Narrowing the selection to 29 autoantigens using pooled sera first and individual sera afterwards allowed a discrimination of CP and AIP from PDAC. For validation, predictive models based on the identified antigens were generated which enabled discrimination between PDAC and AIP-1 or AIP-2 yielded high AUC values of 0.940 and 0.925, respectively. A new repertoire of autoantigens was identified and their assembly as a multiplex test will provide a fast and cost-effective tool for differential diagnosis of pancreatic diseases with high clinical relevance.

The combination of lentiviruses with techniques such as CRISPR-Cas9 has resulted in efficient and precise processes for targeted genome modification. An often-limiting aspect, however, is the efficiency of cell transduction. Low efficiencies with particular cell types and/or the high complexity of lentiviral libraries can cause insufficient representation. Here, we present a protocol that yielded substantial increases in transduction efficiency in various cell lines in comparison to several other procedures.

Gold compounds have a long history of use as immunosuppressants, but their precise mechanism of action is not completely understood. Using our recently developed liver-on-a-chip platform we now show that gold compounds containing planar N-heterocyclic carbene (NHC) ligands are potent ligands for the aryl hydrocarbon receptor (AHR). Further studies showed that the lead compound (MC3) activates TGFβ1 signaling and suppresses CD4+ T-cell activation in vitro, in human and mouse T cells. Conversely, genetic knockdown or chemical inhibition of AHR activity or of TGFβ1-SMAD-mediated signaling offsets the MC3-mediated immunosuppression. In scurfy mice, a mouse model of human immunodysregulation polyendocrinopathy enteropathy X-linked syndrome, MC3 treatment reduced autoimmune phenotypes and extended lifespan from 24 to 58 days. Our findings suggest that the immunosuppressive activity of gold compounds can be improved by introducing planar NHC ligands to activate the AHR-associated immunosuppressive pathway, thus expanding their potential clinical application for autoimmune diseases., Cheng, Haeberle et al. identify NHC–gold complexes as ligands of the aryl hydrocarbon receptor, which triggers TGFβ production for immune suppression. They find that the MC3 compound activates TGFβ signalling while reducing Il-2 expression and CD4+ T-cell activation and suppresses autoimmune phenotypes in a mouse model of IPEX syndrome.

A total of 2978 patients with validated paired results (SARS-CoV2-antigen and PCR) were identified. Our results show that only 45 antigen tests from 90 patients with positive validated PCR were correctly identified by antigen testing (sensitivity 50%). Roughly 50% of these patients had ongoing respiratory symptoms.

Pre-mRNA splicing is a fundamental process that plays a considerable role in generating protein diversity. Pre-mRNA splicing is also the key to the pathology of numerous diseases, especially cancers. In this review, we discuss how aberrant splicing isoforms precisely regulate three basic functional aspects in cancer: proliferation, metastasis and apoptosis. Importantly, clinical function of aberrant splicing isoforms is also discussed, in particular concerning drug resistance and radiosensitivity. Furthermore, this review discusses emerging strategies how to modulate pathologic aberrant splicing isoforms, which are attractive, novel therapeutic agents in cancer. Last we outline current and future directions of isoforms diagnostic methodologies reported so far in cancer. Thus, it is highlighting significance of aberrant splicing isoforms as markers for cancer and as targets for cancer therapy.

Lung adenocarcinoma (LUAD) is one of the most common cancers and lethal diseases in the world. Recognition of the undetermined lung nodules at an early stage is useful for a favorable prognosis. However, there is no good method to identify the undetermined lung nodules and predict their clinical outcome. DNA methylation alteration is frequently observed in LUAD and may play important roles in carcinogenesis, diagnosis, and prediction. This study took advantage of publicly available methylation profiling resources and a machine learning method to investigate methylation differences between LUAD and adjacent non-malignant tissue. The prediction panel was first constructed using 338 tissue samples from LUAD patients including 149 non-malignant ones. This model was then validated with data from The Cancer Genome Atlas database and clinic samples. As a result, the methylation status of four CpG loci in homeobox A9 (HOXA9), keratin-associated protein 8-1 (KRTAP8-1), cyclin D1 (CCND1), and tubby-like protein 2 (TULP2) were highlighted as informative markers. A random forest classification model with an accuracy of 94.57% and kappa of 88.96% was obtained. To evaluate this panel for LUAD, the methylation levels of four CpG loci in HOXA9, KRTAP8-1, CCND1, and TULP2 of tumor samples and matched adjacent lung samples from 25 patients with LUAD were tested. In these LUAD patients, the methylation of HOXA9 was significantly upregulated, whereas the methylation of KRTAP8-1, CCND1, and TULP2 were downregulated obviously in tumor samples compared with adjacent tissues. Our study demonstrates that the methylation of HOXA9, KRTAP8-1, CCND1, and TULP2 has great potential for the early recognition of LUAD in the undetermined lung nodules. The findings also exhibit that the application of improved mathematic algorithms can yield accurate and particularly robust and widely applicable marker panels. This approach could greatly facilitate the discovery process of biomarkers in various fields.

Using Chlamydia trachomatis (Ct) as a complex model organism, we describe a method to generate bacterial whole-proteome microarrays using cell-free, on-chip protein expression. Expression constructs were generated by two successive PCRs directly from bacterial genomic DNA. Bacterial proteins expressed on microarrays display antigenic epitopes, thereby providing an efficient method for immunoprofiling of patients and allowing de novo identification of disease-related serum antibodies. Through comparison of antibody reactivity patterns, we newly identified antigens recognized by known Ct-seropositive samples, and antigens reacting only with samples from cervical cancer (CxCa) patients. Large-scale validation experiments using high-throughput suspension bead array serology confirmed their significance as markers for either general Ct infection or CxCa, supporting an association of Ct infection with CxCa. In conclusion, we introduce a method for generation of fast and efficient proteome immunoassays which can be easily adapted for other microorganisms in all areas of infection research.

BACKGROUND Physical exercise has been shown to be an effective therapy for non-specific low back pain. The study investigated if swimming exercise is a means to reduce the spinal sensitization in an animal model of non-specific low back pain. METHODS In deeply anesthetized rats, dorsal horn neurons were recorded in spinal segment L2. To induce sensitization of dorsal horn neurons, two injections of nerve growth factor were made into the lumbar multifidus muscle at an interval of 5 days. Swimming exercise for 30 min was performed on the 5 days between both NGF injections. A control group received the NGF injections without exercise treatment. RESULTS Swimming exercise caused a significant decrease in the NGF-induced hyperexcitability of dorsal horn neurons. Compared to control, the proportion of neurons with input from deep somatic tissues and of convergent neurons with input from at least two types of different tissues decreased significantly (50% vs. 25% and 37% vs. 15%; both p

// Tamara Garcia-Salum 1, 2 , Andrea Villablanca 1, 2 , Franziska Matthaus 3 , Andres Tittarelli 1, 2 , Mauricio Baeza 4 , Cristian Pereda 1, 2 , M. Alejandra Gleisner 1, 2 , Fermin E. Gonzalez 2, 5 , Mercedes N. Lopez 1, 2 , Jorg D. Hoheisel 6 , Johannes Norgauer 7 , Peter J. Gebicke-Haerter 1, 8 and Flavio Salazar-Onfray 1, 2 1 Disciplinary Program of Immunology, Institute of Biomedical Sciences, Faculty of Medicine, Universidad de Chile, 8380453 Santiago, Chile 2 Millennium Institute on Immunology and Immunotherapy, Faculty of Medicine, Universidad de Chile, 8380453 Santiago, Chile 3 Faculty of Biological Sciences and FIAS, University of Frankfurt, Ruth-Moufang-Strase 1, 60438 Frankfurt am Main, Germany 4 Laboratory of Periodontal Biology, Faculty of Dentistry, Universidad de Chile, 8380492 Santiago, Chile 5 Laboratory of Experimental Immunology and Cancer, Faculty of Dentistry, Universidad de Chile, 8380492 Santiago, Chile 6 Functional Genome Analysis, German Cancer Research Centre (DKFZ), Im Neuenheimer Feld 580, 69120 Heidelberg, Germany 7 Department of Dermatology, Jena University Hospital D-07743 Jena, Germany 8 Institute of Psychopharmacology, Central Institute of Mental Health, University of Heidelberg, J5, 68159 Mannheim, Germany Correspondence to: Flavio Salazar-Onfray, email: fsalazar@u.uchile.cl Keywords: molecular signatures; immunotherapy; melanoma; CXCR4; CD32 Received: July 28, 2017 Accepted: February 26, 2018 Published: March 30, 2018 ABSTRACT Purpose: We previously showed that autologous dendritic cells (DCs) loaded with an allogeneic heat shock (HS)-conditioned melanoma cell-derived lysate, called TRIMEL, induce T-cell-mediated immune responses in stage IV melanoma patients. Importantly, a positive delayed-type hypersensitivity (DTH) reaction against TRIMEL after vaccination, correlated with patients prolonged survival. Furthermore, we observed that DTH reaction was associated with a differential response pattern reflected in the presence of distinct cell subpopulations in peripheral blood. Detected variations in patient responses encouraged molecular studies aimed to identify gene expression profiles induced after vaccination in treated patients, allowing the identification of new molecular predictive markers. Methods: Gene expression patterns were analyzed by microarrays during vaccination, and some of them confirmed by quantitative real-time reverse transcriptase PCR (qRT-PCR) in the total leukocyte population of a representative group of responder and non-responder patients. New candidates for biomarkers with predictive value were identified using bioinformatics, molecular analysis, and flow cytometry. Results: Seventeen genes overexpressed in responder patients after vaccination respect to non-responders were identified after a mathematical analysis, from which ten were linked to immune responses and five related to cell cycle control and signal transduction. In immunological responder patients, increased protein levels of the chemokine receptor CXCR4 and the Fc-receptor CD32 were observed on cell membranes of CD8+ T and B cells and the monocyte population, respectively, confirming gene expression results. Conclusions: Our study contributes to finding new molecular markers associated with clinical outcome and better understanding of clinically relevant immunological responses induced by anti-tumor DC-vaccines.

Pancreatic ductal adenocarcinoma (PDAC) is the most common type of pancreatic cancer, affecting 85% of patients. It is the fourth most frequent cause of cancer-related death and bound to become sec...

Background: Large-scale international collaboration is essential to decipher relevant information in the context of omics-scale interrogations in cancer research. This is even more important for rare and fatal diseases like pancreas cancer (PC). Methods: The COST Action BM1204 is a unique platform to facilitate the collaboration of a broad range of European and international PC multidisciplinary research groups in order to: (1) integrate knowledge and experience in a multidisciplinary way ‘from cell to society', (2) promote the application of uniform study tools and protocols, (3) foster their optimal use by early-stage researchers, (4) enhance the mobility and training of researchers, and (5) disseminate the results produced to the broader society. Results: This Action will develop novel interdisciplinary tools for collaborative research to improve our understanding of PC and its prevention, diagnosis and treatment. It also aims to answer questions related to the etiology, early detection, evidence-based and personalized treatment, and health management for PC. Furthermore, the Action will contribute to new insights into PC personalized medicine and beyond as well as to the understanding of complex and rare diseases taking PC as a best practice example. The Action aims at attracting young scholars across a range of disciplines in collaboration with more experienced researchers and enhancing active European participation in the international scenario of PC research. Conclusion: The ultimate aim is to foster PC research in Europe and to coordinate this effort with other international initiatives to reduce disease mortality.