[New guidelines for the diagnosis of irreversible loss of brain function : Concept and limitations, organizational demands, and implementation]. / Neue Richtlinien zur Diagnostik des irreversiblen Hirnfunktionsausfalls : Konzept und Limitationen, organisatorische Vorgaben und Ausführung.

In 2015, the German Medical Association (Bundesärztekammer) issued new guidelines on the diagnosis of the "irreversible loss of brain function" (ILBF). ILBF replaced the colloquial term "brain death" in order to leave the notion that concepts of death might vary such as "cardiac death" or "apparent death" and stress the objective medical-scientific matter. The German Transplantation Law describes ILBF as "the final, irreversible loss of all function of the cerebrum, cerebellum, and brainstem." The new guidelines are to be followed closely. They demand higher qualifications of physicians involved in the diagnosis of ILBF and emphasize at the same time the interdisciplinary approach and the mandatory involvement of at least one specialist in the neurological field. Several technical methods were added as additional tools to support the ILBF diagnosis such as CT-angiography and duplex ultrasound of brain and neck vessels. The new guidelines thereby raise the impact of demonstrating complete cerebral circulatory arrest but leave other options to prove irreversibility. Many procedures, such as the apnea test, were specified in more detail. This article summarizes the new features of the new guideline with a practical overview on who must be involved in the diagnosis of ILBF, how often, how the diagnosis is achieved stepwise from stage I to III and how it is secured as well as what technical methods may be involved at what stage of the procedure.

[PROtocol-based MObilizaTION on intensive care units : Design of a cluster randomized pilot study]. / PROtokollbasierte MObilisierung auf IntensivstaTIONen : Design einer clusterrandomisierten Pilotstudie.

BACKGROUND: Despite convincing evidence for early mobilization of patients on intensive care units (ICU), implementation in practice is limited. Protocols for early mobilization, including in- and exclusion criteria, assessments, safety criteria, and step schemes may increase the rate of implementation and mobilization. HYPOTHESIS: Patients (population) on ICUs with a protocol for early mobilization (intervention), compared to patients on ICUs without protocol (control), will be more frequently mobilized (outcome). METHODS: A multicenter, stepped-wedge, cluster-randomized pilot study is presented. Five ICUs will receive an adapted, interprofessional protocol for early mobilization in randomized order. Before and after implementation, mobilization of ICU patients will be evaluated by randomized monthly one-day point prevalence surveys. Primary outcome is the percentage of patients mobilized out of bed, operationalized as a score of ≥3 on the ICU Mobility Scale. Secondary outcome parameters will be presence and/or length of mechanical ventilation, delirium, stay on ICU and in hospital, barriers to early mobilization, adverse events, and process parameters as identified barriers, used strategies, and adaptions to local conditions. EXPECTED RESULTS: Exploratory evaluation of study feasibility and estimation of effect sizes as the basis for a future explanatory study.

Metabolic plasticity in CLL: adaptation to the hypoxic niche.

Metabolic transformation in cancer is increasingly well understood. However, little is known about the metabolic responses of cancer cells that permit their survival in different microenvironments. We have used a nuclear magnetic resonance based approach to monitor metabolism in living primary chronic lymphoid leukemia (CLL) cells and to interrogate their real-time metabolic responses to hypoxia. Our studies demonstrate considerable metabolic plasticity in CLL cells. Despite being in oxygenated blood, circulating CLL cells are primed for hypoxia as measured by constitutively low level hypoxia-inducible factor (HIF-1α) activity and modest lactate production from glycolysis. Upon entry to hypoxia we observed rapid upregulation of metabolic rates. CLL cells that had adapted to hypoxia returned to the 'primed' state when re-oxygenated and again showed the same adaptive response upon secondary exposure to hypoxia. We also observed HIF-1α independent differential utilization of pyruvate in oxygenated and hypoxic conditions. When oxygenated, CLL cells released pyruvate, but in hypoxia imported pyruvate to protect against hypoxia-associated oxidative stress. Finally, we identified a marked association of slower resting glucose and glutamine consumption, and lower alanine and lactate production with Binet A0 stage samples indicating that CLL may be divided into tumors with higher and lower metabolic states that reflect disease stage.

High prevalence of NMDA receptor IgA/IgM antibodies in different dementia types.

OBJECTIVE: To retrospectively determine the frequency of N-Methyl-D-Aspartate (NMDA) receptor (NMDAR) autoantibodies in patients with different forms of dementia. METHODS: Clinical characterization of 660 patients with dementia, neurodegenerative disease without dementia, other neurological disorders and age-matched healthy controls combined with retrospective analysis of serum or cerebrospinal fluid (CSF) for the presence of NMDAR antibodies. Antibody binding to receptor mutants and the effect of immunotherapy were determined in a subgroup of patients. RESULTS: Serum NMDAR antibodies of IgM, IgA, or IgG subtypes were detected in 16.1% of 286 dementia patients (9.5% IgM, 4.9% IgA, and 1.7% IgG) and in 2.8% of 217 cognitively healthy controls (1.9% IgM and 0.9% IgA). Antibodies were rarely found in CSF. The highest prevalence of serum antibodies was detected in patients with "unclassified dementia" followed by progressive supranuclear palsy, corticobasal syndrome, Parkinson's disease-related dementia, and primary progressive aphasia. Among the unclassified dementia group, 60% of 20 patients had NMDAR antibodies, accompanied by higher frequency of CSF abnormalities, and subacute or fluctuating disease progression. Immunotherapy in selected prospective cases resulted in clinical stabilization, loss of antibodies, and improvement of functional imaging parameters. Epitope mapping showed varied determinants in patients with NMDAR IgA-associated cognitive decline. INTERPRETATION: Serum IgA/IgM NMDAR antibodies occur in a significant number of patients with dementia. Whether these antibodies result from or contribute to the neurodegenerative disorder remains unknown, but our findings reveal a subgroup of patients with high antibody levels who can potentially benefit from immunotherapy.

Selective AKR1C3 inhibitors do not recapitulate the anti-leukaemic activities of the pan-AKR1C inhibitor medroxyprogesterone acetate.

BACKGROUND: We and others have identified the aldo-keto reductase AKR1C3 as a potential drug target in prostate cancer, breast cancer and leukaemia. As a consequence, significant effort is being invested in the development of AKR1C3-selective inhibitors. METHODS: We report the screening of an in-house drug library to identify known drugs that selectively inhibit AKR1C3 over the closely related isoforms AKR1C1, 1C2 and 1C4. This screen initially identified tetracycline as a potential AKR1C3-selective inhibitor. However, mass spectrometry and nuclear magnetic resonance studies identified that the active agent was a novel breakdown product (4-methyl(de-dimethylamine)-tetracycline (4-MDDT)). RESULTS: We demonstrate that, although 4-MDDT enters AML cells and inhibits their AKR1C3 activity, it does not recapitulate the anti-leukaemic actions of the pan-AKR1C inhibitor medroxyprogesterone acetate (MPA). Screens of the NCI diversity set and an independently curated small-molecule library identified several additional AKR1C3-selective inhibitors, none of which had the expected anti-leukaemic activity. However, a pan AKR1C, also identified in the NCI diversity set faithfully recapitulated the actions of MPA. CONCLUSIONS: In summary, we have identified a novel tetracycline-derived product that provides an excellent lead structure with proven drug-like qualities for the development of AKR1C3 inhibitors. However, our findings suggest that, at least in leukaemia, selective inhibition of AKR1C3 is insufficient to elicit an anticancer effect and that multiple AKR1C inhibition may be required.

Evaluation of stable isotope fingerprinting techniques for the assessment of the predominant methanogenic pathways in anaerobic digesters.

Laboratory biogas reactors were operated under various conditions using maize silage, chicken manure, or distillers grains as substrate. In addition to the standard process parameters, the hydrogen and carbon stable isotopic composition of biogas was analyzed to estimate the predominant methanogenic pathways as a potential process control tool. The isotopic fingerprinting technique was evaluated by parallel analysis of mcrA genes and their transcripts to study the diversity and activity of methanogens. The dominant hydrogenotrophs were Methanomicrobiales, while aceticlastic methanogens were represented by Methanosaeta and Methanosarcina at low and high organic loading rates, respectively. Major changes in the relative abundance of mcrA transcripts were observed compared to the results obtained from DNA level. In agreement with the molecular results, the isotope data suggested the predominance of the hydrogenotrophic pathway in one reactor fed with chicken manure, while both pathways were important in the other reactors. Short-term changes in the isotopic composition were followed, and a significant change in isotope values was observed after feeding a reactor digesting maize silage. This ability of stable isotope fingerprinting to follow short-term activity changes shows potential for indicating process failures and makes it a promising technology for process control.

PT symmetry and spontaneous symmetry breaking in a microwave billiard.

We demonstrate the presence of parity-time (PT) symmetry for the non-Hermitian two-state Hamiltonian of a dissipative microwave billiard in the vicinity of an exceptional point (EP). The shape of the billiard depends on two parameters. The Hamiltonian is determined from the measured resonance spectrum on a fine grid in the parameter plane. After applying a purely imaginary diagonal shift to the Hamiltonian, its eigenvalues are either real or complex conjugate on a curve, which passes through the EP. An appropriate basis choice reveals its PT symmetry. Spontaneous symmetry breaking occurs at the EP.

Diagnostic value of confocal endomicroscopy in celiac disease.

BACKGROUND: Improved endoscopic screening with targeted biopsies might enhance diagnostic yield in celiac disease. Confocal endomicroscopy (CEM) allows high-resolution in vivo histological analysis. We compared the endomicroscopic findings during ongoing endoscopy with the histological findings graded according to the Marsh classification. METHODS: Twenty-four patients with celiac disease and six patients with celiac disease that was refractory on a gluten-free diet were examined using CEM. The duodenal mucosa was evaluated by CEM and by conventional histological analysis in respect of villous atrophy, crypt hyperplasia, and increased numbers of intraepithelial lymphocytes (IELs > 40 / 100 enterocytes). The CEM results were assessed as to sensitivity, specificity, and interobserver variability. A Marsh classification score determined by CEM was compared to that obtained by histology. Thirty patients undergoing routine upper gastrointestinal endoscopy were used as controls. RESULTS: Conventional histology showed villous atrophy and crypt hyperplasia in 23 and increased numbers of IELs in 27 of the 30 patients with celiac disease. With CEM, villous atrophy, crypt hyperplasia, and increased IELs were respectively identified in 17, 12, and 22 of the 30 patients. The agreement of the findings on CEM with those of conventional histology was good in relation to villous atrophy (sensitivity 74 %) and increased numbers of IELs (sensitivity 81 %), but inadequate in relation to crypt hyperplasia (sensitivity 52 %). The kappa values for determination of interobserver variability were 0.90 for villous atrophy, 1.00 for crypt hyperplasia, and 0.84 for IEL detection. In the 30 control patients, normal duodenal architecture was found by both histology and endomicroscopy, indicating an overall specificity of 100 %. CONCLUSION: The assessment of duodenal histology by CEM in patients with celiac disease is sensitive and specific in determining increased numbers of IELs and villous atrophy, but insufficient in respect of crypt hyperplasia. For routine use of CEM in patients with celiac disease, the technique would need to be improved.

Fast targeted multidimensional NMR metabolomics of colorectal cancer.

The study of small molecules in body fluids has become an important tool to monitor the state of biological organisms. Applications range from model studies using cell lines to applications where human body fluids are used to monitor disease states or drug responses. NMR spectroscopy has been an important tool for metabolomics although severe overlap of signals has limited the number of compounds, which can be unambiguously identified and quantified. Therefore, deconvolution of NMR spectra is one of the greatest challenges for NMR-based metabolomics. This has commonly been achieved by using multidimensional spectra that have the disadvantage of requiring significantly longer acquisition times. Recently, a number of methods have been described to record NMR spectra much faster. Here, we explore the use of Hadamard-encoded TOCSY spectra to simultaneously select multiple lines from crowded NMR spectra of blood serum samples to acquire pseudo-two-dimensional spectra in minutes which would otherwise require many hours. The potential of this approach is demonstrated for the detection of a signature for colorectal cancer from human blood samples.

In vivo diagnosis of acute intestinal graft-versus-host disease by confocal endomicroscopy.

BACKGROUND AND STUDY AIMS: Conventional histology with hematoxylin and eosin (H&E) staining is the accepted standard for diagnosing acute intestinal graft-versus-host disease (GvHD). Confocal endomicroscopy (CEM) is a noninvasive method that allows in vivo histology to be performed during endoscopy. The aim of this study was to evaluate CEM for the diagnosis of acute intestinal GvHD. PATIENTS AND METHODS: This observational pilot study conducted between September 2006 and August 2008 included patients with acute diarrhea after stem cell transplantation, infectious diarrhea, or active ulcerative colitis. CEM (EC-3870CIFK, Pentax, Tokyo, Japan) was performed after intravenous injection of fluorescein 10% and topical application of acriflavine 0.05%. RESULTS: A total of 35 patients with acute diarrhea after stem cell transplantation were examined. In 16 patients, CEM and histology showed no evidence of GvHD. In 14/19 patients with histologically confirmed GvHD, the diagnosis could already be established by CEM during ongoing endoscopy. In GvHD grade IV, near complete destruction of the colonic crypts ("flat mucosa") was visible. Control patients with infectious colitis (N = 15) or ulcerative colitis (N = 15) displayed inflammatory changes but no evidence of GvHD. Altogether, sensitivity of CEM was 74% and specificity was 100 %. CONCLUSIONS: CEM improves rapid diagnosis of acute intestinal GvHD with high accuracy while performing endoscopy. Platelet transfusions and unnecessary biopsy acquisition can be avoided once acute intestinal GvHD has been diagnosed in vivo.

[Nodular regenerative hyperplasia as a side effect of azathioprine in a patient with Crohn's disease]. / Noduläre regenerative Hyperplasie als Nebenwirkung von Azathioprin bei Morbus Crohn.

HISTORY: A 48-year-old patient with Crohn's disease was admitted to our hospital with fatigue, icterus, hepatosplenomegaly and ascites. INVESTIGARTIOS: The whole blood count revealed a pancytopenia, hyperbilirubinemia and slightly elevated transaminases. Examination of the liver histology showed areas of enlarged hyperplastic hepatocytes adjacent to areas of atrophic hepatocytes and dilated sinusoids. DIAGNOSIS, TREATMENT AND COURSE: Pancytopenia was most likely azathioprine-related. Analysis of the liver histology was highly suggestive of an azathioprine-related, nodular regenerative hyperplasia (NRH). After discontinuation of azathioprine the patient's condition improved substantially. CONCLUSIONS: NRH is a rare but potentially serious complication of azathioprine therapy. Other causes include various rheumatological, vascular and myeloproliferative diseases. When azathioprine is prescribed it must be borne in mind that it can cause NRH as a potential adverse effect, and liver enzymes should be measured at regular follow-up examinations.

NMR in the SPINE Structural Proteomics project.

This paper describes the developments, role and contributions of the NMR spectroscopy groups in the Structural Proteomics In Europe (SPINE) consortium. Focusing on the development of high-throughput (HTP) pipelines for NMR structure determinations of proteins, all aspects from sample preparation, data acquisition, data processing, data analysis to structure determination have been improved with respect to sensitivity, automation, speed, robustness and validation. Specific highlights are protonless (13)C-direct detection methods and inferential structure determinations (ISD). In addition to technological improvements, these methods have been applied to deliver over 60 NMR structures of proteins, among which are five that failed to crystallize. The inclusion of NMR spectroscopy in structural proteomics pipelines improves the success rate for protein structure determinations.

NMR structure of the N-SH2 of the p85 subunit of phosphoinositide 3-kinase complexed to a doubly phosphorylated peptide reveals a second phosphotyrosine binding site.

The N-terminal src homology 2 (SH2) domain of the p85 subunit of phosphoinositide 3-kinase (PI3K) has a higher affinity for a peptide with two phosphotyrosines than for the same peptide with only one. This unexpected result was not observed for the C-terminal SH2 from the same protein. NMR structural analysis has been used to understand the behavior of the N-SH2. The structure of the free SH2 domain has been compared to that of the SH2 complexed with a doubly phosphorylated peptide derived from polyomavirus middle T antigen (MT). The structure of the free SH2 domain shows some differences from previous NMR and X-ray structures. In the N-SH2 complexed with a doubly phosphorylated peptide, a second site for phosphotyrosine interaction has been identified. Further, line shapes of NMR signals showed that the SH2 protein-ligand complex is subject to temperature-dependent conformational mobility. Conformational mobility is also supported by the spectra of the ligand peptide. A binding model which accounts for these results is developed.

Serum ferritin iron in iron overload and liver damage: correlation to body iron stores and diagnostic relevance.

The iron content of serum ferritin has been determined in groups of patients with normal or increased iron stores by using a technique of ferritin immunoprecipitation followed by iron quantitation with atomic absorption spectroscopy. The results were correlated to individual liver iron concentrations, measured non-invasively by superconducting quantum interference device (SQUID) biomagnetometry. A close correlation between serum concentrations of ferritin protein and ferritin iron was found (r = 0.92) in all groups of patients. However, the correlation between ferritin iron concentration and individual liver iron concentration was poor in patients with hemochromatosis (r = 0.63) and patients with beta-thalassemia major (r = 0.57). The degree of ferritin iron saturation was about 5% in iron-loaded patients, which contrasts with results in two recent studies but confirms older observations. In patients with liver cell damage, the ferritin iron saturation in serum was significantly higher than that found in groups with iron overload disease, probably indicating the release of intracellular iron-rich ferritin into the blood. The monitoring of patients undergoing bone marrow transplantation indicated that the release of iron-rich and iron-poor ferritin occurred during phases of hepatocellular damage and inflammation, respectively. We find the benefits of serum ferritin iron measurement to be marginal in patients with iron overload disease.

Fibrogenesis and fibrolysis in collagenous colitis. Patterns of procollagen types I and IV, matrix-metalloproteinase-1 and -13, and TIMP-1 gene expression.

Collagenous colitis is characterized by the deposition of a superficial subepithelial collagenous layer, the pathogenesis of which is unknown. Because the excess matrix deposition is potentially reversible, a labile imbalance between fibrogenesis and fibrolysis may be suspected. Expression of procollagen alpha1(I) and alpha1(IV), matrix-metalloproteinase (MMP)-1 and -13, and tissue inhibitor of metalloproteinase (TIMP)-1 genes was semiquantitated by in situ hybridization on serial biopsies of 12 patients with collagenous colitis and compared to controls. Collagen types I, III, IV, and VI, tenascin, undulin/collagen XIV, and alpha-actin were localized by immunohistology. The superficial collagen layer stained strongly for collagen types I, III, and VI, and particularly for tenascin, but not for undulin. Elevated procollagen alpha1(I), procollagen alpha1(IV), and TIMP-1 transcript levels were found in alpha-actin-positive cells with linear distribution underneath the superficial collagenous layer, whereas MMP-1 RNA expression was variable and restricted to cell clusters. MMP-13 expression was undetectable. The patterns of procollagen alpha1(I)- and alpha1(IV)-specific labeling, combined with an intense tenascin- but absent undulin-specific staining, indicate deposition of an immature interstitial matrix that may be susceptible to degradation. The restricted MMP-1 RNA expression, counteracted by increased TIMP-1 expression, suggests locally impaired fibrolysis as a relevant factor in the pathogenesis of collagenous colitis.

Suppression of T cell-mediated injury in human gut by interleukin 10: role of matrix metalloproteinases.

BACKGROUND & AIMS: Activation of lamina propria T cells with pokeweed mitogen in human fetal gut explant cultures results in severe mucosal injury. In the same system, Staphylococcus aureus enterotoxin B produces villus atrophy and crypt cell hyperplasia. The aim of this study was to examine the ability of interleukin (IL)-10 to modify these changes. METHODS: Mucosal pathology and lamina propria glycosaminoglycans were assessed histologically, and CD3(+), CD25(+) and alpha-actin smooth muscle-positive cells were determined by immunohistochemistry. Reverse plaque assays and quantitative reverse-transcriptase polymerase chain reaction were used to analyze the cytokine response. Matrix metalloproteinase production was analyzed by Western blotting, in situ hybridization, and zymography. RESULTS: Both experimental enteropathies produced mucosal damage, although injury was greater after pokeweed mitogen activation than S. aureus enterotoxin B. In both cases, the increase in cytokine-secreting cells and transcripts observed after T-cell activation was inhibited by IL-10. IL-10 also inhibited the increase in collagenase and stromelysin-1 in the explant culture supernatants and the loss of glycosaminoglycans. IL-10 decreased metalloproteinase production in control explants and increased matrix. In mesenchymal cells, IL-10 had a minor effect on metalloproteinase production. CONCLUSIONS: IL-10 down-regulates mucosal T-cell activation, metalloproteinase production, and loss of extracellular matrix and prevents mucosal damage in the gut.

NMR analysis of interactions of a phosphatidylinositol 3'-kinase SH2 domain with phosphotyrosine peptides reveals interdependence of major binding sites.

The interactions of the N-terminal src homology (SH2) domain (N-SH2) of the 85 kDa subunit of phosphatidylinositol 3'-kinase (PI-3K) with phosphotyrosine (ptyr) and a series of ptyr-containing peptides have been examined by NMR spectroscopy. HSQC (heteronuclear single-quantum coherence) NMR spectra of 15N-labeled SH2 were used to evaluate its interactions with ptyr-containing ligands. The ability of ligands to cause chemical shift changes was compared to their potency as competitors in in vitro binding experiments using polyoma virus middle T antigen (MT). The results suggest the interdependence of SH2 binding elements. Chemical shifts of residues involved in the ptyr binding were altered by variations of the sequence of the bound peptide, suggesting that the ptyr fit can be adjusted by the peptide sequence. Perturbations of chemical shifts of residues coordinating the methionine three residues C-terminal to the ptyr (the +3 residue) were affected by substitution in the binding peptide at +1 and vice versa. Such results show synergistic interplay between regions of the SH2 binding residues C-terminal to the ptyr.