CRP/Neopterin Ratio and Neuropsychiatric Symptoms in Patients with Different Forms of Pneumonia: Results of a Pilot Study.

BACKGROUND: Pneumonia is one of the most common infectious diseases, mostly caused by viruses or bacteria. In response to bacteria or viruses which are different but which also are partly overlapping, innate and adaptive immune responses are induced, which can be quantified using the determination of specific biomarkers. Among these, C-reactive protein (CRP) has been established as a marker of innate immune function, whereas Neopterin, which is mainly produced upon stimulation with interferon-gamma, reflects cellular immune activation. AIM: We investigated inflammation markers in patients with microbiologically confirmed viral or bacterial pneumonia, and studied the potential of CRP, Neopterin, and the CRP/Neopterin ratio to distinguish between viral and bacterial pathogenesis. Furthermore, we examined, how often neuropsychiatric symptoms occur in patients suffering from different kinds of pneumonia. PATIENTS AND METHOD: A total of 194 patients diagnosed with either coronavirus disease 2019 (COVID-19) (n = 63), bacterial pneumonia (n = 58), Influenza infection (n = 10), Influenza and a bacterial superinfection (n = 9), and COVID-19 patients with a bacterial superinfection (n = 54) were included in our pilot study. Clinical as well as laboratory parameters were determined shortly after admission. RESULTS: We found significantly higher CRP/Neopterin ratios in patients with bacterial pneumonia (median: 0.34) and lower CRP/Neopterin ratios in patients hospitalized with COVID-19 infection (median: 0.03; p < 0.001). Both in men and in women, the CRP/Neopterin ratio was able to distinguish between viral and bacterial pathogens, but also was able to detect bacterial super-infection (BSI) in subjects with initial viral pneumonia (p < 0.001). Patients with BSI presented with significantly lower CRP/Neopterin ratios (median 0.08) than patients with bacterial infection only (median 0.34; p < 0.001). Interestingly, COVID-19 patients had a decreased physical functioning (as reflected in the ECOG score) and a higher frequency of fatigue (84.1%) and neurological symptoms (54.8%) than patients with pneumonia, due to other underlying pathogens. Patients that reported fatigue during viral and bacterial pneumonia presented with lower CRP concentrations than patients without it. CONCLUSIONS: The CRP/Neopterin ratio is useful to differentiate between viral and bacterial pathogenesis. The occurrence of neuropsychiatric symptoms in pneumonia appears to depend on the kind of pathogen causing the infection. Lower CRP concentrations at admission appear to be related to fatigue during acute viral and bacterial infection.

Effects of an inpatient rehabilitation programme on functional capacity, quality of life and psychological distress in patients with post covid-19 condition: an observational study.

OBJECTIVES: To examine changes in functional capacity, health-related quality of life and psychological distress in patients with post-COVID-19 condition following a multidisciplinary rehabilitation programme. In addition, to explore whether additional respiratory muscle training for more impaired patients might support their recovery process. DESIGN: Retrospective observational cohort study. PATIENTS: A total of 779 patients with post-COVID-19 condition (47.9% female, mean age 56.6 years). METHODS: Measures assessed were: 6-minute walk test (6MWT), 5-level EQ-5D (EQ-5D-5L) including EQ Visual Analogue Scale (EQ-VAS) and Patient Health Questionnaire-4 (PHQ-4). Data were provided pre- and post-rehabilitation from 2 cohorts: (i) patients participating in a regular multidisciplinary rehabilitation programme; and (ii) patients receiving additional respiratory muscle training due to an initially greater level of impairment. Dependent t-tests and general linear mixed models were used for data analysis. RESULTS: A series of dependent t-tests revealed mean overall improvement for both groups in 6-minute walk test distance (6MWD), EQ-5D-5L index, EQ-VAS and PHQ-4 following the rehabilitation programme. General linear mixed models showed significant interaction effects between groups and time for the EQ-5D-5L index and 6MWD. CONCLUSION: A multidisciplinary rehabilitation programme appears to have a beneficial impact on the recovery process of patients with post-COVID-19 condition.

Identification of Hetero-aggregates in Antibody Co-formulations by Multi-dimensional Liquid Chromatography Coupled to Mass Spectrometry.

Antibody combination therapies have become viable therapeutic treatment options for certain severe diseases such as cancer. The co-formulation production approach is intrinsically associated with more complex drug product variant profiles and creates more challenges for analytical control of drug product quality. In addition to various individual quality attributes, those arising from the interactions between the antibodies also potentially emerge through co-formulation. In this study, we describe the development of a widely applicable multi-dimensional liquid chromatography coupled to tandem mass spectrometry method for antibody homo- versus hetero-aggregate characterization. The co-formulation of trastuzumab and pertuzumab was used, a challenging model system, comprising two monoclonal antibodies with very similar physicochemical properties. The data presented demonstrate the high stability of the co-formulation, where only minor aggregate formation is observed upon product storage and accelerated temperature or light-stress conditions. The results also show that the homo- and hetero-aggregates, formed in low and comparable proportions, are only marginally impacted by the formulation and product storage conditions. No preferential formation of hetero-aggregates, in comparison to the already existing pertuzumab and trastuzumab homo-aggregates, was observed.

Comprehensive Multidimensional Liquid Chromatography-Mass Spectrometry for the Characterization of Charge Variants of a Bispecific Antibody.

Identification and further characterization of antibody charge variants is a crucial step during biopharmaceutical drug development, particularly with regard to the increasing complexity of novel antibody formats. As a standard analytical approach, manual offline fractionation of charge variants by cation-exchange chromatography followed by comprehensive analytical testing is applied. These conventional workflows are time-consuming and labor-intensive and overall reach their limits in terms of chromatographic separation of enhanced structural heterogeneities raised from new antibody formats. For these reasons, we aimed to develop an alternative online characterization strategy for charge variant characterization of a therapeutic bispecific antibody by online mD-LC-MS at middle-up (2D-LC-MS) and bottom-up (4D-LC-MS) level. Using the implemented online mD-LC-MS approach, all medium- and even low-abundant product variants previously identified by offline fraction experiments and liquid chromatography mass spectrometry could be monitored. The herein reported automated online mD-LC-MS methodology therefore represents a complementary and in part alternative approach for analytical method validation including multiattribute monitoring (MAM) strategies by mass spectrometry, offering various benefits including increased throughput and reduced sample handling and combined protein information at intact protein and peptide level.

Phytohormones and volatile organic compounds, like geosmin, in the ectomycorrhiza of Tricholoma vaccinum and Norway spruce (Picea abies).

The ectomycorrhizospheric habitat contains a diverse pool of organisms, including the host plant, mycorrhizal fungi, and other rhizospheric microorganisms. Different signaling molecules may influence the ectomycorrhizal symbiosis. Here, we investigated the potential of the basidiomycete Tricholoma vaccinum to produce communication molecules for the interaction with its coniferous host, Norway spruce (Picea abies). We focused on the production of volatile organic compounds and phytohormones in axenic T. vaccinum cultures, identified the potential biosynthesis genes, and investigated their expression by RNA-Seq analyses. T. vaccinum released volatiles not usually associated with fungi, like limonene and ß-barbatene, and geosmin. Using stable isotope labeling, the biosynthesis of geosmin was elucidated. The geosmin biosynthesis gene ges1 of T. vaccinum was identified, and up-regulation was scored during mycorrhiza, while a different regulation was seen with mycorrhizosphere bacteria. The fungus also released the volatile phytohormone ethylene and excreted salicylic and abscisic acid as well as jasmonates into the medium. The tree excreted the auxin, indole-3-acetic acid, and its biosynthesis intermediate, indole-3-acetamide, as well as salicylic acid with its root exudates. These compounds could be shown for the first time in exudates as well as in soil of a natural ectomycorrhizospheric habitat. The effects of phytohormones present in the mycorrhizosphere on hyphal branching of T. vaccinum were assessed. Salicylic and abscisic acid changed hyphal branching in a concentration-dependent manner. Since extensive branching is important for mycorrhiza establishment, a well-balanced level of mycorrhizospheric phytohormones is necessary. The regulation thus can be expected to contribute to an interkingdom language.

Pseudodiborenes: hydride-bridged diboranes(5) as two-electron reductants of chalcogens.

The reactivity of two nucleophilic neutral sp2-sp3 diboranes towards chalcogens is presented herein. Both diboranes(5) serve as two-electron reductants, incorporating oxygen, sulfur and selenium atoms. Treatment with chalcogen sources results in the oxidative insertion of one chalcogen atom into the B-B single bond, while depending on the negative inductive effect of the chalcogen and the boron bound aryl substituent further N-heterocyclic carbene (NHC) ring expansion and hydride migration can occur. These reactions provide access to unprecedented six- or seven-membered heterocycles and help to illuminate the pseudo-multiple bonding character of hydrogen-bridged B-B single bonds.

The Ectomycorrhizospheric Habitat of Norway Spruce and Tricholoma vaccinum: Promotion of Plant Growth and Fitness by a Rich Microorganismic Community.

The contribution of the mycorrhizospheric microbes in a stand of ectomycorrhizal Norway spruce (Picea abies) featuring mycorrhiza with the basidiomycete Tricholoma vaccinum was addressed by microbiome analysis and in vitro reconstruction of microbial as well as plant-microbe interactions. The protective role of the mycorrhizal fungus with respect to pathogen attack could be validated against Botrytis cinerea and Heterobasidion annosum in co-cultures revealing reduced pathogen growth, higher survival rate of the spruce trees and reduced symptoms on needles upon symbiosis with T. vaccinum. The community structure was shown to yield a high diversity in ECM forming basidiomycetes of Thelephorales and Agaricales associated with a rich bacterial diversity dominated by Rhizobiales with the most abundant Nitrobacter winogradski (3.9%). Isolated bacteria were then used to address plant growth promoting abilities, which included production of the phytohormone indole-3-acetic acid (performed by 74% of the bacterial isolates), siderophores (22%), and phosphate mobilization (23%). Among the isolates, mycorrhiza helper bacteria (MHB) were identified, with Bacillus cereus MRZ-1 inducing hyperbranching in T. vaccinum, supporting tree germination, shoot elongation, and root formation as well as higher mycorrhization rates. Thus, a huge pool of potential MHB and fungal community with widely distributed auxin-production potential extended the ability of T. vaccinum to form ectomycorrhiza. The forest community profited from the mycorrhizal fungus T. vaccinum, with spruce survival enhanced by 33% in microcosms using soil from the native habitat. A higher fungal abundance and diversity in cases where the tree had died during the experiment, showing that decomposition of plant litter from a dead tree supported a different community. T. vaccinum thus actively structured the community of microorganisms in its habitat.

Assessment of susceptible chemical modification sites of trastuzumab and endogenous human immunoglobulins at physiological conditions.

The quality control testing of chemical degradations in the bio-pharmaceutical industry is currently under controversial debate. Here we have systematically applied in vitro and in vivo stress conditions to investigate the influence of protein degradation on structure-function. Extensive purification and characterization enabled identification and functional assessment of the physiological degradation of chemical modification sites in the variable complementarity-determining regions (CDRs) and conserved region of trastuzumab. We demonstrate that the degradation of the solvent-accessible residues located in the CDR and the conserved fragment crystallizable region (Fc) occurs faster in vivo (within days) compared to the levels observed for bio-process and real-time storage conditions. These results hence question the rationality of extreme monitoring of low level alterations in such chemical modifications as critical patient safety parameters in product quality control testing, given that these modifications merely mirror the natural/physiological aging process of endogenous antibodies.

MD-2 is a new predictive biomarker in dilated cardiomyopathy and exerts direct effects in isolated cardiomyocytes.

BACKGROUND: Myeloid differentiation factor-2 (MD-2) has been shown to be an important modulator of the innate immune system, but its role in cardiac diseases is unknown. We investigated whether MD-2 plays a role as risk predictor and contributor in dilated cardiomyopathy (DCM). METHODS AND RESULTS: We included 174 patients with reduced left ventricular (LV) ejection fraction (LVEF <45%) due to DCM. Coronary artery disease and severe valvular diseases were excluded in all patients by angiography or echocardiography. Cardiac inflammation, viral infection and MD-2 expression were analyzed from right ventricular endomyocardial biopsies. MD-2 was quantified by ELISA in serum upon first hospital admission. Myocyte contractility and inflammatory response after stimulation with recombinant MD-2 protein were analyzed in isolated rat cardiomyocytes. Median follow-up of the patients was 3.51 years (2.73; 4.48) with 34 deaths. Absolute mortality risk increases in patients displaying a MD-2 serum concentration greater than the median (302 ng/ml) was 23% (P < 0.0001). Age- and sex-adjusted Cox regression analyses demonstrated that mortality risk was highly related to MD-2 concentrations (P < 0.001), but not to age or sex. An increase of 100 ng/ml in the MD-2 level was associated with an absolute mortality risk increase of 50.4%. Receiver operating characteristic (ROC) analyses showed no difference between MD-2 and nterminal-pro brain natriuretic peptide (NT-pro-BNP), while the combination of both MD-2 and NT-pro-BNP resulted in a significantly increased capability of risk prediction when compared to NT-pro-BNP alone (P = 0.014). In-vitro, recombinant MD-2 decreases cell shortening and modulates cytokine activation in isolated cardiomyocytes. CONCLUSION: MD-2 predicts long-term outcome in DCM patients and improves mortality risk prediction capability compared to NT-pro-BNP alone. In addition, MD-2 exerts direct negative inotropic effects on isolated cardiomyocytes in-vitro. Further randomized trials should confirm MD-2 as a diagnostic and therapeutic target.

[Clinical Validation of the Caregiver-Child Socioemotional and Relationship Rating Scale (SIRS) for Child Behavior in a Preschool-Age Sample]. / Klinische Validierung der Beurteilungsskala Caregiver-Child Socioemotional and Relationship Rating Scale (SIRS) für kindliches Interaktionsverhalten im Kleinkind- und Vorschulalter.

Clinical Validation of the Caregiver-Child Socioemotional and Relationship Rating Scale (SIRS) for Child Behavior in a Preschool-Age Sample The description of child behavior in mother-child-interaction is important in early detection and treatment of psychiatric disorders in preschool children. The Caregiver-Child Socioemotional and Relationship Rating Scale (SIRS) may serve this diagnostic purpose. We aim to examine interrater-reliability of SIRS and concurrent, convergent, and discriminant validity to maternal behavior by Play-PAB, and a measure of mother-child-relationsship by Parent-Infant-Global-Assessment-Scale (PIRGAS). Five raters assessed 47 ten-minute video sequences of parent-child-interaction recorded at the Family Day Hospital for Preschool Children with SIRS, Play-PAB, and PIRGAS. We report psychometric properties of SIRS, and present the association with Play-PAB and PIRGAS. SIRS shows a satisfying interrater-reliability for all items. Positive child behavior e. g. the SIRS' "child responsiveness" shows negative correlation to Play-PAB-scales' parental "hostility" and "intrusiveness", but independence of parental "involvement", "positive emotionality", and "discipline". Child and parental behavior show expected associations with the global relationship measure PIRGAS. The assessment of child behavior in parent-child-interaction with SIRS can be quickly learned and reliably applied without extensive training. SIRS shows meaningful relations to parental behavior and a clinical global measure of the caregiver-child-relationship. We recommend SIRS for clinical diagnostics to describe child behavior in mother-child-interaction.

The hypomorphic TERT A1062T variant is associated with increased treatment-related toxicity in acute myeloid leukemia.

Hypomorphic germline variants in TERT, the gene encoding the reverse transcriptase component of the human telomerase complex, occur with a frequency of 3-5% in acute myeloid leukemia. We analyzed the clinical and prognostic impact of the most common TERT A1062T variant in younger patients with acute myeloid leukemia intensively treated within two prospective multicenter trials. Four hundred and twenty patients (age 17-60 years) were analyzed for the TERT A1062T variant by direct sequencing. Fifteen patients (3.6%) carried the TERT A1062T variant. Patients with the TERT A1062T variant had a trend towards less favorable and more intermediate 2/adverse karyotypes/genotypes according to the European Leukemia Net classification. In univariate and multivariate analysis, patients with the TERT A1062T variant had a significantly inferior overall survival compared to wild-type patients (6-year overall survival 20 vs. 41%, p = 0.005). Patients with the TERT A1062T variant showed a high rate of treatment-related mortality: 5/15 (33%) died during induction therapy or in complete remission as compared to 62/405 (15%) of the wild-type patients. In patients with the TERT variant, 14/15 (93%) suffered from non-hematological/non-infectious grade 3/4 adverse events (mostly hepatic and/or mucosal) as compared to 216/405 (53%) wild-type patients (p = 0.006). In multivariate analysis, the TERT A1062T variant was an independent risk factor predicting for adverse events during induction chemotherapy. In conclusion, the TERT A1062T variant is an independent negative prognostic factor in younger patients with acute myeloid leukemia and seems to predispose those patients to treatment-related toxicity.

Hydrophobins in the Life Cycle of the Ectomycorrhizal Basidiomycete Tricholoma vaccinum.

Hydrophobins-secreted small cysteine-rich, amphipathic proteins-foster interactions of fungal hyphae with hydrophobic surfaces, and are involved in the formation of aerial hyphae. Phylogenetic analyses of Tricholoma vaccinum hydrophobins showed a grouping with hydrophobins of other ectomycorrhizal fungi, which might be a result of co-evolution. Further analyses indicate angiosperms as likely host trees for the last common ancestor of the genus Tricholoma. The nine hydrophobin genes in the T. vaccinum genome were investigated to infer their individual roles in different stages of the life cycle, host interaction, asexual and sexual development, and with respect to different stresses. In aerial mycelium, hyd8 was up-regulated. In silico analysis predicted three packing arrangements, i.e., ring-like, plus-like and sheet-like structure for Hyd8; the first two may assemble to rodlets of hydrophobin covering aerial hyphae, whereas the third is expected to be involved in forming a two-dimensional network of hydrophobins. Metal stress induced hydrophobin gene hyd5. In early steps of mycorrhization, induction of hyd4 and hyd5 by plant root exudates and root volatiles could be shown, followed by hyd5 up-regulation during formation of mantle, Hartig' net, and rhizomorphs with concomitant repression of hyd8 and hyd9. During fruiting body formation, mainly hyd3, but also hyd8 were induced. Host preference between the compatible host Picea abies and the low compatibility host Pinus sylvestris could be linked to a stronger induction of hyd4 and hyd5 by the preferred host and a stronger repression of hyd8, whereas the repression of hyd9 was comparable between the two hosts.

Influence of zygomycete-derived D'orenone on IAA signalling in Tricholoma-spruce ectomycorrhiza.

Despite the rising interest in microbial communication, only few studies relate to mycorrhization and the pool of potential morphogenic substances produced by the surrounding soil community. Here, we investigated the effect exerted by the C18 - ketone ß-apo-13-carotenone, D'orenone, on the ectomycorrhizal basidiomycete Tricholoma vaccinum and its symbiosis with the economically important host tree, spruce (Picea abies). D'orenone is an early intermediate in the biosynthesis of morphogens in sexual development of mucoromycetes, the trisporoids. In the ectomycorrhizal fungus T. vaccinum, D'orenone increased the production and/or release of the phytohormone indole-3-acetic acid (IAA) which had been proposed to be involved in the mutual symbiosis. The induced expression of the fungal aldehyde dehydrogenase, Ald5 is associated with IAA synthesis and excretion. In the host tree, D'orenone modulated root architecture by increasing lateral root length and hypertrophy of root cortex cells, likely via changed IAA concentrations and flux. Thus, we report for the first time on carotenoid metabolites from soil fungi affecting both ectomycorrhizal partners. The data imply a complex network of functions for secondary metabolites which act in an inter-kingdom signalling in soil.

[The Play-PAB and its Validation in a Preschool Psychiatric Population]. / Das Play-PAB-Verfahren und seine Validierung an einer vorschulpsychiatrischen Inanspruchnahmepopulation/ The Play-PAB and its Validation in a Preschool Psychiatric Population.

With community samples, parent behavior can be assessed in standardized mother-child interaction situations with the observational instrument Lab-PAB by Wilson und Durbin (2012) with respect to five dimensions (Involvement, Positive Emotionality, Hostility, Intrusiveness and Discipline). We examined an adaptation of Lab-PAB for non-standardized free-play interaction situations (Play-PAB version) with a preschool psychiatric sample. We examined the internal consistency, interrater reliability and dimensional structure of the Play-PAB and its associations to the clinical relationship assessment scale PIR-GAS from DC:0-3R. Interaction sequences of 47 parent-child dyads who were treated in our Family Day Clinic were evaluated at admission with the Play-PAB, in addition to the PIR-GAS-rating. Each instrument was rated by two independent raters. We report means, standard deviations, internal consistencies and the interrater-reliability for each of the five Play-PAB scales. Furthermore we examine if the scale intercorrelations are reasonable in comparison to the original version. Finally, the associations to PIR-GAS are presented. The Play-PAB scales reflect sufficient variation of parenting, good internal consistencies and satisfactory interrater reliability. The adaptation shows psychometric properties that are comparable to the original version. The scale intercorrelations as well as the associations to PIR-GAS are reasonable. The Play-PAB is a promising instrument for assessing different aspects of parent behavior in a preschool psychiatric sample, with meaningful associations to parent-child-relationship quality.

Tricholoma vaccinum host communication during ectomycorrhiza formation.

The genome sequence of Tricholoma vaccinum was obtained to predict its secretome in order to elucidate communication of T. vaccinum with its host tree spruce (Picea abies) in interkingdom signaling. The most prominent protein domains within the 206 predicted secreted proteins belong to energy and nutrition (52%), cell wall degradation (19%) and mycorrhiza establishment (9%). Additionally, we found small secreted proteins that show typical features of effectors potentially involved in host communication. From the secretome, 22 proteins could be identified, two of which showed higher protein abundances after spruce root exudate exposure, while five were downregulated in this treatment. The changes in T. vaccinum protein excretion with first recognition of the partner were used to identify small secreted proteins with the potential to act as effectors in the mutually beneficial symbiosis. Our observations support the hypothesis of a complex communication network including a cocktail of communication molecules induced long before physical contact of the partners.

Comparison of methods for the analysis of therapeutic immunoglobulin G Fc-glycosylation profiles-Part 2: Mass spectrometric methods.

To monitor the Fc glycosylation of therapeutic immunoglobulin G in bioprocess development, product characterization and release analytics, reliable techniques for glycosylation analysis are needed. Several analytical methods are suitable for this application. We recently presented results comparing detection methods for glycan analysis that are separation-based, but did not include mass spectrometry (MS). In the study reported here, we comprehensively compared MS-based methods for Fc glycosylation profiling of an IgG biopharmaceutical. A therapeutic antibody reference material was analyzed 6-fold on 2 different days, and the methods investigated were compared with respect to precision, accuracy, throughput and analysis time. Emphasis was put on the detection and quantitation of sialic acid-containing glycans. Eleven MS methods were compared to hydrophilic interaction liquid chromatography of 2-aminobenzamide labeled glycans with fluorescence detection, which served as a reference method and was also used in the first part of the study. The methods compared include electrospray MS of the heavy chain and Fc part after limited digestion, liquid chromatography MS of a tryptic digest, porous graphitized carbon chromatography MS of released glycans, electrospray MS of glycopeptides, as well as matrix assisted laser desorption ionization MS of glycans and glycopeptides. Most methods showed excellent precision and accuracy. Some differences were observed with regard to the detection and quantitation of low abundant glycan species like the sialylated glycans and the amount of artefacts due to in-source decay.

Expression of Hedgehog Pathway Mediator GLI Represents a Negative Prognostic Marker in Human Acute Myeloid Leukemia and Its Inhibition Exerts Antileukemic Effects.

PURPOSE: The Hedgehog pathway plays an important role in stem-cell biology and malignant transformation. Therefore, we investigated the expression and prognostic impact of Hedgehog pathway members in acute myeloid leukemia (AML). EXPERIMENTAL DESIGN: Pretreatment samples from 104 newly diagnosed AML patients (AMLSG 07-04 trial) were analyzed by qPCR, and expression of Hedgehog family members was correlated with clinical outcome. Inhibition of GLI by GANT61 or shRNA was investigated in AML cells in vitro and in vivo. RESULTS: Expression of receptors Smoothened and Patched-1 and their downstream mediators, GLI1, GLI2, and GLI3, was found in AML patients in contrast to Hedgehog ligands. GLI2 expression had a significant negative influence on event-free survival (EFS), relapse-free survival (RFS), and overall survival (OS; P = 0.037, 0.026, and 0.013, respectively) and was correlated with FLT3 mutational status (P < 0.001). Analysis of a second, independent patient cohort confirmed the negative impact of GLI2 on EFS and OS (P = 0.007 and 0.003, respectively; n = 290). Within this cohort, GLI1 had a negative prognostic impact (P < 0.001 for both EFS and OS). Although AML cells did not express Hedgehog ligands by qPCR, AML patients had significantly increased Desert Hedgehog (DHH) plasma levels compared with healthy subjects (P = 0.002), in whom DHH was presumably provided by bone marrow niche cells. Moreover, the GLI inhibitor GANT61 or knockdown of GLI1/2 by shRNA caused antileukemic effects, including induction of apoptosis, reduced proliferation, and colony formation in AML cells, and a survival benefit in mice. CONCLUSIONS: GLI expression is a negative prognostic factor and might represent a novel druggable target in AML.

Innate immune receptors in heart failure: Side effect or potential therapeutic target?

Heart failure (HF) is a leading cause of mortality and morbidity in western countries and occasions major expenses for public health systems. Although optimal medical treatment is widely available according to current guidelines, the prognosis of patients with HF is still poor. Despite the etiology of the disease, increased systemic or cardiac activation of the innate immune system is well documented in several types of HF. In some cases there is evidence of an association between innate immune activation and clinical outcome of patients with this disease. However, the few large trials conducted with the use of anti-inflammatory medication in HF have not revealed its benefits. Thus, greater understanding of the relationship between alteration in the immune system and development and progression of HF is urgently necessary: prior to designing therapeutic interventions that target pathological inflammatory processes in preventing harmful cardiac effects of immune modulatory therapy. In this regard, relatively recently discovered receptors of the innate immune system, i.e., namely toll-like receptors (TLRs) and nod-like receptors (NLRs)-are the focus of intense cardiovascular research. These receptors are main up-stream regulators of cytokine activation. This review will focus on current knowledge of the role of TLRs and NLRs, as well as on downstream cytokine activation, and will discuss potential therapeutic implications.

Lack of noncanonical RAS mutations in cytogenetically normal acute myeloid leukemia.

Transforming mutations in RAS genes are commonly found in human malignancies, including myeloid leukemias. To investigate the incidence, spectrum, and distribution of activating K- and N-RAS mutations in cytogenetically normal acute myeloid leukemia (CN-AML) patients, 204 CN-AML patients were screened. Activating K- and N-RAS mutations were detected in 3 of 204 (1.5 %) and 22 of 204 (10.8 %) CN-AML samples, respectively. RAS mutated patients presented with a lower percentage of bone marrow blasts (65 vs 80 %, P = 0.022). RAS mutations tended to occur with nucleophosmin-1 (NPM1) mutations (P = 0.079), and all three samples containing K-RAS mutations had concomitant NPM1 mutations. There was no significant overlap between K-RAS mutations and N-RAS, FLT3, CEBPA, IDH1/2, WT1 or MLL mutations. RAS mutation status did not impact relapse-free or overall survival of CN-AML patients. In contrast to reports of noncanonical RAS mutations in other cancers, including some leukemia subtypes, we only observed K- and N-RAS mutations in codons 12, 13, or 61 in CN-AML samples. Our findings suggest that while K-RAS mutations are infrequent in CN-AML, activating K-RAS mutations may cooperate with mutated NPM1 to induce leukemia.

Deficits in dopaminergic transmission precede neuron loss and dysfunction in a new Parkinson model.

The pathological end-state of Parkinson disease is well described from postmortem tissue, but there remains a pressing need to define early functional changes to susceptible neurons and circuits. In particular, mechanisms underlying the vulnerability of the dopamine neurons of the substantia nigra pars compacta (SNc) and the importance of protein aggregation in driving the disease process remain to be determined. To better understand the sequence of events occurring in familial and sporadic Parkinson disease, we generated bacterial artificial chromosome transgenic mice (SNCA-OVX) that express wild-type α-synuclein from the complete human SNCA locus at disease-relevant levels and display a transgene expression profile that recapitulates that of endogenous α-synuclein. SNCA-OVX mice display age-dependent loss of nigrostriatal dopamine neurons and motor impairments characteristic of Parkinson disease. This phenotype is preceded by early deficits in dopamine release from terminals in the dorsal, but not ventral, striatum. Such neurotransmission deficits are not seen at either noradrenergic or serotoninergic terminals. Dopamine release deficits are associated with an altered distribution of vesicles in dopaminergic axons in the dorsal striatum. Aged SNCA-OVX mice exhibit reduced firing of SNc dopamine neurons in vivo measured by juxtacellular recording of neurochemically identified neurons. These progressive changes in vulnerable SNc neurons were observed independently of overt protein aggregation, suggesting neurophysiological changes precede, and are not driven by, aggregate formation. This longitudinal phenotyping strategy in SNCA-OVX mice thus provides insights into the region-specific neuronal disturbances preceding and accompanying Parkinson disease.