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In acute promyelocytic leukemia (APL), hematopoietic differentiation is blocked and immature blasts accumulate in the bone marrow and blood. APL is associated with chromosomal aberrations, including t(15;17) and t(11;17). For these two translocations, the retinoic acid receptor alpha (RARα) is fused to the promyelocytic leukemia (PML) gene or the promyelocytic zinc finger (PLZF) gene, respectively. Both fusion proteins lead to the formation of a high-molecular-weight complex. High-molecular-weight complexes are caused by the “coiled-coil” domain of PML or the BTB/POZ domain of PLZF. PML/RARα without the “coiled-coil” fails to block differentiation and mediates an all-trans retinoic acid-response. Similarly, mutations in the BTB/POZ domain disrupt the high-molecular-weight complex, abolishing the leukemic potential of PLZF/RARα. Specific interfering polypeptides were used to target the oligomerization domain of PML/RARα or PLZF/RARα. PML/RARα and PLZF/RARα were analyzed for the ability to form high-molecular-weight complexes, the protein stability and the potential to induce a leukemic phenotype in the presence of the interfering peptides. Expression of these interfering peptides resulted in a reduced replating efficiency and overcame the differentiation block induced by PML/RARα and PLZF/RARα in murine hematopoietic stem cells. This expression also destabilized the PLZF/RARα-induced high-molecular-weight complex formation and caused the degradation of the fusion protein. Targeting fusion proteins through interfering peptides is a promising approach to further elucidate the biology of leukemia.
Dendritic cells (DCs) play a pivotal role in the development of cutaneous contact hypersensitivity (CHS) and atopic dermatitis as they capture and process antigen and present it to T lymphocytes in the lymphoid organs. Recently, it has been indicated that a topical application of the sphingolipid sphingosine 1-phosphate (S1P) prevents the inflammatory response in CHS, but the molecular mechanism is not fully elucidated. Here we indicate that treatment of mice with S1P is connected with an impaired antigen uptake by Langerhans cells (LCs), the initial step of CHS. Most of the known actions of S1P are mediated by a family of five specific G protein-coupled receptors. Our results indicate that S1P inhibits macropinocytosis of the murine LC line XS52 via S1P2 receptor stimulation followed by a reduced phosphatidylinositol 3-kinase (PI3K) activity. As down-regulation of S1P2 not only diminished S1P-mediated action but also enhanced the basal activity of LCs on antigen capture, an autocrine action of S1P has been assumed. Actually, S1P is continuously produced by LCs and secreted via the ATP binding cassette transporter ABCC1 to the extracellular environment. Consequently, inhibition of ABCC1, which decreased extracellular S1P levels, markedly increased the antigen uptake by LCs. Moreover, stimulation of sphingosine kinase activity, the crucial enzyme for S1P formation, is connected not only with enhanced S1P levels but also with diminished antigen capture. These results indicate that S1P is essential in LC homeostasis and influences skin immunity. This is of importance as previous reports suggested an alteration of S1P levels in atopic skin lesions.
During postnatal development hippocampal dentate granule cells (GCs) often extend dendrites from the basal pole of their cell bodies into the hilar region. These so-called hilar basal dendrites (hBD) usually regress with maturation. However, hBDs may persist in a subset of mature GCs under certain conditions (both physiological and pathological). The functional role of these hBD-GCs remains not well understood. Here, we have studied hBD-GCs in mature (≥18 days in vitro) mouse entorhino-hippocampal slice cultures under control conditions and have compared their basic functional properties (basic intrinsic and synaptic properties) and structural properties (dendritic arborisation and spine densities) to those of neighboring GCs without hBDs in the same set of cultures. Except for the presence of hBDs, we did not detect major differences between the two GC populations. Furthermore, paired recordings of neighboring GCs with and without hBDs did not reveal evidence for a heavy aberrant GC-to-GC connectivity. Taken together, our data suggest that in control cultures the presence of hBDs on GCs is neither sufficient to predict alterations in the basic functional and structural properties of these GCs nor indicative of a heavy GC-to-GC connectivity between neighboring GCs.
Tumor-associated macrophages (TAM) are a major supportive component within neoplasms and by their plasticity promote all phases of tumor development. Mechanisms of macrophage (M Phi) attraction and differentiation to a tumor-promoting phenotype, defined among others by distinct cytokine patterns such as pronounced immunosuppressive interleukin 10 (IL-10) production, are largely unknown. However, a high apoptosis index within tumors and strong M Phi infiltration correlate with poor prognosis. Thus, I aimed at identifying signaling pathways contributing to generation of TAM-like M Phi by using supernatant of apoptotic cancer cells (ACM) as stimulus.
To distinguish novel factors involved in generating TAM-like M Phi, I used an adenoviral RNAi-based approach. The primary read-out was production of IL-10. However, mediators modulating IL-10 were re-validated for their impact on regulation of the cytokines IL-6, IL-8 and IL-12. Following assay development, optimization and down-scaling to a 384-well format, primary human M Phi were transduced with 8495 constructs of the adenoviral shRNA SilenceSelect® library of Galapagos BV, followed by activation to a TAM-like phenotype using ACM. I identified 96 genes involved in IL-10 production in response to ACM and observed a pronounced cluster of 22 targets regulating IL-10 and IL-6. Principal validation of five targets of the IL-10/IL-6 cluster was performed using siRNA or pharmacological inhibitors. Among those, IL-4 receptor-alpha and cannabinoid receptor 2 were confirmed as regulators of IL-10 and IL-6 secretion.
One protein identified in the screen, the nerve growth factor (NGF) receptor TRKA was chosen for in-depth validation, based on its involvement in IL-10, IL-6 and IL-12 secretion from ACM-stimulated human M Phi. TRKA possesses a cardinal role in neuronal development, but compelling evidence emerges suggesting participation of TRKA in cancer development. First experiments using pharmacological inhibitors principally confirmed the involvement of TRKA in IL-10 secretion by ACM-stimulated M Phi and revealed PI3K/AKT and to a lesser extend MAPK p38 as important signaling molecules downstream of TRKA activation. Signaling through TRKA required the presence of its ligand NGF, as indicated by NGF neutralization experiments. NGF was not induced by or present in ACM, but was constitutively secreted by M Phi. Interestingly, M Phi responded to authentic NGF with neither AKT and p38 phosphorylation nor IL-10 production. TRKA is well known to be transactivated by other receptors and in neurons its cellular localization is decisive for its function. Inhibitors of common transactivation partners did not influence IL-10 production by human M Phi. Rather, ACM-treatment provoked pronounced translocation of TRKA to the plasma membrane within 10 minutes as observed by immunofluorescence staining. Consequently, I was intrigued to clarify mechanisms of TRKA trafficking in response to ACM.
The bioactive lipid sphingosine-1-phosphate (S1P) has been previously identified as important apoptotic cell-derived mediator involved in TAM-like M Phi polarization. Indeed, I observed S1P and src kinase involvement in ACM-mediated IL-10 induction. Furthermore, inhibition of S1P receptor (S1PR) signaling or src kinase activity prevented TRKA translocation, whereas a TRKA inhibitor or anti-NGF did not block TRKA trafficking to the plasma membrane in response to ACM. Thus, autocrine secreted NGF activated TRKA to promote IL-10 secretion, which required previous S1PR/src-dependent translocation of TRKA to the plasma membrane. Following the detailed analysis of IL-10 regulation, I was interested whether other TAM phenotype markers were influenced by ACM and whether their expression was regulated through TRKA-dependent signaling. Five of six markers were up-regulated on mRNA level by ACM, and secretion of IL-6, IL-8 and TNF-alpha was triggered. S1PR-signaling was essential for induction of all but one marker, whereas TRKA signaling was only required for cytokine secretion. Interestingly, none of the investigated TAM markers was regulated identically to IL-10, emphasizing a tight and exclusive regulation machinery of this potent immunosuppressive cytokine.
Finally, I aimed to validate the in vitro findings in human ACM-stimulated M Phi. Therefore, I isolated murine TAM as well as other major mononuclear phagocyte populations from primary oncogene-induced breast cancer tissue. Indeed, TRKA-dependent signaling was required for spontaneous cytokine production selectively by primary murine TAM. Besides IL-10, the TRKA pathway was decisive for secretion of IL-6, TNF-alpha and monocyte chemotactic protein-1, indicating its relevance in cancer-associated inflammation.
In summary, my findings highlight a fine-tuned regulatory system of S1P-dependent TRKA trafficking and autocrine NGF signaling in TAM biology. Both factors, S1P as well as NGF, might be interesting targets for future cancer therapy.
Ubiquitination relies on a subtle balance between selectivity and promiscuity achieved through specific interactions between ubiquitin-conjugating enzymes (E2s) and ubiquitin ligases (E3s). Here, we report how a single aspartic to glutamic acid substitution acts as a dynamic switch to tip the selectivity balance of human E2s for interaction toward E3 RING-finger domains. By combining molecular dynamic simulations, experimental yeast-two-hybrid screen of E2-E3 (RING) interactions and mutagenesis, we reveal how the dynamics of an internal salt-bridge network at the rim of the E2-E3 interaction surface controls the balance between an “open”, binding competent, and a “closed”, binding incompetent state. The molecular dynamic simulations shed light on the fine mechanism of this molecular switch and allowed us to identify its components, namely an aspartate/glutamate pair, a lysine acting as the central switch and a remote aspartate. Perturbations of single residues in this network, both inside and outside the interaction surface, are sufficient to switch the global E2 interaction selectivity as demonstrated experimentally. Taken together, our results indicate a new mechanism to control E2-E3 interaction selectivity at an atomic level, highlighting how minimal changes in amino acid side-chain affecting the dynamics of intramolecular salt-bridges can be crucial for protein-protein interactions. These findings indicate that the widely accepted sequence-structure-function paradigm should be extended to sequence-structure-dynamics-function relationship and open new possibilities for control and fine-tuning of protein interaction selectivity.
Background: Chronic myelogenous leukemia (CML) and Philadelphia chromosome-positive (Ph+) acute lymphatic leukemia (Ph + ALL) are caused by the t(9;22), which fuses BCR to ABL resulting in deregulated ABL-tyrosine kinase activity. The constitutively activated BCR/ABL-kinase "escapes" the auto-inhibition mechanisms of c-ABL, such as allosteric inhibition. The ABL-kinase inhibitors (AKIs) Imatinib, Nilotinib or Dasatinib, which target the ATP-binding site, are effective in Ph + leukemia. Another molecular therapy approach targeting BCR/ABL restores allosteric inhibition. Given the fact that all AKIs fail to inhibit BCR/ABL harboring the 'gatekeeper' mutation T315I, we investigated the effects of AKIs in combination with the allosteric inhibitor GNF2 in Ph + leukemia.
Methods: The efficacy of this approach on the leukemogenic potential of BCR/ABL was studied in Ba/F3 cells, primary murine bone marrow cells, and untransformed Rat-1 fibroblasts expressing BCR/ABL or BCR/ABL-T315I as well as in patient-derived long-term cultures (PDLTC) from Ph + ALL-patients.
Results: Here, we show that GNF-2 increased the effects of AKIs on unmutated BCR/ABL. Interestingly, the combination of Dasatinib and GNF-2 overcame resistance of BCR/ABL-T315I in all models used in a synergistic manner.
Conclusions: Our observations establish a new approach for the molecular targeting of BCR/ABL and its resistant mutants using a combination of AKIs and allosteric inhibitors.
Background: Cognitive-behavioral therapy (CBT) is generally known to be efficacious in the treatment of social phobia when applied in RCT's, namely when the treatment manual is based on the Clark-Wells approach. However, little is known about the efficacy of manualized treatments in routine clinical practice (Phase IV of psychotherapy research). The present study (SOPHO-PRAX) is a continuation of a large multi-centre randomized clinical trial (SOPHO-NET) and analyses the extent to which additional training practitioners in manualized procedures enhances treatment effect.
Methods: N = 36 private practitioners will be included in three treatment centres and randomly designated to either training in manualized CBT or no specific training. The treatment effects of the therapies conducted by both groups of therapists will be compared. A total of 162 patients (N = 116 completers; N = 58 per condition) will be enrolled. Liebowitz Social Anxiety Scale (LSAS) will serve as primary outcome measure. Remission from social phobia is defined as LSAS total [less than or equal to] 30 points. Data will be collected at treatment begin, after 8, 15, and 25 sessions (50 mins. each), at treatment completion, as well at 6 and 12 months post-treatment.
Discussion: The present CBT trial combines elements of randomized-controlled trials and naturalistic studies in an innovative way. It will directly inform about the incremental effects of procedures established in a controlled trial into clinical practice. Study results are relevant to health care decisions and policy. They may serve to improve quality of treatment, and shorten the timeframe between the development and widespread dissemination of effective methods, thereby reducing health cost expenditures. The results of this study will not only inform about the degree to which the new methods lead to an improvement of treatment course and outcome, but also about whether the effects of routine psychotherapeutic treatment are comparable to those of the controlled, strictly manualized treatments of the SOPHO-NET study. Trial Registration: ClinicalTrials.gov identifier: NCT01388231. This study was funded by the German Federal Ministry of Education and Research (SOPHO-NET: BMBF 01GV0607; SOPHO-PRAX: BMBF 01GV1001).
Background: Hodkin s lymphoma is one of the most frequent lymphoma in western world. Despite an overall good prognosis some patients suffer relapsing tumors which are difficult to cure. Over a long period Vitamin D has been shown to be a potential treatment for cancer. Vitamin D acts via the vitamin D receptor, a nuclear receptor, acting as an inducible transcription factor. We aimed to investigate the expression of vitamin D receptor as potential therapeutic target structure in Hodgkin s lymphoma as well as in non Hodgkin s lymphoma.
Methods: We used a panel of 193 formalin fixed tissues of lymphoma cases consisting of 55 cases of Hodgkin s lymphoma and 138 cases on several non Hodgkin s lymphoma entities.
Results: Vitamin D receptor is strongly expressed in Hodgkin s lymphoma, regardless of the subentity with an overall positivity of 80% of all Hodgkin lymphoma cases. In contrast, only about 17% of the analyzed non Hodgkin s lymphoma of B-cell origin showed positivity for vitamin D receptor. Predominant nuclear localization of vitamin D receptor in Hodgkin s lymphoma suggests activated status of the vitamin D receptor.
Conclusions: From this study, we conclude that vitamin D receptor plays a potentially important role in pathogenesis of Hodgkin s lymphoma but not in non Hodgkin s lymphoma. Further investigations of mutational status and functional studies may shed some light in functional relevance of vitamin D receptor signaling in Hodgkin s lymphoma.
kurz und kn@pp news : Nr. 25
(2012)
* "Evidence based medicine meets multimorbidity: a blind date?"
* Aarhus Statement zur Frühdiagnose von Krebserkrankungen veröffentlicht
* Auszeichnungen
* ForN geht auf die 100 zu ... – Weiterer Aufbau des Frankfurter Netzes Akademischer Forschungspraxen
* 5. Frankfurter Tag der Allgemeinmedizin: Hochwertige Fortbildung für Hausarztpraxen
kurz und kn@pp news : Nr. 24
(2012)
Einleitung: Lokoregionäre Rezidivtumore der Kopf-Hals-Region können häufig nicht mehr kurativ operativ oder radiotherapeutisch behandelt werden, so dass neue Therapiekonzepte erforderlich sind. Es konnte gezeigt werden, dass statische Magnetfelder (SMF) Tumorwachstum und -angiogenese signifikant beeinflussen und zu einem intratumoralen Ödem führen. Das Ziel der vorliegenden Studie war die Evaluation des Effektes von SMF auf die Permeabilität von Tumorblutgefäßen und die therapeutische Nutzbarkeit in Kombination mit einer konventionellen Chemotherapie.
Methoden: Zellen eines syngenen amelanotischen Melanoms wurden in transparente Rückenhautkammern bei Goldhamstern implantiert. Unter SMF-Exposition von 587 mT wurde fluoreszenzmikroskopisch die Extravasation von rhodaminmarkiertem Albumin zur Errechnung der Gefäßpermeabilität gemessen und intratumorale Leukozyten-Endothelzell-Interaktionen quantifiziert. Für die anschließende Therapiestudie wurden die antitumoralen Effekte einer Kombinationstherapie von Paclitaxel und SMF-Exposition verglichen mit drei Kontrollgruppen (Glucose, Paclitaxel allein, SMF allein; je n=6).
Ergebnisse: SMF führen zu einer signifikanten Erhöhung der Tumorblutgefäßpermeabilität bei unveränderten Leukozyten-Endothelzell-Interaktionen. Die Kombinationstherapie von SMF und Paclitaxel ist – bezogen auf Tumorwachstum und Angiogenese – Monotherapien überlegen.
Schlussfolgerung: Eine SMF-induzierte Steigerung der Gefäßpermeabilität kann die Blut-Tumor-Schranke beeinflussen und somit die Effektivität einer Chemotherapie mit kleinmolekularen Substanzen wie Paclitaxel deutlich steigern. Bei Verwendung von Kopfspulen erscheint eine derartige adjuvante Kombinationstherapie für lokoregionäre Karzinomrezidive der Kopf-Hals-Region besonders geeignet.
The key cognitive impairments of children with attention deficit/-hyperactivity disorder (ADHD) include executive control functions such as inhibitory control, task-switching, and working memory (WM). In this training study we examined whether task-switching training leads to improvements in these functions. Twenty children with combined type ADHD and stable methylphenidate medication performed a single-task and a task-switching training in a crossover training design. The children were randomly assigned to one of two groups. One group started with the single-task training and then performed the task-switching training and the other group vice versa. The effectiveness of the task-switching training was measured as performance improvements (relative to the single-task training) on a structurally similar but new switching task and on other executive control tasks measuring inhibitory control and verbal WM as well as on fluid intelligence (reasoning). The children in both groups showed improvements in task-switching, that is, a reduction of switching costs, but not in performing the single-tasks across four training sessions. Moreover, the task-switching training lead to selective enhancements in task-switching performance, that is, the reduction of task-switching costs was found to be larger after task-switching than after single-task training. Similar selective improvements were observed for inhibitory control and verbal WM, but not for reasoning. Results of this study suggest that task-switching training is an effective cognitive intervention that helps to enhance executive control functioning in children with ADHD.
Mitochondria form a dynamic tubular reticulum within eukaryotic cells. Currently, quantitative understanding of its morphological characteristics is largely absent, despite major progress in deciphering the molecular fission and fusion machineries shaping its structure. Here we address the principles of formation and the large-scale organization of the cell-wide network of mitochondria. On the basis of experimentally determined structural features we establish the tip-to-tip and tip-to-side fission and fusion events as dominant reactions in the motility of this organelle. Subsequently, we introduce a graph-based model of the chondriome able to encompass its inherent variability in a single framework. Using both mean-field deterministic and explicit stochastic mathematical methods we establish a relationship between the chondriome structural network characteristics and underlying kinetic rate parameters. The computational analysis indicates that mitochondrial networks exhibit a percolation threshold. Intrinsic morphological instability of the mitochondrial reticulum resulting from its vicinity to the percolation transition is proposed as a novel mechanism that can be utilized by cells for optimizing their functional competence via dynamic remodeling of the chondriome. The detailed size distribution of the network components predicted by the dynamic graph representation introduces a relationship between chondriome characteristics and cell function. It forms a basis for understanding the architecture of mitochondria as a cell-wide but inhomogeneous organelle. Analysis of the reticulum adaptive configuration offers a direct clarification for its impact on numerous physiological processes strongly dependent on mitochondrial dynamics and organization, such as efficiency of cellular metabolism, tissue differentiation and aging.
Molecular biology of hearing
(2012)
The inner ear is our most sensitive sensory organ and can be subdivided into three functional units: organ of Corti, stria vascularis and spiral ganglion. The appropriate stimulus for the organ of hearing is sound, which travels through the external auditory canal to the middle ear where it is transmitted to the inner ear. The inner ear houses the hair cells, the sensory cells of hearing. The inner hair cells are capable of mechanotransduction, the transformation of mechanical force into an electrical signal, which is the basic principle of hearing. The stria vascularis generates the endocochlear potential and maintains the ionic homeostasis of the endolymph. The dendrites of the spiral ganglion form synaptic contacts with the hair cells. The spiral ganglion is composed of neurons that transmit the electrical signals from the cochlea to the central nervous system. In recent years there has been significant progress in research on the molecular basis of hearing. An increasing number of genes and proteins related to hearing are being identified and characterized. The growing knowledge of these genes contributes not only to greater appreciation of the mechanism of hearing but also to a deeper understanding of the molecular basis of hereditary hearing loss. This basic research is a prerequisite for the development of molecular diagnostics and novel therapies for hearing loss.
Background: Despite limited effectiveness of short-term psychotherapy for chronic depression, there is a lack of trials of long-term psychotherapy. Our study is the first to determine the effectiveness of controlled long-term psychodynamic and cognitive-behavioral (CBT) treatments and to assess the effects of preferential vs. randomized assessment.
Methods/design: Patients are assigned to treatment according to their preference or randomized (if they have no clear preference). Up to 80 sessions of psychodynamic or psychoanalytically oriented treatments (PAT) or up to 60 sessions of CBT are offered during the first year in the study. After the first year, PAT can be continued according to the ‘naturalistic’ usual method of treating such patients within the system of German health care (normally from 240 up to 300 sessions over two to three years). CBT therapists may extend their treatment up to 80 sessions, but focus mainly maintenance and relapse prevention. We plan to recruit a total of 240 patients (60 per arm). A total of 11 assessments are conducted throughout treatment and up to three years after initiation of treatment. The primary outcome measures are the Quick Inventory of Depressive Symptoms (QIDS, independent clinician rating) and the Beck Depression Inventory (BDI) after the first year.
Discussion: We combine a naturalistic approach with randomized controlled trials(RCTs)to investigate how effectively chronic depression can be treated on an outpatient basis by the two forms of treatment reimbursed in the German healthcare system and we will determine the effects of treatment preference vs. randomization.
Staphylococcus aureus proteins Sbi and Efb recruit human plasmin to degrade complement C3 and C3b
(2012)
Upon host infection, the human pathogenic microbe Staphylococcus aureus (S. aureus) immediately faces innate immune reactions such as the activated complement system. Here, a novel innate immune evasion strategy of S. aureus is described. The staphylococcal proteins surface immunoglobulin-binding protein (Sbi) and extracellular fibrinogen-binding protein (Efb) bind C3/C3b simultaneously with plasminogen. Bound plasminogen is converted by bacterial activator staphylokinase or by host-specific urokinase-type plasminogen activator to plasmin, which in turn leads to degradation of complement C3 and C3b. Efb and to a lesser extend Sbi enhance plasmin cleavage of C3/C3b, an effect which is explained by a conformational change in C3/C3b induced by Sbi and Efb. Furthermore, bound plasmin also degrades C3a, which exerts anaphylatoxic and antimicrobial activities. Thus, S. aureus Sbi and Efb comprise platforms to recruit plasmin(ogen) together with C3 and its activation product C3b for efficient degradation of these complement components in the local microbial environment and to protect S. aureus from host innate immune reactions.
Background: The human ATP-binding cassette, subfamily B, member 11 (ABCB11) gene encodes the bile salt export pump, which is exclusively expressed at the canalicular membrane of hepatocytes. A frequent variant in the coding region, c.1331 T > C, leading to the amino acid exchange p.V444A, has been associated with altered serum bile salt levels in healthy individuals and predisposes homozygous carriers of the [C] allele for obstetric cholestasis. Recently, elevated bile salt levels were shown to be significantly associated with rates and risk of cirrhosis in patients with chronic hepatitis C virus (HCV) infection treated with pegylated interferon-alpha2 and ribavirin, suggesting a potential role for bile salt levels in HCV treatment outcomes and in the fibrogenic evolution of HCV-related liver disease. The aim of this study was to investigate a possible association of ABCB11 c.1331 T > C with hepatitis C virus (HCV) infection and fibrosis stages as assessed by non-invasive transient elastography in a German cohort of patients.
Methods: ABCB11 c.1331 T > C genotype was determined by allelic discrimination assay in 649 HCV infected cases and 413 controls. Overall, 444 cases were staged for fibrotic progression by measurement of liver stiffness.
Results: Homo- or heterozygous presence of the frequent [C] allele was associated with HCV positivity (OR = 1.41, CI = 1.02 - 1.95, p = 0.037). No association was detectable between the ABCB11 c.1331 T > C genotype and increased liver stiffness.
Conclusions: Our data confirm that homozygous presence of the major [C] allele of ABCB11 c.1331 T > C is a genetic susceptibility factor for HCV infection, but not for liver fibrosis.
Background: Hereditary angioedema (HAE) is a rare but serious disease marked by swelling attacks in the extremities, face, trunk, airway, or abdominal areas that can be spontaneous or the result of trauma and other triggers. It can be life-threatening due to the risk of asphyxiation. While there have been major advancements in our understanding of the immunogenetics of HAE, there are significant gaps in the literature regarding understanding of the humanistic and economic impact of the disease, particularly in Europe. The purpose of the HAE Burden of Illness Study-Europe (HAE-BOIS-Europe), the development and methodology of which is described here, is to better understand the management and impact of HAE from the patient perspective in Europe.
Methods/Design: This is a cross-sectional study in which retrospective data were also collected being conducted in Denmark, Germany and Spain. The study is open to patients ages 12 and older with a diagnosis of HAE-I or HAE-II. Data collection includes: (i) a survey on individuals’ health care resource use, direct and indirect medical costs, impact on work and school, treatment satisfaction, and emotional functioning (via the Hospital Anxiety and Depression Scale); and (ii) one-on-one interviews to collect detailed descriptive data and patient testimonials on the impact of HAE on patients’ health-related quality of life.
Discussion: The present manuscript describes the development and plans for implementing a multi-country European study with the aim of characterizing the humanistic and economic burden of HAE from the patient perspective. This study will help raise awareness of HAE as a rare but debilitating condition with wide-ranging impacts.
Background: Evidence from animal studies suggests that leptin metabolism is associated with zinc (Zn) status. However, research investigating this relationship in adolescents and young adults with anorexia nervosa (AN) is scarce; the present study aims to fill that gap.
Methods: Serum concentrations of leptin, the soluble leptin receptor (sOB-R) and the free leptin index (FLI) were obtained in healthy control subjects (n=19), acutely ill individuals (n=14) and recovered patients with AN (n=15). Serum Zn concentrations noted in previous research data were also incorporated for all groups.
Results: Leptin, FLI and Zn concentrations were higher in recovered subjects with AN when compared with acutely ill AN patients. Remitted patients showed higher sOB-R concentrations but no difference in FLI compared with the control group. Leptin and FLI were lower in the acutely ill patients compared with the control subjects, who showed no differences in Zn concentrations. Zn concentrations were not correlated with leptin, sOB-R or FLI concentrations in any of the three investigated subgroups.
Conclusions: The present investigation does not entirely support an association between Zn, Leptin and FLI concentrations in subjects with AN, possibly due to limited statistical power. Further research and replication of the present findings related to the interaction between leptin and Zn is warranted. However, with respect to serum leptin levels the data of the present investigation indicate that acutely ill and remitted patients with AN differ as regards serum leptin concentrations and FLI, which is in line with previous research.
Small RNAs have been implicated in numerous cellular processes, including effects on chromatin structure and the repression of transposons. We describe the generation of a small RNA response at DNA ends in Drosophila that is analogous to the recently reported double-strand break (DSB)-induced RNAs or Dicer- and Drosha-dependent small RNAs in Arabidopsis and vertebrates. Active transcription in the vicinity of the break amplifies this small RNA response, demonstrating that the normal messenger RNA contributes to the endogenous small interfering RNAs precursor. The double-stranded RNA precursor forms with an antisense transcript that initiates at the DNA break. Breaks are thus sites of transcription initiation, a novel aspect of the cellular DSB response. This response is specific to a double-strand break since nicked DNA structures do not trigger small RNA production. The small RNAs are generated independently of the exact end structure (blunt, 3′- or 5′-overhang), can repress homologous sequences in trans and may therefore—in addition to putative roles in repair—exert a quality control function by clearing potentially truncated messages from genes in the vicinity of the break.
Background: The diagnostic and pathophysiological relevance of antibodies to aquaporin-4 (AQP4-Ab) in patients with neuromyelitis optica spectrum disorders (NMOSD) has been intensively studied. However, little is known so far about the clinical impact of AQP4-Ab seropositivity.
Objective: To analyse systematically the clinical and paraclinical features associated with NMO spectrum disorders in Caucasians in a stratified fashion according to the patients' AQP4-Ab serostatus.
Methods: Retrospective study of 175 Caucasian patients (AQP4-Ab positive in 78.3%).
Results: Seropositive patients were found to be predominantly female (p < 0.0003), to more often have signs of co-existing autoimmunity (p < 0.00001), and to experience more severe clinical attacks. A visual acuity of ≤ 0.1 during acute optic neuritis (ON) attacks was more frequent among seropositives (p < 0.002). Similarly, motor symptoms were more common in seropositive patients, the median Medical Research Council scale (MRC) grade worse, and MRC grades ≤ 2 more frequent, in particular if patients met the 2006 revised criteria (p < 0.005, p < 0.006 and p < 0.01, respectively), the total spinal cord lesion load was higher (p < 0.006), and lesions ≥ 6 vertebral segments as well as entire spinal cord involvement more frequent (p < 0.003 and p < 0.043). By contrast, bilateral ON at onset was more common in seronegatives (p < 0.007), as was simultaneous ON and myelitis (p < 0.001); accordingly, the time to diagnosis of NMO was shorter in the seronegative group (p < 0.029). The course of disease was more often monophasic in seronegatives (p < 0.008). Seropositives and seronegatives did not differ significantly with regard to age at onset, time to relapse, annualized relapse rates, outcome from relapse (complete, partial, no recovery), annualized EDSS increase, mortality rate, supratentorial brain lesions, brainstem lesions, history of carcinoma, frequency of preceding infections, oligoclonal bands, or CSF pleocytosis. Both the time to relapse and the time to diagnosis was longer if the disease started with ON (p < 0.002 and p < 0.013). Motor symptoms or tetraparesis at first myelitis and > 1 myelitis attacks in the first year were identified as possible predictors of a worse outcome.
Conclusion: This study provides an overview of the clinical and paraclinical features of NMOSD in Caucasians and demonstrates a number of distinct disease characteristics in seropositive and seronegative patients
The cerebellum is crucially important for motor control and adaptation. Recent non-invasive brain stimulation studies have indicated the possibility to alter the excitability of the cerebellum and its projections to the contralateral motor cortex, with behavioral consequences on motor control and adaptation. Here we sought to induce bidirectional spike-timing dependent plasticity (STDP)-like modifications of motor cortex (M1) excitability by application of paired associative stimulation (PAS) in healthy subjects. Conditioning stimulation over the right lateral cerebellum (CB) preceded focal transcranial magnetic stimulation (TMS) of the left M1 hand area at an interstimulus interval of 2 ms (CB→M1 PAS(2 ms)), 6 ms (CB→M1 PAS(6 ms)) or 10 ms (CB→M1 PAS(10 ms)) or randomly alternating intervals of 2 and 10 ms (CB→M1 PAS(Control)). Effects of PAS on M1 excitability were assessed by the motor-evoked potential (MEP) amplitude, short-interval intracortical inhibition (SICI), intracortical facilitation (ICF) and cerebellar-motor cortex inhibition (CBI) in the first dorsal interosseous muscle of the right hand. CB→M1 PAS(2 ms) resulted in MEP potentiation, CB→M1 PAS(6 ms) and CB→M1 PAS(10 ms) in MEP depression, and CB→M1 PAS(Control) in no change. The MEP changes lasted for 30-60 min after PAS. SICI and CBI decreased non-specifically after all PAS protocols, while ICF remained unaltered. The physiological mechanisms underlying these MEP changes are carefully discussed. Findings support the notion of bidirectional STDP-like plasticity in M1 mediated by associative stimulation of the cerebello-dentato-thalamo-cortical pathway and M1. Future studies may investigate the behavioral significance of this plasticity.
Aim: Ventilation with pure oxygen (hyperoxic ventilation: HV) is thought to decrease whole body oxygen consumption (VO(2)). However, the validity and impact of this phenomenon remain ambiguous; until now, under hyperoxic conditions, VO(2) has only been determined by the reverse Fick principle, a method with inherent methodological problems. The goal of this study was to determine changes of VO(2), carbon dioxide production (VCO(2)), and the respiratory quotient (RQ) during normoxic and hyperoxic ventilation, using a metabolic monitor.
Methods: After providing signed informed consent and institutional acceptance, 14 healthy volunteers were asked to sequentially breathe room air, pure oxygen, and room air again. VO(2), VCO(2), RQ, and energy expenditure (EE) were determined by indirect calorimetry using a modified metabolic monitor during HV.
Results: HV reduced VO(2) from 3.4 (3.0/4.0) mL/kg/min to 2.8 (2.5/3.6) mL/kg/min (P < 0.05), whereas VCO(2) remained constant (3.0 [2.6/3.6] mL/kg/min versus 3.0 [2.6/3.5] mL/kg/min, n.s.). After onset of HV, RQ increased from 0.9 (0.8/0.9) to 1.1 (1.0/1.1). Most changes during HV were immediately reversed during subsequent normoxic ventilation.
Conclusion: HV not only reduces VO(2), but also increases the respiratory quotient. This might be interpreted as an indicator of the substantial metabolic changes induced by HV. However, the impact of this phenomenon requires further study.
Hemorrhagic shock leads to hepatic hypoperfusion and activation of mitogen-activated stress kinases (MAPK) like c-Jun N-terminal kinase (JNK) 1 and 2. Our aim was to determine whether mitochondrial dysfunction leading to hepatic necrosis and apoptosis after hemorrhage/resuscitation (H/R) was dependent on JNK2. Under pentobarbital anesthesia, wildtype (WT) and JNK2 deficient (KO) mice were hemorrhaged to 30 mm Hg for 3 h and then resuscitated with shed blood plus half the volume of lactated Ringer's solution. Serum alanine aminotransferase (ALT), necrosis, apoptosis and oxidative stress were assessed 6 h after resuscitation. Mitochondrial polarization was assessed by intravital microscopy. After H/R, ALT in WT-mice increased from 130 U/L to 4800 U/L. In KO-mice, ALT after H/R was blunted to 1800 U/l (P < 0.05). Necrosis, caspase-3 activity and ROS were all substantially decreased in KO compared to WT mice after H/R. After sham operation, intravital microscopy revealed punctate mitochondrial staining by rhodamine 123 (Rh123), indicating normal mitochondrial polarization. At 4 h after H/R, Rh123 staining became dim and diffuse in 58% of hepatocytes, indicating depolarization and onset of the mitochondrial permeability transition (MPT). By contrast, KO mice displayed less depolarization after H/R (23%, P < 0.05). In conclusion, JNK2 contributes to MPT-mediated liver injury after H/R.
Orthotopic liver transplantation in human-immunodeficiency-virus-positive patients in Germany
(2012)
Objectives: This summary evaluates the outcomes of orthotopic liver transplantation (OLT) of HIV-positive patients in Germany.
Methods: Retrospective chart analysis of HIV-positive patients, who had been liver-transplanted in Germany between July 1997 and July 2011.
Results: 38 transplantations were performed in 32 patients at 9 German transplant centres. The reasons for OLT were end-stage liver disease (ESLD) and/or liver failure due to hepatitis C (HCV) (n = 19), hepatitis B (HBV) (n = 10), multiple viral infections of the liver (n = 2) and Budd-Chiari-Syndrome. In July 2011 19/32 (60%) of the transplanted patients were still alive with a median survival of 61 months (IQR (interquartile range): 41-86 months). 6 patients had died in the early post-transplantation period from septicaemia (n = 4), primary graft dysfunction (n = 1), and intrathoracal hemorrhage (n = 1). Later on 7 patients had died from septicaemia (n = 2), delayed graft failure (n = 2), recurrent HCC (n = 2), and renal failure (n = 1). Recurrent HBV infection was efficiently prevented in 11/12 patients; HCV reinfection occurred in all patients and contributed considerably to the overall mortality.
Conclusions: Overall OLT is a feasible approach in HIV-infected patients with acceptable survival rates in Germany. Reinfection with HCV still remains a major clinical challenge in HIV/HCV coinfection after OLT.
Objective. To examine the effects of clinical hypnosis versus NLP intervention on the success rate of ECV procedures in comparison to a control group.
Methods. A prospective off-centre randomised trial of a clinical hypnosis intervention against NLP of women with a singleton breech fetus at or after 370/7 (259 days) weeks of gestation and normal amniotic fluid index. All 80 participants heard a 20-minute recorded intervention via head phones. Main outcome assessed was success rate of ECV. The intervention groups were compared with a control group with standard medical care alone (n=122).
Results. A total of 42 women, who received a hypnosis intervention prior to ECV, had a 40.5% (n=17), successful ECV, whereas 38 women, who received NLP, had a 44.7% (n=17) successful ECV (P > 0.05). The control group had similar patient characteristics compared to the intervention groups (P > 0.05). In the control group (n = 122) 27.3% (n = 33) had a statistically significant lower successful ECV procedure than NLP (P = 0.05) and hypnosis and NLP (P = 0.03).
Conclusions. These findings suggest that prior clinical hypnosis and NLP have similar success rates of ECV procedures and are both superior to standard medical care alone.
To compare the development of the auditory system in hearing and completely acoustically deprived animals, naive congenitally deaf white cats (CDCs) and hearing controls (HCs) were investigated at different developmental stages from birth till adulthood. The CDCs had no hearing experience before the acute experiment. In both groups of animals, responses to cochlear implant stimulation were acutely assessed. Electrically evoked auditory brainstem responses (E-ABRs) were recorded with monopolar stimulation at different current levels. CDCs demonstrated extensive development of E-ABRs, from first signs of responses at postnatal (p.n.) day 3 through appearance of all waves of brainstem response at day 8 p.n. to mature responses around day 90 p.n.. Wave I of E-ABRs could not be distinguished from the artifact in majority of CDCs, whereas in HCs, it was clearly separated from the stimulus artifact. Waves II, III, and IV demonstrated higher thresholds in CDCs, whereas this difference was not found for wave V. Amplitudes of wave III were significantly higher in HCs, whereas wave V amplitudes were significantly higher in CDCs. No differences in latencies were observed between the animal groups. These data demonstrate significant postnatal subcortical development in absence of hearing, and also divergent effects of deafness on early waves II–IV and wave V of the E-ABR.
In our previous work we showed that NGAL, a protein involved in the regulation of proliferation and differentiation, is overexpressed in human breast cancer (BC) and predicts poor prognosis. In neoadjuvant chemotherapy (NACT) pathological complete response (pCR) is a predictor for outcome. The aim of this study was to evaluate NGAL as a predictor of response to NACT and to validate NGAL as a prognostic factor for clinical outcome in patients with primary BC. Immunohistochemistry was performed on tissue microarrays from 652 core biopsies from BC patients, who underwent NACT in the GeparTrio trial. NGAL expression and intensity was evaluated separately. NGAL was detected in 42.2% of the breast carcinomas in the cytoplasm. NGAL expression correlated with negative hormone receptor (HR) status, but not with other baseline parameters. NGAL expression did not correlate with pCR in the full population, however, NGAL expression and staining intensity were significantly associated with higher pCR rates in patients with positive HR status. In addition, strong NGAL expression correlated with higher pCR rates in node negative patients, patients with histological grade 1 or 2 tumors and a tumor size <40 mm. In univariate survival analysis, positive NGAL expression and strong staining intensity correlated with decreased disease-free survival (DFS) in the entire cohort and different subgroups, including HR positive patients. Similar correlations were found for intense staining and decreased overall survival (OS). In multivariate analysis, NGAL expression remained an independent prognostic factor for DFS. The results show that in low-risk subgroups, NGAL was found to be a predictive marker for pCR after NACT. Furthermore, NGAL could be validated as an independent prognostic factor for decreased DFS in primary human BC.
Pre-publication peer review of scientific literature in its present state suffers from a lack of evaluation validity and transparency to the community. Inspired by social networks, we propose a framework for the open exchange of post-publication evaluation to complement the current system. We first formulate a number of necessary conditions that should be met by any design dedicated to perform open scientific evaluation. To introduce our framework, we provide a basic data standard and communication protocol. We argue for the superiority of a provider-independent framework, over a few isolated implementations, which allows the collection and analysis of open evaluation content across a wide range of diverse providers like scientific journals, research institutions, social networks, publishers websites, and more. Furthermore, we describe how its technical implementation can be achieved by using existing web standards and technology. Finally, we illustrate this with a set of examples and discuss further potential.
The junctional adhesion molecule (JAM)-C is a widely expressed adhesion molecule regulating cell adhesion, cell polarity and inflammation. JAM-C expression and function in the central nervous system (CNS) has been poorly characterized to date. Here we show that JAM-C−/− mice backcrossed onto the C57BL/6 genetic background developed a severe hydrocephalus. An in depth immunohistochemical study revealed specific immunostaining for JAM-C in vascular endothelial cells in the CNS parenchyma, the meninges and in the choroid plexus of healthy C57BL/6 mice. Additional JAM-C immunostaining was detected on ependymal cells lining the ventricles and on choroid plexus epithelial cells. Despite the presence of hemorrhages in the brains of JAM-C−/− mice, our study demonstrates that development of the hydrocephalus was not due to a vascular function of JAM-C as endothelial re-expression of JAM-C failed to rescue the hydrocephalus phenotype of JAM-C−/− C57BL/6 mice. Evaluation of cerebrospinal fluid (CSF) circulation within the ventricular system of JAM-C−/− mice excluded occlusion of the cerebral aqueduct as the cause of hydrocephalus development but showed the acquisition of a block or reduction of CSF drainage from the lateral to the 3rd ventricle in JAM-C−/− C57BL/6 mice. Taken together, our study suggests that JAM-C−/− C57BL/6 mice model the important role for JAM-C in brain development and CSF homeostasis as recently observed in humans with a loss-of-function mutation in JAM-C.
Background: Liver cirrhosis is associated with high morbidity and mortality. MicroRNAs (miRs) circulating in the blood are an emerging new class of biomarkers. In particular, the serum level of the liver-specific miR-122 might be a clinically useful new parameter in patients with acute or chronic liver disease.
Aim: Here we investigated if the serum level of miR-122 might be a prognostic parameter in patients with liver cirrhosis.
Methods: 107 patients with liver cirrhosis in the test cohort and 143 patients in the validation cohort were prospectively enrolled into the present study. RNA was extracted from the sera obtained at the time of study enrollment and the level of miR-122 was assessed. Serum miR-122 levels were assessed by quantitative reverse-transcription PCR (RT-PCR) and were compared to overall survival time and to different complications of liver cirrhosis.
Results: Serum miR-122 levels were reduced in patients with hepatic decompensation in comparison to patients with compensated liver disease. Patients with ascites, spontaneous bacterial peritonitis and hepatorenal syndrome had significantly lower miR-122 levels than patients without these complications. Multivariate Cox regression analysis revealed that the miR-122 serum levels were associated with survival independently from the MELD score, sex and age.
Conclusions: Serum miR-122 is a new independent marker for prediction of survival of patients with liver cirrhosis.
Einleitung: Beim allergischen Asthma bronchiale handelt es sich um eine weltweit zunehmende Erkrankung, für die es bislang nur wenige kausale Therapien gibt. In der Therapie chronisch-entzündlicher Erkrankungen wie der chronischen Rhinitis, Sinusitis, Akne vulgaris oder auch dem Asthma bronchiale, werden seit vielen Jahrzehnten autologe Autovaccine eingesetzt und stellen eine effektive Behandlungsoption dar.
Methoden: In einer prospektiven, randomisierten, doppelblinden Studie wurden 31 Probanden mit einem Asthma GINA I° sowie einem positiven Prick-Hauttest und einer positiven bronchialen Allergenprovokation mit Hausstaubmilbe entweder mit einer autologen E. coli Autovaccine (AV) oder einem Placebo über 24 Wochen behandelt, woraufhin eine abschließende Provokationsphase folgte. Als primären Zielparameter bestimmten wir das exhalative Stickstoffmonoxid (eNO) als Marker der bronchialen Entzündung. Die sekundären Zielparameter waren die bronchiale Hyperreagibilität vor und nach Behandlung sowie nach Provokation, das Allergie-Labor (Gesamt-IgE, spezifisches IgE gegen Hausstaubmilbe), die klinische Verträglichkeit sowie die Spätreaktion und der Medikamentenverbrauch in der abschließenden Provokationswoche.
Ergebnisse: Die Patienten der AV-Gruppe (36,7 ppb) fielen unter Behandlung signifikant auf 24,2 ppb ab, während die Placebo-Gruppe (26,8 ppb) mit 30,6 ppb eher angestiegen war. Bezüglich der bronchialen Hyperreagibilität ergab sich in der AV-Gruppe ebenfalls eine deutliche Verbesserung im Vergleich zur Placebo-Gruppe: Placebo 1,17 mg vs. AV 0,51 mg vor Behandlung auf Placebo 1,03 mg vs. AV 0,99 mg nach Behandlung. Sowohl bezüglich der BHR als auch der eNO-Werte konnten diese Ergebnisse unter Provokation nicht bestätigt werden. In beiden Gruppen stieg das eNO unter den abschließenden bronchialen Allergenprovokationen signifikant auf 104,4 ppb in der Placebo-Gruppe und 91,1 ppb in der AV-Gruppe an. Während der abschließenden Provokationsphase zeigten sich in der AV-Gruppe ein signifikant geringerer Salbutamol-Bedarf sowie ein signifikant geringeres Auftreten von Spätreaktionen (LAR) im Vergleich zur Placebo-Gruppe. Es traten nur leichte, selbst limitierende Lokalreaktionen auf.
Diskussion: Die Anwendung einer autologen E. coli Autovaccine war sicher und gut verträglich. Die Ergebnisse zeigten einen positiven Einfluss auf die bronchiale Entzündung und Hyperreagibilität bei leichten Asthmatikern mit Hausstaubmilben-Allergie und stellen so eine neue Behandlungsoption dar. Zum genauen Wirkmechanismus der AV bedarf es weiterer Studien.
Due to recent technical developments, a high number of long non-coding RNAs (lncRNAs) have been discovered in mammals. Although it has been shown that lncRNAs are regulated differently among tissues and disease statuses, functions of these transcripts are still unknown in most cases. GeneChip Exon 1.0 ST Arrays (exon arrays) from Affymetrix, Inc. have been used widely to profile genome-wide expression changes and alternative splicing of protein-coding genes. Here, we demonstrate that re-annotation of exon array probes can be used to profile expressions of tens of thousands of lncRNAs. With this annotation, a detailed inspection of lncRNAs and their isoforms is possible. To allow for a general usage to the research community, we developed a user-friendly web interface called 'noncoder'. By uploading CEL files from exon arrays and with a few mouse clicks and parameter settings, exon array data will be normalized and analysed to identify differentially expressed lncRNAs. Noncoder provides the detailed annotation information of lncRNAs and is equipped with unique features to allow for an efficient search for interesting lncRNAs to be studied further. The web interface is available at http://noncoder.mpi-bn.mpg.de.
Cytokines play an important role in ischemic injury and repair. However, little is known about their prognostic value in cardiovascular disease. The aim of this study was to investigate the prognostic importance of chemokines CCL3/MIP-1α, CCL5/RANTES and CCL18/PARC for the risk of future cardiovascular events in patients with acute coronary syndromes (ACS). Baseline levels of CCL3/MIP-1α, CCL5/RANTES and CCL18/PARC were determined in ACS patients from the Bad Nauheim ACS II registry (n = 609). During the following 200 days, patients were monitored for the occurrence of fatal and non-fatal cardiovascular events. Patients with CCL3/MIP1α, CCL5/RANTES and CCL18/PARC concentrations in the highest tertile were associated with an increased risk of a fatal event during follow-up (HR: 2.19, 95%CI: 1.04–4.61 for CCL3/MIP1α, HR: 3.45, 95%CI: 1.54–7.72 for CCL5/RANTES and HR: 3.14, 95%CI: 1.33–7.46 for CCL18/PARC). This risk was highest for patients with all three biomarkers concentrations in the upper tertile (HR: 2.52, 95%CI: 1.11–5.65). Together with known risk predictors of cardiovascular events, CCL3/MIP-1α, CCL5/RANTES and CCL18/PARC combined improved the c-statistics from 0.74 to 0.81 (p = 0.007). In conclusion, CCL3/MIP-1α, CCL5/RANTES and CCL18/PARC are independently associated with the risk of short-term mortality in ACS patients. Combining all three biomarkers further increased their prognostic value.
In the systemic circulation, 11,12-epoxyeicosatrienoic acid (11,12-EET) elicits nitric oxide (NO)- and prostacyclin-independent vascular relaxation, partially through the activation of large conductance Ca2+-activated potassium (BK) channels. However, in the lung 11,12-EET contributes to hypoxia-induced pulmonary vasoconstriction. Since pulmonary artery smooth muscle cells also express BK channels, we assessed the consequences of BKβ1 subunit deletion on pulmonary responsiveness to 11,12-EET as well as to acute hypoxia. In buffer-perfused mouse lungs, hypoxia increased pulmonary artery pressure and this was significantly enhanced in the presence of NO synthase (NOS) and cyclooxygenase (COX) inhibitors. Under these conditions the elevation of tissue EET levels using an inhibitor of the soluble epoxide hydrolase (sEH-I), further increased the hypoxic contraction. Direct administration of 11,12-EET also increased pulmonary artery pressure, and both the sEH-I and 11,12-EET effects were prevented by iberiotoxin and absent in BKβ1−/− mice. In pulmonary artery smooth muscle cells treated with NOS and COX inhibitors and loaded with the potentiometric dye, di-8-ANEPPS, 11,12-EET induced depolarization while the BK channel opener NS1619 elicited hyperpolarization indicating there was no effect of the EET on classical plasma membrane BK channels. In pulmonary artery smooth muscle cells a subpopulation of BK channels is localized in mitochondria. In these cells, 11,12-EET elicited an iberiotoxin-sensitive loss of mitochondrial membrane potential (JC-1 fluorescence) leading to plasma membrane depolarization, an effect not observed in BKβ1−/− cells. Mechanistically, stimulation with 11,12-EET time-dependently induced the association of the BK α and β1 subunits. Our data indicate that in the absence of NO and prostacyclin 11,12-EET contributes to pulmonary vasoconstriction by stimulating the association of the α and β1 subunits of mitochondrial BK channels. The 11,12-EET-induced activation of BK channels results in loss of the mitochondrial membrane potential and depolarization of the pulmonary artery smooth muscle cells.
Background: Vesicular stomatitis virus (VSV) is a potent candidate vaccine vector for various viral diseases (e.g. HIV, HCV, RSV). The biggest limitation of VSV, however, is its neurotoxicity, which limits application in humans. The second drawback is that VSV induces neutralizing antibodies rapidly and is thus ineffective as a vaccine vector upon repeated applications. Our group has recently shown that VSV pseudotyped with the glycoprotein (GP) of the lymphocytic choriomeningitis virus (LCMV), VSV-GP, is not neurotoxic. The aim of this project was to evaluate the potential of VSV-GP as a vaccine vector.
Methods: For this purpose, we used Ovalbumin (OVA) as a model antigen and analyzed immunogenicity of GP-pseudotyped and wildtype VSV containing OVA (VSV-GP-OVA and VSV-OVA) in vitro and in vivo in mouse models.
Results: We showed that both vectors infected murine bone marrow-derived dendritic cells (bmDCs) in vitro. These bmDCs were able to activate OVA specific CD8+ and CD4+ T cells. Immunization experiments in mice revealed that both VSV-OVA and VSV-GP-OVA induced functional OVA-specific cytotoxic T cells (CTLs) after a single immunization. In addition, with both viruses, mice generated antibodies against OVA. However, boosting with the same virus was only possible for the GP-pseudotyped virus but not for wild type VSV. The efficacy of repeated immunization with VSV-OVA was most likely limited by high levels of neutralizing antibodies, which we detected after the first immunization. In contrast, no neutralizing antibodies against VSV-GP were induced even after boosting.
Conclusion: Taken together, we showed that the non-neurotoxic VSV-GP is able to induce specific T cell and B cell responses against the model antigen OVA to the same level as the wild type VSV vector. However, in contrast to wild type VSV, VSV-GP-OVA boosted the immune response upon repeated applications. Thus, VSV-GP is a promising novel vaccine vector.
Background: Both standard and low-dose allergen provocations are an established tool in asthma research to improve our understanding of the pathophysiological mechanism of allergic asthma. However, clinical symptoms are less likely to be induced. Therefore, we designed a protocol for repetitive high-dose bronchial allergen challenges to generate clinical symptoms and airway inflammation.
Methods: A total of 27 patients aged 18 to 40 years with positive skin-prick tests and mild asthma underwent repetitive high-dose allergen challenges with household dust mites for four consecutive days. Pulmonary function and exhaled NO were measured at every visit. Induced sputum was analysed before and after the allergen challenges for cell counts, ECP, IL-5, INF-γ, IL-8, and the transcription factor Foxp3.
Results: We found a significant decrease in pulmonary function, an increased use of salbutamol and the development of a late asthmatic response and bronchial hyperresponsiveness, as well as a significant induction of eNO, eosinophils, and Th-2 cytokines. Repeated provocation was feasible in the majority of patients. Two subjects had severe adverse events requiring prednisolone to cope with nocturnal asthma symptoms.
Conclusions: Repeated high-dose bronchial allergen challenges resulted in severe asthma symptoms and marked Th-2-mediated allergic airway inflammation. The high-dose challenge model is suitable only in an attenuated form in diseased volunteers for proof-of-concept studies and in clinical settings to reduce the risk of severe asthma exacerbations.
Trial registration: ClinicalTrials.govNCT00677209
Decorin, a member of the small leucine-rich proteoglycan gene family, exists and functions wholly within the tumor microenvironment to suppress tumorigenesis by directly targeting and antagonizing multiple receptor tyrosine kinases, such as the EGFR and Met. This leads to potent and sustained signal attenuation, growth arrest, and angiostasis. We thus sought to evaluate the tumoricidal benefits of systemic decorin on a triple-negative orthotopic breast carcinoma xenograft model. To this end, we employed a novel high-density mixed expression array capable of differentiating and simultaneously measuring gene signatures of both Mus musculus (stromal) and Homo sapiens (epithelial) tissue origins. We found that decorin protein core modulated the differential expression of 374 genes within the stromal compartment of the tumor xenograft. Further, our top gene ontology classes strongly suggests an unexpected and preferential role for decorin protein core to inhibit genes necessary for immunomodulatory responses while simultaneously inducing expression of those possessing cellular adhesion and tumor suppressive gene properties. Rigorous verification of the top scoring candidates led to the discovery of three genes heretofore unlinked to malignant breast cancer that were reproducibly found to be induced in several models of tumor stroma. Collectively, our data provide highly novel and unexpected stromal gene signatures as a direct function of systemic administration of decorin protein core and reveals a fundamental basis of action for decorin to modulate the tumor stroma as a biological mechanism for the ascribed anti-tumorigenic properties.
Ischemia/reperfusion (I/R) is at the basis of renal transplantation and acute kidney injury. Molecular mechanisms underlying proximal tubule response to I/R will allow the identification of new therapeutic targets for both clinical settings. microRNAs have emerged as crucial and tight regulators of the cellular response to insults including hypoxia. Here, we have identified several miRNAs involved in the response of the proximal tubule cell to I/R. Microarrays and RT-PCR analysis of proximal tubule cells submitted to I/R mimicking conditions in vitro demonstrated that miR-127 is induced during ischemia and also during reperfusion. miR-127 is also modulated in a rat model of renal I/R. Interference approaches demonstrated that ischemic induction of miR-127 is mediated by Hypoxia Inducible Factor-1alpha (HIF-1α) stabilization. Moreover, miR-127 is involved in cell-matrix and cell-cell adhesion maintenance, since overexpression of miR-127 maintains focal adhesion complex assembly and the integrity of tight junctions. miR-127 also regulates intracellular trafficking since miR-127 interference promotes dextran-FITC uptake. In fact, we have identified the Kinesin Family Member 3B (KIF3B), involved in cell trafficking, as a target of miR-127 in rat proximal tubule cells. In summary, we have described a novel role of miR-127 in cell adhesion and its regulation by HIF-1α. We also identified for the first time KIF3B as a miR-127 target. Both, miR-127 and KIF3B appear as key mediators of proximal epithelial tubule cell response to I/R with potential al application in renal ischemic damage management.
nefficient intracellular protein trafficking is a critical issue in the pathogenesis of a variety of diseases and in recombinant protein production. Here we investigated the trafficking of factor VIII (FVIII), which is affected in the coagulation disorder hemophilia A. We hypothesized that chemical chaperones may be useful to enhance folding and processing of FVIII in recombinant protein production, and as a therapeutic approach in patients with impaired FVIII secretion. A tagged B-domain-deleted version of human FVIII was expressed in cultured Chinese Hamster Ovary cells to mimic the industrial production of this important protein. Of several chemical chaperones tested, the addition of betaine resulted in increased secretion of FVIII, by increasing solubility of intracellular FVIII aggregates and improving transport from endoplasmic reticulum to Golgi. Similar results were obtained in experiments monitoring recombinant full-length FVIII. Oral betaine administration also increased FVIII and factor IX (FIX) plasma levels in FVIII or FIX knockout mice following gene transfer. Moreover, in vitro and in vivo applications of betaine were also able to rescue a trafficking-defective FVIII mutant (FVIIIQ305P). We conclude that chemical chaperones such as betaine might represent a useful treatment concept for hemophilia and other diseases caused by deficient intracellular protein trafficking.
A subgroup of pediatric acute T-lymphoblastic leukemia (T-ALL) was characterized by a gene expression profile comparable to that of early T-cell precursors (ETPs) with a highly unfavorable outcome. We have investigated clinical and molecular characteristics of the ETP-ALL subgroup in adult T-ALL. As ETP-ALL represents a subgroup of early T-ALL we particularly focused on this cohort and identified 178 adult patients enrolled in the German Acute Lymphoblastic Leukemia Multicenter studies (05/93–07/03). Of these, 32% (57/178) were classified as ETP-ALL based on their characteristic immunophenotype. The outcome of adults with ETP-ALL was poor with an overall survival of only 35% at 10 years, comparable to the inferior outcome of early T-ALL with 38%. The molecular characterization of adult ETP-ALL revealed distinct alterations with overexpression of stem cell-related genes (BAALC, IGFBP7, MN1, WT1). Interestingly, we found a low rate of NOTCH1 mutations and no FBXW7 mutations in adult ETP-ALL. In contrast, FLT3 mutations, rare in the overall cohort of T-ALL, were very frequent and nearly exclusively found in ETP-ALL characterized by a specific immunophenotype. These molecular characteristics provide biologic insights and implications with respect to innovative treatment strategies (for example, tyrosine kinase inhibitors) for this high-risk subgroup of adult ETP-ALL.
Spinocerebellar Ataxia Type 2 (SCA2) is caused by expansion of a polyglutamine encoding triplet repeat in the human ATXN2 gene beyond (CAG)31. This is thought to mediate toxic gain-of-function by protein aggregation and to affect RNA processing, resulting in degenerative processes affecting preferentially cerebellar neurons. As a faithful animal model, we generated a knock-in mouse replacing the single CAG of murine Atxn2 with CAG42, a frequent patient genotype. This expansion size was inherited stably. The mice showed phenotypes with reduced weight and later motor incoordination. Although brain Atxn2 mRNA became elevated, soluble ATXN2 protein levels diminished over time, which might explain partial loss-of-function effects. Deficits in soluble ATXN2 protein correlated with the appearance of insoluble ATXN2, a progressive feature in cerebellum possibly reflecting toxic gains-of-function. Since in vitro ATXN2 overexpression was known to reduce levels of its protein interactor PABPC1, we studied expansion effects on PABPC1. In cortex, PABPC1 transcript and soluble and insoluble protein levels were increased. In the more vulnerable cerebellum, the progressive insolubility of PABPC1 was accompanied by decreased soluble protein levels, with PABPC1 mRNA showing no compensatory increase. The sequestration of PABPC1 into insolubility by ATXN2 function gains was validated in human cell culture. To understand consequences on mRNA processing, transcriptome profiles at medium and old age in three different tissues were studied and demonstrated a selective induction of Fbxw8 in the old cerebellum. Fbxw8 is encoded next to the Atxn2 locus and was shown in vitro to decrease the level of expanded insoluble ATXN2 protein. In conclusion, our data support the concept that expanded ATXN2 undergoes progressive insolubility and affects PABPC1 by a toxic gain-of-function mechanism with tissuespecific effects, which may be partially alleviated by the induction of FBXW8.
Background: Acoustic Radiation Force Impulse (ARFI)-imaging is an ultrasound-based elastography method enabling quantitative measurement of tissue stiffness. The aim of the present study was to evaluate sensitivity and specificity of ARFI-imaging for differentiation of thyroid nodules and to compare it to the well evaluated qualitative real-time elastography (RTE).
Methods: ARFI-imaging involves the mechanical excitation of tissue using acoustic pulses to generate localized displacements resulting in shear-wave propagation which is tracked using correlation-based methods and recorded in m/s. Inclusion criteria were: nodules $5 mm, and cytological/histological assessment. All patients received conventional ultrasound, real-time elastography (RTE) and ARFI-imaging.
Results: One-hundred-fifty-eight nodules in 138 patients were available for analysis. One-hundred-thirty-seven nodules were benign on cytology/histology, and twenty-one nodules were malignant. The median velocity of ARFI-imaging in the healthy thyroid tissue, as well as in benign and malignant thyroid nodules was 1.76 m/s, 1.90 m/s, and 2.69 m/s, respectively. While no significant difference in median velocity was found between healthy thyroid tissue and benign thyroid nodules, a significant difference was found between malignant thyroid nodules on the one hand and healthy thyroid tissue (p = 0.0019) or benign thyroid nodules (p = 0.0039) on the other hand. No significant difference of diagnostic accuracy for the diagnosis of malignant thyroid nodules was found between RTE and ARFI-imaging (0.74 vs. 0.69, p = 0.54). The combination of RTE with ARFI did not improve diagnostic accuracy.
Conclusions: ARFI can be used as an additional tool in the diagnostic work up of thyroid nodules with high negative predictive value and comparable results to RTE.
Purpose: To investigate the prognostic value of survivin expression in pretreatment specimens from patients with anal cancer treated with concurrent 5-FU and mitomycin C-based chemoradiation (CRT).
Material and methods: Immunohistochemical staining for survivin was performed in pretreatment biopsies of 62 patients with anal carcinoma. Survivin expression was correlated with clinical and histopathological characteristics as well as local failure free- (LFFS), distant metastases free- (DMFS), cancer specific- (CSS), and overall survival (OS).
Results: Survivin staining intensity was weak in 10%, intermediate in 48% and intense in 42% of the patients. No association between survivin expression and clinicopathologic factors (tumor stage, age and HIV status) could be shown. In univariate analysis, the level of survivin staining was significantly correlated with DMFS (low survivin vs. high survivin: 94% vs. 74%, p=0.04). T-stage, N-stage and the tumor grading were significantly associated with OS and CSS and with DMFS and LFFS, respectively. In multivariate analysis, survivin was confirmed as independent prognostic parameter for DMFS (RR, 0.04; p=0.02) and for OS (RR, 0.27; p=0.04).
Conclusion: Our results demonstrated that the level of pretreatment survivin is correlated with the clinical outcome in patients with anal carcinoma treated with concurrent CRT. Further studies are warranted to elucidate the complex role of survivin for the oncologic treatment and to exploit the protein as a therapeutic target in combined modality treatment of anal cancer.
SHARPIN (SHANK-associated RH domain interacting protein) is part of a large multi-protein E3 ubiquitin ligase complex called LUBAC (linear ubiquitin chain assembly complex), which catalyzes the formation of linear ubiquitin chains and regulates immune and apoptopic signaling pathways. The C-terminal half of SHARPIN contains ubiquitin-like domain and Npl4-zinc finger domains that mediate the interaction with the LUBAC subunit HOIP and ubiquitin, respectively. In contrast, the N-terminal region does not show any homology with known protein interaction domains but has been suggested to be responsible for self-association of SHARPIN, presumably via a coiled-coil region. We have determined the crystal structure of the N-terminal portion of SHARPIN, which adopts the highly conserved pleckstrin homology superfold that is often used as a scaffold to create protein interaction modules. We show that in SHARPIN, this domain does not appear to be used as a ligand recognition domain because it lacks many of the surface properties that are present in other pleckstrin homology fold-based interaction modules. Instead, it acts as a dimerization module extending the functional applications of this superfold.
Tumor progression and pregnancy share many common features, such as immune tolerance and invasion. The invasion of trophoblasts in the placenta into the uterine wall is essential for fetal development, and is thus precisely regulated. Its deregulation has been implicated in preeclampsia, a leading cause for maternal and perinatal mortality and morbidity. Pathogenesis of preeclampsia remains to be defined. Microarray-based gene profiling has been widely used for identifying genes responsible for preeclampsia. In this review, we have summarized the recent data from the microarray studies with preeclamptic placentas. Despite the complex of gene signatures, suggestive of the heterogeneity of preeclampsia, these studies identified a number of differentially expressed genes associated with preeclampsia. Interestingly, most of them have been reported to be tightly involved in tumor progression. We have discussed these interesting genes and analyzed their potential molecular functions in preeclampsia, compared with their roles in malignancy development. Further investigations are warranted to explore the involvement in molecular network of each identified gene, which may provide not only novel strategies for prevention and therapy for preeclampsia but also a better understanding of cancer cells. The trophoblastic cells, with their capacity for proliferation and differentiation, apoptosis and survival, migration, angiogenesis and immune modulation by exploiting similar molecular pathways, make them a compelling model for cancer research.
Activation of TRPC6 channels is essential for lung ischaemia–reperfusion induced oedema in mice
(2012)
Lung ischaemia–reperfusion-induced oedema (LIRE) is a life-threatening condition that causes pulmonary oedema induced by endothelial dysfunction. Here we show that lungs from mice lacking nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox2y/−) or the classical transient receptor potential channel 6 (TRPC6−/−) are protected from LIR-induced oedema (LIRE). Generation of chimeric mice by bone marrow cell transplantation and endothelial-specific Nox2 deletion showed that endothelial Nox2, but not leukocytic Nox2 or TRPC6, are responsible for LIRE. Lung endothelial cells from Nox2- or TRPC6-deficient mice showed attenuated ischaemia-induced Ca2+ influx, cellular shape changes and impaired barrier function. Production of reactive oxygen species was completely abolished in Nox2y/− cells. A novel mechanistic model comprising endothelial Nox2-derived production of superoxide, activation of phospholipase C-γ, inhibition of diacylglycerol (DAG) kinase, DAG-mediated activation of TRPC6 and ensuing LIRE is supported by pharmacological and molecular evidence. This mechanism highlights novel pharmacological targets for the treatment of LIRE.
Previous PET and MRI studies have indicated that the degree to which pathology translates into clinical symptoms is strongly dependent on sex with women more likely to express pathology as a diagnosis of AD, whereas men are more resistant to clinical symptoms in the face of the same degree of pathology. Here we use DTI to investigate the difference between male and female white matter tracts in healthy older participants (24 women, 16 men) and participants with mild cognitive impairment (21 women, 12 men). Differences between control and MCI participants were found in fractional anisotropy (FA), radial diffusion (DR), axial diffusion (DA) and mean diffusion (MD). A significant main effect of sex was also reported for FA, MD and DR indices, with male control and male MCI participants having significantly more microstructural damage than their female counterparts. There was no sex by diagnosis interaction. Male MCIs also had significantly less normalised grey matter (GM) volume than female MCIs. However, in terms of absolute brain volume, male controls had significantly more brain volume than female controls. Normalised GM and WM volumes were found to decrease significantly with age with no age by sex interaction. Overall, these data suggest that the same degree of cognitive impairment is associated with greater structural damage in men compared with women.
Background: In patients with genotype 1 chronic hepatitis C infection, telaprevir (TVR) in combination with peginterferon and ribavirin (PR) significantly increased sustained virologic response (SVR) rates compared with PR alone. However, genotypic changes could be observed in TVR-treated patients who did not achieve an SVR.
Methods: Population sequence analysis of the NS3•4A region was performed in patients who did not achieve SVR with TVR-based treatment.
Results: Resistant variants were observed after treatment with a telaprevir-based regimen in 12% of treatment-naïve patients (ADVANCE; T12PR arm), 6% of prior relapsers, 24% of prior partial responders, and 51% of prior null responder patients (REALIZE, T12PR48 arms). NS3 protease variants V36M, R155K, and V36M+R155K emerged frequently in patients with genotype 1a and V36A, T54A, and A156S/T in patients with genotype 1b. Lower-level resistance to telaprevir was conferred by V36A/M, T54A/S, R155K/T, and A156S variants; and higher-level resistance to telaprevir was conferred by A156T and V36M+R155K variants. Virologic failure during telaprevir treatment was more common in patients with genotype 1a and in prior PR nonresponder patients and was associated with higher-level telaprevir-resistant variants. Relapse was usually associated with wild-type or lower-level resistant variants. After treatment, viral populations were wild-type with a median time of 10 months for genotype 1a and 3 weeks for genotype 1b patients.
Conclusions: A consistent, subtype-dependent resistance profile was observed in patients who did not achieve an SVR with telaprevir-based treatment. The primary role of TVR is to inhibit wild-type virus and variants with lower-levels of resistance to telaprevir. The complementary role of PR is to clear any remaining telaprevir-resistant variants, especially higher-level telaprevir-resistant variants. Resistant variants are detectable in most patients who fail to achieve SVR, but their levels decline over time after treatment.
Introduction: Acute kidney injury (AKI) after cardiac surgery increases length of hospital stay and in-hospital mortality. A significant number of patients undergoing cardiac surgical procedures require perioperative intra-aortic balloon pump (IABP) support. Use of an IABP has been linked to an increased incidence of perioperative renal dysfunction and death. This might be due to dislodgement of atherosclerotic material in the descending thoracic aorta (DTA). Therefore, we retrospectively studied the correlation between DTA atheroma, AKI and in-hospital mortality.
Methods: A total of 454 patients were retrospectively matched to one of four groups: -IABP/-DTA atheroma, +IABP/-DTA atheroma, -IABP/+DTA atheroma, +IABP/+DTA atheroma. Patients were then matched according to presence/absence of DTA atheroma, presence/absence of IABP, performed surgical procedure, age, gender and left ventricular ejection fraction (LVEF). DTA atheroma was assessed through standard transesophageal echocardiography (TEE) imaging studies of the descending thoracic aorta.
Results: Basic patient characteristics, except for age and gender, did not differ between groups. Perioperative AKI in patients with -DTA atheroma/+IABP was 5.1% versus 1.7% in patients with -DTA atheroma/-IABP. In patients with +DTA atheroma/+IABP the incidence of AKI was 12.6% versus 5.1% in patients with +DTA atheroma/-IABP. In-hospital mortality in patients with +DTA atheroma/-IABP was 3.4% versus 8.4% with +DTA atheroma/+IABP. In patients with +DTA atheroma/+IABP in hospital mortality was 20.2% versus 6.4% with +DTA atheroma/-IABP. Multivariate logistic regression identified DTA atheroma > 1 mm (P = *0.002, odds ratio (OR) = 4.13, confidence interval (CI) = 1.66 to 10.30), as well as IABP support (P = *0.015, OR = 3.04, CI = 1.24 to 7.45) as independent predictors of perioperative AKI and increased in-hospital mortality. DTA atheroma in conjunction with IABP significantly increased the risk of developing acute kidney injury (P = 0.0016) and in-hospital mortality (P = 0.0001) when compared to control subjects without IABP and without DTA atheroma.
Conclusions: Perioperative IABP and DTA atheroma are independent predictors of perioperative AKI and in-hospital mortality. Whether adding an IABP in patients with severe DTA calcification increases their risk of developing AKI and mortality postoperatively cannot be clearly answered in this study. Nevertheless, when IABP and DTA are combined, patients are more likely to develop AKI and to die postoperatively in comparison to patients without IABP and DTA atheroma.
Expansions of the polyglutamine (polyQ) domain (≥34) in Ataxin-2 (ATXN2) are the primary cause of spinocerebellar ataxia type 2 (SCA2). Recent studies reported that intermediate-length (27–33) expansions increase the risk of Amyotrophic Lateral Sclerosis (ALS) in 1–4% of cases in diverse populations. This study investigates the Turkish population with respect to ALS risk, genotyping 158 sporadic, 78 familial patients and 420 neurologically healthy controls. We re-assessed the effect of ATXN2 expansions and extended the analysis for the first time to cover the ATXN2 locus with 18 Single Nucleotide Polymorphisms (SNPs) and their haplotypes. In accordance with other studies, our results confirmed that 31–32 polyQ repeats in the ATXN2 gene are associated with risk of developing ALS in 1.7% of the Turkish ALS cohort (p = 0.0172). Additionally, a significant association of a 136 kb haplotype block across the ATXN2 and SH2B3 genes was found in 19.4% of a subset of our ALS cohort and in 10.1% of the controls (p = 0.0057, OR: 2.23). ATXN2 and SH2B3 encode proteins that both interact with growth receptor tyrosine kinases. Our novel observations suggest that genotyping of SNPs at this locus may be useful for the study of ALS risk in a high percentage of individuals and that ATXN2 and SH2B3 variants may interact in modulating the disease pathway.
Visual perception is highly variable and can be influenced by the surrounding world. Previous research has revealed that body perception can be biased due to adaptation to thin or fat body shapes. The aim of the present study was to show that adaptation to certain body shapes and the resulting perceptual biases transfer across different identities of adaptation and test stimuli. We designed two similar adaptation experiments in which healthy female participants adapted to pictures of either thin or fat bodies and subsequently compared more or less distorted pictures of their own body to their actual body shape. In the first experiment (n = 16) the same identity was used as adaptation and test stimuli (i.e. pictures of the participant’s own body) while in the second experiment (n = 16) we used pictures of unfamiliar thin or fat bodies as adaptation stimuli. We found comparable adaptation effects in both experiments: After adaptation to a thin body, participants rated a thinner than actual body picture to be the most realistic and vice versa. We therefore assume that adaptation to certain body shapes transfers across different identities. These results raise the questions of whether some type of natural adaptation occurs in everyday life. Natural and predominant exposure to certain bodily features like body shape – especially the thin ideal in Western societies – could bias perception for these features. In this regard, further research might shed light on aspects of body dissatisfaction and the development of body image disturbances in terms of eating disorders.
Background: With increasing numbers of lamellar keratoplasties, eye banks are challenged to deliver precut lamellar donor tissue. In Europe, the most common technique of corneal storage is organ culture which requires a deswelling process before surgical processing. The aim of this study was to investigate the influence of different deswelling times on the cutting plane quality after microkeratome-assisted lamellar dissection.
Methods: Eight paired donor corneas (16 specimens) not suitable for transplantation were organ cultured under standard conditions at the Eye Bank of the Ludwig-Maximilians Universität, Munich, Germany. Pairs of corneal buttons were analyzed during the deswelling process in dextrane-containing medium. While one cornea was cut at an early time point during the deswelling process and put back into deswelling medium thereafter, the partner cornea was completely deswollen and dissected after 72 hours. Specimens were then further processed for scanning electron microscopy. Surface quality was assessed both digitally using Scanning Probe Imaging Processing software, and manually by three blinded graders.
Results: The corneal buttons processed at the beginning of the deswelling process had a smoother surface when compared to the partner cornea that was cut at the end of the deswelling process. In our setting, no relevant difference was detectable between manual and automated microkeratome dissection.
Conclusion: For lamellar keratoplasty, organ-cultured corneas should be processed at an early stage during the deswelling process. We interpret the smoother dissection plane during early deswelling as a result of mechanical properties in a highly hydrated cornea.
Chronic kidney disease (CKD) is an important cause of morbidity and mortality in HIV-positive individuals. Hepatitis C (HCV) co-infection has been associated with increased risk of CKD, but prior studies lack information on potential mechanisms. We evaluated the association between HCV or hepatitis B (HBV) co-infection and progressive CKD among 3,441 antiretroviral-treated clinical trial participants. Progressive CKD was defined as the composite of end-stage renal disease, renal death, or significant glomerular filtration rate (eGFR) decline (25% decline to eGFR <60 mL/min/1.73 m2 or 25% decline with a baseline <60). Generalized Estimating Equations were used to model the odds of progressive CKD. At baseline, 13.8% and 3.3% of participants were co-infected with HCV and HBV, respectively. Median eGFR was 111, and 3.7% developed progressive CKD. After adjustment, the odds of progressive CKD were increased in participants with HCV (OR 1.72, 95% CI 1.07–2.76) or HBV (OR 2.26, 95% CI 1.15–4.44). Participants with undetectable or low HCV-RNA had similar odds of progressive CKD as HCV seronegative participants, while participants with HCV-RNA >800,000 IU/ml had increased odds (OR 3.07; 95% CI 1.60–5.90). Interleukin-6, hyaluronic acid, and the FIB-4 hepatic fibrosis index were higher among participants who developed progressive CKD, but were no longer associated with progressive CKD after adjustment. Future studies should validate the relationship between HCV viremia and CKD.
Background: To perform a comprehensive study on the relationship between vitamin D metabolism and the response to interferon-α-based therapy of chronic hepatitis C.
Methodology/Principal Findings: Associations between a functionally relevant polymorphism in the gene encoding the vitamin D 1α-hydroxylase (CYP27B1-1260 rs10877012) and the response to treatment with pegylated interferon-α (PEG-IFN-α) and ribavirin were determined in 701 patients with chronic hepatitis C. In addition, associations between serum concentrations of 25-hydroxyvitamin D3 (25[OH]D3) and treatment outcome were analysed. CYP27B1-1260 rs10877012 was found to be an independent predictor of sustained virologic response (SVR) in patients with poor-response IL28B genotypes (15% difference in SVR for rs10877012 genotype AA vs. CC, p = 0.02, OR = 1.52, 95% CI = 1.061–2.188), but not in patients with favourable IL28B genotype. Patients with chronic hepatitis C showed a high prevalence of vitamin D insufficiency (25[OH]D3<20 ng/mL) during all seasons, but 25(OH)D3 serum levels were not associated with treatment outcome.
Conclusions/Significance: Our study suggests a role of bioactive vitamin D (1,25[OH]2D3, calcitriol) in the response to treatment of chronic hepatitis C. However, serum concentration of the calcitriol precursor 25(OH)D3 is not a suitable predictor of treatment outcome.
Background and Aims: The prevalence of hepatitis C virus (HCV) antibodies in Germany has been estimated to be in the range of 0.4–0.63%. Screening for HCV is recommended in patients with elevated ALT levels or significant risk factors for HCV transmission only. However, 15–30% of patients report no risk factors and ALT levels can be normal in up to 20–30% of patients with chronic HCV infection. The aim of this study was to assess the HCV seroprevalence in patients visiting two tertiary care emergency departments in Berlin and Frankfurt, respectively.
Methods: Between May 2008 and March 2010, a total of 28,809 consecutive patients were screened for the presence of anti-HCV antibodies. Anti-HCV positive sera were subsequently tested for HCV-RNA.
Results: The overall HCV seroprevalence was 2.6% (95% CI: 2.4–2.8; 2.4% in Berlin and 3.5% in Frankfurt). HCV-RNA was detectable in 68% of anti-HCV positive cases. Thus, the prevalence of chronic HCV infection in the overall study population was 1.6% (95% CI 1.5–1.8). The most commonly reported risk factor was former/current injection drug use (IDU; 31.2%) and those with IDU as the main risk factor were significantly younger than patients without IDU (p<0.001) and the male-to-female ratio was 72% (121 vs. 46 patients; p<0.001). Finally, 18.8% of contacted HCV-RNA positive patients had not been diagnosed previously.
Conclusions: The HCV seroprevalence was more than four times higher compared to current estimates and almost one fifth of contacted HCV-RNA positive patients had not been diagnosed previously.
Recent evidence has demonstrated additional roles for the neuronal guidance protein receptor UNC5B outside the nervous system. Given the fact that ischemia reperfusion injury (IRI) of the liver is a common source of liver dysfunction and the role of UNC5B during an acute inflammatory response we investigated the role of UNC5B on acute hepatic IRI. We report here that UNC5B+/− mice display reduced hepatic IRI and neutrophil (PMN) infiltration compared to WT controls. This correlated with serum levels of lactate dehydrogenase (LDH), aspartate- (AST) and alanine- (ALT) aminotransferase, the presence of PMN within ischemic hepatic tissue, and serum levels of inflammatory cytokines. Moreover, injection of an anti-UNC5B antibody resulted in a significant reduction of hepatic IR injury. This was associated with reduced parameters of liver injury (LDH, ALT, AST) and accumulation of PMN within the injured hepatic tissue. In conclusion our studies demonstrate a significant role for UNC5B in the development of hepatic IRI and identified UNC5B as a potential drug target to prevent liver dysfunction in the future.
Background: Dopamine plays an important role in orienting and the regulation of selective attention to relevant stimulus characteristics. Thus, we examined the influences of functional variants related to dopamine inactivation in the dopamine transporter (DAT1) and catechol-O-methyltransferase genes (COMT) on the time-course of visual processing in a contingent negative variation (CNV) task.
Methods: 64-channel EEG recordings were obtained from 195 healthy adolescents of a community-based sample during a continuous performance task (A-X version). Early and late CNV as well as preceding visual evoked potential components were assessed.
Results: Significant additive main effects of DAT1 and COMT on the occipito-temporal early CNV were observed. In addition, there was a trend towards an interaction between the two polymorphisms. Source analysis showed early CNV generators in the ventral visual stream and in frontal regions. There was a strong negative correlation between occipito-temporal visual post-processing and the frontal early CNV component. The early CNV time interval 500–1000 ms after the visual cue was specifically affected while the preceding visual perception stages were not influenced.
Conclusions: Late visual potentials allow the genomic imaging of dopamine inactivation effects on visual post-processing. The same specific time-interval has been found to be affected by DAT1 and COMT during motor post-processing but not motor preparation. We propose the hypothesis that similar dopaminergic mechanisms modulate working memory encoding in both the visual and motor and perhaps other systems.
Staurosporine induces necroptotic cell death under caspase-compromised conditions in U937 cells
(2012)
For a long time necrosis was thought to be an uncontrolled process but evidences recently have revealed that necrosis can also occur in a regulated manner. Necroptosis, a type of programmed necrosis is defined as a death receptor-initiated process under caspase-compromised conditions. The process requires the kinase activity of receptor-interacting protein kinase 1 and 3 (RIPK1 and RIPK3) and mixed lineage kinase domain-like protein (MLKL), as a substrate of RIPK3. The further downstream events remain elusive. We applied known inhibitors to characterize the contributing enzymes in necroptosis and their effect on cell viability and different cellular functions were detected mainly by flow cytometry. Here we report that staurosporine, the classical inducer of intrinsic apoptotic pathway can induce necroptosis under caspase-compromised conditions in U937 cell line. This process could be hampered at least partially by the RIPK1 inhibitor necrotstin-1 and by the heat shock protein 90 kDa inhibitor geldanamycin. Moreover both the staurosporine-triggered and the classical death ligand-induced necroptotic pathway can be effectively arrested by a lysosomal enzyme inhibitor CA-074-OMe and the recently discovered MLKL inhibitor necrosulfonamide. We also confirmed that the enzymatic role of poly(ADP-ribose)polymerase (PARP) is dispensable in necroptosis but it contributes to membrane disruption in secondary necrosis. In conclusion, we identified a novel way of necroptosis induction that can facilitate our understanding of the molecular mechanisms of necroptosis. Our results shed light on alternative application of staurosporine, as a possible anticancer therapeutic agent. Furthermore, we showed that the CA-074-OMe has a target in the signaling pathway leading to necroptosis. Finally, we could differentiate necroptotic and secondary necrotic processes based on participation of PARP enzyme.
Background: Dabigatran etexilate (DE) is a new oral direct thrombin inhibitor. Clinical trials point towards a favourable risk-to-benefit profile of DE compared to warfarin. In this study, we evaluated whether hemorrhagic transformation (HT) occurs after experimental stroke under DE treatment as we have shown for warfarin.
Methods: 44 male C57BL/6 mice were pretreated orally with 37.5 mg/kg DE, 75 mg/kg DE or saline and diluted thrombin time (dTT) and DE plasma concentrations were monitored. Ischemic stroke was induced by transient middle cerebral artery occlusion (tMCAO) for 1 h or 3 h. We assessed functional outcome and HT blood volume 24 h and 72 h after tMCAO.
Results: After 1 h tMCAO, HT blood volume did not differ significantly between mice pretreated with DE 37.5 mg/kg and controls (1.5±0.5 µl vs. 1.8±0.5 µl, p>0.05). After 3 h tMCAO, DE-anticoagulated mice did also not show an increase in HT, neither at the dose of 37.5 mg/kg equivalent to anticoagulant treatment in the therapeutic range (1.3±0.9 µl vs. control 2.3±0.5 µl, p>0.05) nor at 75 mg/kg, clearly representing supratherapeutic anticoagulation (1.8±0.8 µl, p>0.05). Furthermore, no significant increase in HT under continued anticoagulation with DE 75 mg/kg could be found at 72 h after tMCAO for 1 h (1.7±0.9 µl vs. control 1.6±0.4 µl, p>0.05).
Conclusion: Our experimental data suggest that DE does not significantly increase hemorrhagic transformation after transient focal cerebral ischemia in mice. From a translational viewpoint, this indicates that a continuation of DE anticoagulation in case of an ischemic stroke might be safe, but clearly, clinical data on this question are warranted.
Background: Adaptation to low oxygen by changing gene expression is vitally important for cell survival and tissue development. The sprouting of new blood vessels, initiated from endothelial cells, restores the oxygen supply of ischemic tissues. In contrast to the transcriptional response induced by hypoxia, which is mainly mediated by members of the HIF family, there are only few studies investigating alternative splicing events. Therefore, we performed an exon array for the genome-wide analysis of hypoxia-related changes of alternative splicing in endothelial cells.
Methodology/Principal findings: Human umbilical vein endothelial cells (HUVECs) were incubated under hypoxic conditions (1% O(2)) for 48 h. Genome-wide transcript and exon expression levels were assessed using the Affymetrix GeneChip Human Exon 1.0 ST Array. We found altered expression of 294 genes after hypoxia treatment. Upregulated genes are highly enriched in glucose metabolism and angiogenesis related processes, whereas downregulated genes are mainly connected to cell cycle and DNA repair. Thus, gene expression patterns recapitulate known adaptations to low oxygen supply. Alternative splicing events, until now not related to hypoxia, are shown for nine genes: six which are implicated in angiogenesis-mediated cytoskeleton remodeling (cask, itsn1, larp6, sptan1, tpm1 and robo1); one, which is involved in the synthesis of membrane-anchors (pign) and two universal regulators of gene expression (cugbp1 and max).
Conclusions/Significance: For the first time, this study investigates changes in splicing in the physiological response to hypoxia on a genome-wide scale. Nine alternative splicing events, until now not related to hypoxia, are reported, considerably expanding the information on splicing changes due to low oxygen supply. Therefore, this study provides further knowledge on hypoxia induced gene expression changes and presents new starting points to study the hypoxia adaptation of endothelial cells.
Adaptation of wild-type p53 expressing UKF-NB-3 cancer cells to the murine double minute 2 inhibitor nutlin-3 causes de novo p53 mutations at high frequency (13/20) and multi-drug resistance. Here, we show that the same cells respond very differently when adapted to RITA, a drug that, like nutlin-3, also disrupts the p53/Mdm2 interaction. All of the 11 UKF-NB-3 sub-lines adapted to RITA that we established retained functional wild-type p53 although RITA induced a substantial p53 response. Moreover, all RITA-adapted cell lines remained sensitive to nutlin-3, whereas only five out of 10 nutlin-3-adapted cell lines retained their sensitivity to RITA. In addition, repeated adaptation of the RITA-adapted sub-line UKF-NB-3rRITA10 μM to nutlin-3 resulted in p53 mutations. The RITA-adapted UKF-NB-3 sub-lines displayed no or less pronounced resistance to vincristine, cisplatin, and irradiation than nutlin-3-adapted UKF-NB-3 sub-lines. Furthermore, adaptation to RITA was associated with fewer changes at the expression level of antiapoptotic factors than observed with adaptation to nutlin-3. Transcriptomic analyses indicated the RITA-adapted sub-lines to be more similar at the gene expression level to the parental UKF-NB-3 cells than nutlin-3-adapted UKF-NB-3 sub-lines, which correlates with the observed chemotherapy and irradiation sensitivity phenotypes. In conclusion, RITA-adapted cells retain functional p53, remain sensitive to nutlin-3, and display a less pronounced resistance phenotype than nutlin-3-adapted cells.
Urban health is potentially affected by particle emissions. The potential toxicity of nanoparticles is heavily debated and there is an enormous global increase in research activity in this field. In this respect, it is commonly accepted that nanoparticles may also be generated in processes occurring while driving vehicles. So far, a variety of studies addressed traffic-related particulate matter emissions, but only few studies focused on potential nanoparticles. Therefore, the present study analyzed the literature with regard to nanoparticles and cars. It can be stated that, to date, only a limited amount of research has been conducted in this area and more studies are needed to 1) address kind and sources of nanoparticles within automobiles and to 2) analyse whether there are health effects caused by these nanoparticles.
Bicycle traumata are very common and especially neurologic complications lead to disability and death in all stages of the life. This review assembles the most recent findings concerning research in the field of bicycle traumata combined with the factor of bicycle helmet use. The area of bicycle trauma research is by nature multidisciplinary and relevant not only for physicians but also for experts with educational, engineering, judicial, rehabilitative or public health functions. Due to this plurality of global publications and special subjects, short time reviews help to detect recent research directions and provide also information from neighbour disciplines for researchers. It can be stated that to date, that although a huge amount of research has been conducted in this area more studies are needed to evaluate and improve special conditions and needs in different regions, ages, nationalities and to create successful prevention programs of severe head and face injuries while cycling. Focus was explicit the bicycle helmet use, wherefore sledding, ski and snowboard studies were excluded and only one study concerning electric bicycles remained due to similar motion structures within this review. The considered studies were all published between January 2010 and August 2011 and were identified via the online databases Medline PubMed and ISI Web of Science.
Increasing evidence about the central nervous representation of pain in the brain suggests that the operculo-insular cortex is a crucial part of the pain matrix. The pain-specificity of a brain region may be tested by administering nociceptive stimuli while controlling for unspecific activations by administering non-nociceptive stimuli. We applied this paradigm to nasal chemosensation, delivering trigeminal or olfactory stimuli, to verify the pain-specificity of the operculo-insular cortex. In detail, brain activations due to intranasal stimulation induced by non-nociceptive olfactory stimuli of hydrogen sulfide (5 ppm) or vanillin (0.8 ppm) were used to mask brain activations due to somatosensory, clearly nociceptive trigeminal stimulations with gaseous carbon dioxide (75% v/v). Functional magnetic resonance (fMRI) images were recorded from 12 healthy volunteers in a 3T head scanner during stimulus administration using an event-related design. We found that significantly more activations following nociceptive than non-nociceptive stimuli were localized bilaterally in two restricted clusters in the brain containing the primary and secondary somatosensory areas and the insular cortices consistent with the operculo-insular cortex. However, these activations completely disappeared when eliminating activations associated with the administration of olfactory stimuli, which were small but measurable. While the present experiments verify that the operculo-insular cortex plays a role in the processing of nociceptive input, they also show that it is not a pain-exclusive brain region and allow, in the experimental context, for the interpretation that the operculo-insular cortex splay a major role in the detection of and responding to salient events, whether or not these events are nociceptive or painful.
1 Purpose of the Study:
The purpose of this retrospective study was to assess the volumetric changes of our institutional pediatric neuroblastoma in response to various therapeutic protocols.
2 Materials and Methods:
A retrospective study was conducted on children with neuroblastoma from different anatomical locations including suprarenal, paraspinal, pelvic, mediastinal and cervical neuroblastoma primaries. These children underwent tumor-stage based therapeutic protocols in Johann Wolfgang Goethe University Hospital, Frankfurt am Main, Germany, between January 1996 and July 2008. The study included 72 patients (44 males and 28 females). Patient demographics (age and gender), disease-related symptoms, laboratory results (tumor biomarkers including ferritin, neuron specific enolase, and urine catecholamine) and histopathological reports were collected from the electronic medical archiving system and subsequently analyzed.
Patients were classified into following groups according the anatomical origin of the primary neuroblastoma into:
1) Suprarenal neuroblastoma Group: This group included patients with neuroblastoma arising from the suprarenal gland. This group composed of 54 patients with male to female ratio (32:22).
2) Paravertebral neuroblastoma Group: This group composed of 6 male patients.
3) Mediastinal neuroblastoma Group: This group included patients with mediastinal neuroblastoma and composed of 3 patients (1 male and 2 females).
4) Pelvic neuroblastoma Group: This group included patients with pelvic neuroblastoma and composed of 6 patients (3 males and 3 females).
5) Cervical neuroblastoma Group: This group included patients with cervical neuroblastoma and composed of 2 male patients.
3 Results:
The mean volume of all suprarenal neuroblastoma group involved in the study before therapy was 176.62 cm3 (SD: 234.15) range: 239.4-968.9cm3. The mean initial volume of all suprarenal neuroblastoma group who underwent observation protocol was 86.0378 cm3 (SD: 114.44) range: 5.2-347.94cm3. Volumetric evaluation of suprarenal neuroblastoma following observation (Wait and See) protocol revealed continuous reduction of the tumor volumes in a statistically significant manner during the follow up periods up to 12 months with p value of less than 0.05. The volumetric changes afterwards were statistically insignificant.
The mean initial volume of all suprarenal neuroblastoma group who underwent primary surgery protocol was 42.4 cm3 (SD: 28.5) range: 7.5-90cm3. Complete surgical resection of the tumor was not feasible in all lesions due to local tumor extension and / or infiltration with the associated risk of injury of nearby organs or structures. However statistical analysis of the volumetric changes in the successive follow up periods did not reveal statistical significance.
Volumetric estimation of the tumor in the subsequent follow up periods revealed significant changes within the period first (3-9 month periods). The changes afterwards were statistically non significant. On the other hand, the mean initial volume of all suprarenal neuroblastoma group who underwent combined chemotherapy and Stem cell transplantation protocol only without surgical interference was 99.98cm3 (SD:46.2) range: 48.48-160.48 cm3. In this group the volumetric changes were variable and difference in volumes in follow up was statistically non significant during the follow up period.
The mean initial volume of all abdominal paravertebral neuroblastoma group was 249.197cm3 (SD: 249.63) range: 9.6-934cm3. The mean initial volume of all pelvic neuroblastoma group was 118.88cm3 (SD: 50.61) range: 73.4-173.4cm3. The mean initial volume of all mediastinal neuroblastoma group was 189.7cm3 (SD: 139.057) range: 10.7-415 cm3. The mean initial volume of all cervical neuroblastoma group was 189.7cm3 (SD: 139.057) range: 10.7-415 cm3. The volumetric measurements in the corresponding follow up periods according to the therapeutic protocol of abdominal paravertebral neuroblastoma, pelvic neuroblastoma, mediastinal and cervical neuroblastoma revealed significant change in the tumor volume within the early 3-6 months from the initial therapy while subsequently the tumor volumetric changes were statistically non significant.
4 Conclusion:
In conclusion, the role of MRI volumetry in the evaluation of tumor response is dependent on the risk adapted concept of neuroblastoma with the combination of different imaging modalities as well the therapeutic protocol. MRI Volumetry in addition to new protocols such as Whole-body imaging and 3D visualization techniques are gaining more importance and acceptance.
Mitochondrial dynamics and mitophagy play a key role in ensuring mitochondrial quality control. Impairment thereof was proposed to be causative to neurodegenerative diseases, diabetes, and cancer. Accumulation of mitochondrial dysfunction was further linked to aging. Here we applied a probabilistic modeling approach integrating our current knowledge on mitochondrial biology allowing us to simulate mitochondrial function and quality control during aging in silico. We demonstrate that cycles of fusion and fission and mitophagy indeed are essential for ensuring a high average quality of mitochondria, even under conditions in which random molecular damage is present. Prompted by earlier observations that mitochondrial fission itself can cause a partial drop in mitochondrial membrane potential, we tested the consequences of mitochondrial dynamics being harmful on its own. Next to directly impairing mitochondrial function, pre-existing molecular damage may be propagated and enhanced across the mitochondrial population by content mixing. In this situation, such an infection-like phenomenon impairs mitochondrial quality control progressively. However, when imposing an age-dependent deceleration of cycles of fusion and fission, we observe a delay in the loss of average quality of mitochondria. This provides a rational why fusion and fission rates are reduced during aging and why loss of a mitochondrial fission factor can extend life span in fungi. We propose the ‘mitochondrial infectious damage adaptation’ (MIDA) model according to which a deceleration of fusion–fission cycles reflects a systemic adaptation increasing life span.
Background: Valvular aortic stenosis is a common disease in the elderly, often in multimorbid patients. It is often associated with coronary artery disease and peripheral artery disease. In this situation, the risk of conventional open-heart surgery is too high, and other treatment strategies have to be evaluated.
Case report: A 79-year-old female patient with severe aortic stenosis, coronary artery disease and end-stage chronic obstructive pulmonary disease suffering from dyspnea at rest and permanently dependent on oxygen was treated in three steps. Firstly, her pulmonary infection was treated with antibiotics for 7 days. Then, the left anterior descending artery was stented (bare-metal stent). In the same session, valvuloplasty of the aortic valve was performed. She was sent to rehabilitation to improve her pulmonary condition and took clopidogrel for 4 weeks. Finally, she underwent transapical aortic valve replacement. She was released to rehabilitation on postoperative day 12.
Conclusion: A combination of modern interventional and minimally invasive surgical techniques to treat aortic stenosis and coronary heart disease can be a viable option for multimorbid patients with extremely high risk in conventional open-heart surgery.
Objective: Sensorineural hearing loss leads to the progressive degeneration of spiral ganglion cells (SGC). Next to postoperative fibrous tissue growth, which should be suppressed to assure a close nerve–electrode interaction, the density of healthy SGC is one factor that influences the efficiency of cochlear implants (CI), the choice of treatment for affected patients. Rolipram, a phosphodiesterase-4 inhibitor, has proven neuroprotective and anti-inflammatory effects and might also reduce SGC degeneration and fibrosis, but it has to pass the cellular membrane to be biologically active.
Methods: Lipidic nanocapsules (LNC) can be used as biodegradable drug carriers to increase the efficacy of conventional application methods. We examined the biological effects of rolipram and LNC's core encapsulated rolipram on SGC and dendritic cell (DC) tumor necrosis factor-α (TNF-α) production in vitro and on SGC survival in systemically-deafened guinea pigs in vivo.
Results: Our results prove that rolipram does not have a beneficial effect on cultured SGC. Incorporation of rolipram in LNC increased the survival of SGC significantly. In the DC study, rolipram significantly inhibited TNF-α in a dose-dependent manner. The rolipram-loaded LNC provided a significant cytokine inhibition as well. In vivo data do not confirm the in vitro results.
Conclusion: By transporting rolipram into the SGC cytoplasm, LNC enabled the neuroprotective effect of rolipram in vitro, but not in vivo. This might be due to dilution of test substances by perilymph or an inadequate release of rolipram based on differing in vivo and in vitro conditions. Nevertheless, based on in vitro results, proving a significantly increased neuronal survival when using LNC-rolipram compared to pure rolipram and pure LNC application, we believe that the combination of rolipram and LNC can potentially reduce neuronal degeneration and fibrosis after CI implantation. We conclude that rolipram is a promising drug that can be used in inner ear therapy and that LNC have potential as an inner ear drug-delivery system. Further experiments with modified conditions might reveal in vivo biological effects.
The synthesis of the recently characterized depsipeptide szentiamide (1), which is produced by the entomopathogenic bacterium Xenorhabdus szentirmaii, is described. Whereas no biological activity was previously identified for 1, the material derived from the efficient synthesis enabled additional bioactivity tests leading to the identification of a notable activity against insect cells and Plasmodium falciparum, the causative agent of malaria.
Objective: Betahistine is a histamine H1-receptor agonist and H3-receptor antagonist that is administered to treat Menière’s disease. Despite widespread use, its pharmacological mode of action has not been entirely elucidated. This study investigated the effect of betahistine on guinea pigs at dosages corresponding to clinically used doses for cochlear microcirculation.
Methods: Thirty healthy Dunkin-Hartley guinea pigs were randomly assigned to five groups to receive betahistine dihydrochloride in a dose of 1,000 mg/kg b. w. (milligram per kilogram body weight), 0.100 mg/kg b. w., 0.010 mg/kg b. w., 0.001 mg/kg b. w. in NaCl 0.9% or NaCl 0.9% alone as placebo. Cochlear blood flow and mean arterial pressure were continuously monitored by intravital fluorescence microscopy and invasive blood pressure measurements 3 minutes before and 15 minutes after administration of betahistine.
Results: When betahistine was administered in a dose of 1.000 mg/kg b. w. cochlear blood flow was increased to a peak value of 1.340 arbitrary units (SD: 0.246; range: 0.933–1.546 arb. units) compared to baseline (p<0.05; Two Way Repeated Measures ANOVA/Bonferroni t-test). The lowest dosage of 0.001 mg/kg b. w. betahistine or NaCl 0.9% had the same effect as placebo. Nonlinear regression revealed that there was a sigmoid correlation between increase in blood flow and dosages.
Conclusions: Betahistine has a dose-dependent effect on the increase of blood flow in cochlear capillaries. The effects of the dosage range of betahistine on cochlear microcirculation corresponded well to clinically used single dosages to treat Menière’s disease. Our data suggest that the improved effects of higher doses of betahistine in the treatment of Menière’s disease might be due to a corresponding increase of cochlear blood flow.
The main goal of adequate organ preservation is to avoid further cellular metabolism during the phase of ischemia. However, modern preservation solutions do rarely achieve this target. In donor organs hypoxia and ischemia induce a broad spectrum of pathologic molecular mechanisms favoring primary graft dysfunction (PGD) after transplantation. Increased hypoxia-induced transcriptional activity leads to increased vascular permeability which in turn is the soil of a reperfusion edema and the enhancement of a pro-inflammatory response in the graft after reperfusion. We hypothesize that inhibition of the respiration chain in mitochondria and thus inhibition of the hypoxia induced mechanisms might reduce reperfusion edema and consecutively improve survival in vivo. In this study we demonstrate that the rotenoid Deguelin reduces the expression of hypoxia induced target genes, and especially VEGF-A, dose-dependently in hypoxic human lung derived cells. Furthermore, Deguelin significantly suppresses the mRNA expression of the HIF target genes VEGF-A, the pro-inflammatory CXCR4 and ICAM-1 in ischemic lungs vs. control lungs. After lung transplantation, the VEGF-A induced reperfusion-edema is significantly lower in Deguelin-treated animals than in controls. Deguelin-treated rats exhibit a significantly increased survival-rate after transplantation. Additionally, a downregulation of the pro-inflammatory molecules ICAM-1 and CXCR4 and an increase in the recruitment of immunomodulatory monocytes (CD163+ and CD68+) to the transplanted organ involving the IL4 pathway was observed. Therefore, we conclude that ischemic periods preceding reperfusion are mainly responsible for the increased vascular permeability via upregulation of VEGF. Together with this, the resulting endothelial dysfunction also enhances inflammation and consequently lung dysfunction. Deguelin significantly decreases a VEGF-A induced reperfusion edema, induces the recruitment of immunomodulatory monocytes and thus improves organ function and survival after lung transplantation by interfering with hypoxia induced signaling.
Prostate cancer is one of the most common tumor diseases worldwide. Often being non-aggressive, prostate tumors in these cases do not need immediate treatment. However, about 20% of diagnosed prostate cancers tend to metastasize and require treatment. Existing diagnostic methods may fail to accurately recognize the transition of a dormant, non-aggressive tumor into highly malignant prostate cancer. Therefore, new diagnostic tools are needed to improve diagnosis and therapy of prostate carcinoma. This review evaluates existing methods to diagnose prostate carcinoma, such as the biochemical marker prostate-specific antigen (PSA), but also discusses the possibility to use the altered expression of integrins and laminin-332 in prostate carcinomas as diagnostic tools and therapeutic targets of prostate cancer.
VEGF (vascular endothelial growth factor) promotes vascularization and remodeling of bone substitutes. The aim of this study was to examine the effect of distinct resorbable ceramic carriers on bone forming capacities of VEGF transfected bone marrow stromal cells (BMSC). A critical size defect of the radius in rabbits was filled either by a low surface scaffold called beta-TCP (tricalciumphsphate) or the high surface scaffold CDHA (calcium deficient hydroxy-apatite) loaded with autologous BMSC, which were either transfected with a control plasmid or a plasmid coding for phVEGF165. They were compared to unloaded scaffolds. Thus, six treatment groups (n = 6 in each group) were followed by X-ray over 16 weeks. After probe retrieval, the volume of new bone was measured by micro-CT scans and vascularization was assessed in histology. While only minor bone formation was found in both carriers when implanted alone, BMSC led to increased osteogenesis in both carriers. VEGF promoted vascularization of the scaffolds significantly in contrast to BMSC alone. Bone formation was increased in the beta-TCP group, whereas it was inhibited in the CDHA group that showed faster scaffold degradation. The results indicate that the interaction of VEGF transfected BMSC with resorbable ceramic carrier influences the ability to promote bone healing.
Speech production involves widely distributed brain regions. This MEG study focuses on the spectro-temporal dynamics that contribute to the setup of this network. In 21 participants performing a cue-target reading paradigm, we analyzed local oscillations during preparation for overt and covert reading in the time-frequency domain and localized sources using beamforming. Network dynamics were studied by comparing different dynamic causal models of beta phase coupling in and between hemispheres. While a broadband low frequency effect was found for any task preparation in bilateral prefrontal cortices, preparation for overt speech production was specifically associated with left-lateralized alpha and beta suppression in temporal cortices and beta suppression in motor-related brain regions. Beta phase coupling in the entire speech production network was modulated by anticipation of overt reading. We propose that the processes underlying the setup of the speech production network connect relevant brain regions by means of beta synchronization and prepare the network for left-lateralized information routing by suppression of inhibitory alpha and beta oscillations.
Introduction: In this article the personal study and life situation of parents who are also medical students at the Medical School of the Goethe University Frankfurt am Main is discussed. There is a special focus on the topics "studying with children" and "family-friendly university", which have been present in discussions about university development and in the daily life of academics, especially during the last decade. The workgroup "Individual Student Services" at the medical faculty at the Goethe University tries to meet the necessities of the individual study courses and to support the study success with a new counselling and student service concept.
Methods: The experience of parents studying medicine was recorded in semi-structured interviews (Date: April 2010), which were held as part of the sponsored pilot project on part-time medical studies ("Pilot Project Part-time Medical Studies"). Additionally, study results from the Medical School of the Goethe University Frankfurt am Main were integrated as well as a literature analysis.
Results: It was found that the teaching demands and support services, which have been suggested and needed for years now, have been partially implemented and are without sufficient support at the faculty level to date. Thus the current situation of medical students with children is still difficult and seems a big challenge for everyone involved.
Solution: As part of the "Individual Student Services" a new pilot project on part-time medical studies was established in November 2009. Only the use of new, unconventional and innovative ideas allows universities to adequately support the changing and heterogeneous student population and support them to successfully completing their medical studies.
Einleitung: In dem vorliegenden Artikel wird die persönliche Studien- und Lebenssituation von Studierenden mit Kindern am Fachbereich Medizin der Goethe-Universität Frankfurt am Main beleuchtet. Der spezielle Fokus liegt auf den Themen "Studium mit Kind" sowie "familiengerechte Hochschule", welche in Deutschland in der letzten Dekade sehr präsent sowohl in der Hochschulforschung als auch im akademischen Alltag sind. Hier versucht die Arbeitsstelle "Individuelle Studienbegleitung" am FB Medizin der Goethe-Universität durch ein neues Beratungs- und Servicekonzept mit wissenschaftlicher Begleitforschung den individuellen Studienverläufen gerecht zu werden und den Studienerfolg zu fördern.
Methoden: In erster Linie stammen die hier beschriebenen Erfahrungen der Medizinstudierenden mit Kind aus qualitativen Leitfadeninterviews (Stand April 2010), die im Rahmen des Modellprojektes Teilzeitstudium Medizin durchgeführt wurden. Ergänzend wurden die Studienergebnisse des Fachbereichs Medizin der Goethe-Universität Frankfurt am Main herangezogen und eine Literaturanalyse einbezogen.
Ergebnisse: Als Ergebnis kann festgehalten werden, dass die bereits seit mehreren Jahren erhobenen Forderungen und gewünschten Unterstützungsleistungen zwar zum Teil umgesetzt, aber noch nicht ausreichend an allen Fachbereichen implementiert wurden. Somit ist die aktuelle Situation von Studierenden mit Kindern in der Medizin nach wie vor schwierig und stellt für alle Beteiligten eine große Herausforderung dar.
Lösungsansatz: Im Rahmen der Arbeitsstelle "Individuelle Studienbegleitung" konnte im November 2009 das "Modellprojekt Teilzeitstudium Medizin" aufgebaut werden. Nur durch neue, unkonventionelle und innovative Ideen kann man die Sicherstellung der medizinischen Ausbildung erreichen und die heutige heterogene Studierendenschaft adäquat ansprechen sowie Studierende erfolgreich durch ihr Studium begleiten.
In this study, it is demonstrated that moving sounds have an effect on the direction in which one sees visual stimuli move. During the main experiment sounds were presented consecutively at four speaker locations inducing left or rightward auditory apparent motion. On the path of auditory apparent motion, visual apparent motion stimuli were presented with a high degree of directional ambiguity. The main outcome of this experiment is that our participants perceived visual apparent motion stimuli that were ambiguous (equally likely to be perceived as moving left or rightward) more often as moving in the same direction than in the opposite direction of auditory apparent motion. During the control experiment we replicated this finding and found no effect of sound motion direction on eye movements. This indicates that auditory motion can capture our visual motion percept when visual motion direction is insufficiently determinate without affecting eye movements.
Evaluation der SSRI-Medikation bei Kindern und Jugendlichen bezüglich ansteigender Suizidalität
(2012)
Zielsetzung dieser Arbeit war die Klärung der Frage, inwieweit eine SSRI-Medikation
bei Kindern und Jugendlichen mit dem Ansteigen der Suizidalität in Zusammenhang
steht. Es sollte untersucht werden, ob Suizidgedanken und Suizidverhalten im
Verlauf eines stationären Aufenthaltes allgemein ansteigen, diese nur bei
vorliegender Medikation mit SSRI ansteigen, etwa nur bei bereits vorliegender
Suizidalität ansteigen, oder ob suizidales Gedankengut allein unter einer
Wechselwirkung der Faktoren „SSRI-Medikation“ und „vorliegende Suizidalität“
entsteht.
Zur Klärung der Fragen wurden im Rahmen einer klinischen Studie Patienten
zwischen 12 und 19 Jahren über ein Fragebogeninventar befragt. Die Stichprobe
wurde konsekutiv im Zeitraum von Juni 2004 bis Dezember 2006 aus den
aufgenommen Patienten im Zentrum der Kinder- und Jugendpsychiatrie des
Klinikums der Johann Wolfgang Goethe-Universität in Frankfurt am Main rekrutiert.
Sie umfasste 44 Patienten. Ausgeschlossen wurden hierbei hochgradig geistig
retardierte Patienten, Patienten mit ausgeprägter Lese- und Rechtschreibschwäche,
sowie psychotisch erkrankte Patienten. Zur Teilnahme an der Studie war eine
Einverständniserklärung von Seiten der Eltern nötig. Zur Untersuchung der
Fragestellung wurde ein spezielles Fragebogeninventar erstellt zu dem der SBQ,
BSI, JTCI 12-18, YSR, EWL, die Achse V und die wöchentliche Erhebung externaler
Stressoren gehört. Die Suizidalität wurde so hinsichtlich eines Zusammenhangs mit
Depressivität, Impulsivität, Ängstlichkeit, Aggressivität, externalen sozialen Faktoren
und einer SSRI-Medikation in einem Gesamtzeitraum von fünf Wochen untersucht.
Das Gesamtkollektiv teilte sich in 14 männliche und 30 weibliche Patienten. Der
jüngste Patient war 12 Jahre, der älteste 19 Jahre (mittleres Alter 15,68).
Anhand des SBQ (d = 1,83) und des BSI (d = 2,09) Fragebogeninventars konnten
Suizidgedanken und -verhaltensweisen (Erhängen, Vergiften, etc.) sehr gut zwischen
„Suizidalen“ und „Nicht-Suizidalen“ trennen.
Die SSRI haben keinen signifikanten Effekt auf die Befindlichkeit (EWL), auf die
pathologiebezogene Aggressivität und Ängstlichkeit (YSR) und die
persönlichkeitsbezogene „Impulsivität“ und „Ängstlichkeit“ (unter 1%- Niveau).
Abschließend kann somit geschlossen werden, dass kein signifikanter
Zusammenhang zwischen der Gabe von SSRI und Suizidalität besteht.
Patients that survive hemorrhage and resuscitation (H/R) may develop a systemic inflammatory response syndrome (SIRS) that leads to dysfunction of vital organs (multiple organ dysfunction syndrome, MODS). SIRS and MODS may involve mitochondrial dysfunction. Under pentobarbital anesthesia, C57BL6 mice were hemorrhaged to 30 mm Hg for 3 h and then resuscitated with shed blood plus half the volume of lactated Ringer’s solution containing minocycline, tetracycline (both 10 mg/kg body weight) or vehicle. Serum alanine aminotransferase (ALT), necrosis, apoptosis and oxidative stress were assessed 6 h after resuscitation. Mitochondrial polarization was assessed by intravital microscopy. After H/R with vehicle or tetracycline, ALT increased to 4538 U/L and 3999 U/L, respectively, which minocycline decreased to 1763 U/L (P<0.01). Necrosis and TUNEL also decreased from 24.5% and 17.7 cells/field, respectively, after vehicle to 8.3% and 8.7 cells/field after minocycline. Tetracycline failed to decrease necrosis (23.3%) but decreased apoptosis to 9 cells/field (P<0.05). Minocycline and tetracycline also decreased caspase-3 activity in liver homogenates. Minocycline but not tetracycline decreased lipid peroxidation after resuscitation by 70% (P<0.05). Intravital microscopy showed that minocycline preserved mitochondrial polarization after H/R (P<0.05). In conclusion, minocycline decreases liver injury and oxidative stress after H/R by preventing mitochondrial dysfunction.
The human cytomegalovirus (HCMV) is suspected to increase tumour malignancy by infection of cancer and/or stroma cells (oncomodulation). So far, oncomodulatory mechanisms have been attributed to the presence of HCMV and direct action of its gene products on cancer cells. Here, we investigated whether the prolonged presence of HCMV can result in the irreversible selection of a cancer cell population with increased malignancy. The neuroblastoma cell line UKF-NB-4 was long-term (200 passages) infected with the HCMV strain Hi91 (UKF-NB-4Hi) before virus eradication using ganciclovir (UKF-NB-4HiGCV). Global gene expression profiling of UKF-NB-4, UKF-NB-4Hi and UKF-NB-4HiGCV cells and subsequent bioinformatic signal transduction pathway analysis revealed clear differences between UKF-NB-4 and UKF-NB-4Hi, as well as between UKF-NB-4 and UKF-NB-4HiGCV cells, but only minor differences between UKF-NB-4Hi and UKF-NB-4HiGCV cells. Investigation of the expression of a subset of five genes in different chronically HCMV-infected cell lines before and after virus eradication suggested that long-term HCMV infection reproducibly causes specific changes. Array comparative genomic hybridisation showed virtually the same genomic differences for the comparisons UKF-NB-4Hi/UKF-NB-4 and UKF-NB-4HiGCV/UKF-NB-4. UKF-NB-4Hi cells are characterised by an increased invasive potential compared with UKF-NB-4 cells. This phenotype was completely retained in UKF-NB-4HiGCV cells. Moreover, there was a substantial overlap in the signal transduction pathways that differed significantly between UKF-NB-4Hi/UKF-NB-4HiGCV and UKF-NB-4 cells and those differentially regulated between tumour tissues from neuroblastoma patients with favourable or poor outcome. In conclusion, we present the first experimental evidence that long-term HCMV infection can result in the selection of tumour cell populations with enhanced malignancy.
Acute ethanol gavage attenuates hemorrhage/resuscitation-induced hepatic oxidative stress in rats
(2012)
Acute ethanol intoxication increases the production of reactive oxygen species (ROS). Hemorrhagic shock with subsequent resuscitation (H/R) also induces ROS resulting in cellular and hepatic damage in vivo. We examined the role of acute ethanol intoxication upon oxidative stress and subsequent hepatic cell death after H/R. 14 h before H/R, rats were gavaged with single dose of ethanol or saline (5 g/kg, EtOH and ctrl; H/R_EtOH or H/R_ctrl, resp.). Then, rats were hemorrhaged to a mean arterial blood pressure of 30 ± 2 mmHg for 60 min and resuscitated. Two control groups underwent surgical procedures without H/R (sham_ctrl and sham_EtOH, resp.). Liver tissues were harvested at 2, 24, and 72 h after resuscitation. EtOH-gavage induced histological picture of acute fatty liver. Hepatic oxidative (4-hydroxynonenal, 4-HNE) and nitrosative (3-nitrotyrosine, 3-NT) stress were significantly reduced in EtOH-gavaged rats compared to controls after H/R. Proapoptotic caspase-8 and Bax expressions were markedly diminished in EtOH-gavaged animals compared with controls 2 h after resuscitation. EtOH-gavage increased antiapoptotic Bcl-2 gene expression compared with controls 2 h after resuscitation. iNOS protein expression increased following H/R but was attenuated in EtOH-gavaged animals after H/R. Taken together, the data suggest that acute EtOH-gavage may attenuate H/R-induced oxidative stress thereby reducing cellular injury in rat liver.
Autism spectrum disorders (ASD) are a heterogeneous group of neurodevelopmental disorders with a complex inheritance pattern. While many rare variants in synaptic proteins have been identified in patients with ASD, little is known about their effects at the synapse and their interactions with other genetic variations. Here, following the discovery of two de novo SHANK2 deletions by the Autism Genome Project, we identified a novel 421 kb de novo SHANK2 deletion in a patient with autism. We then sequenced SHANK2 in 455 patients with ASD and 431 controls and integrated these results with those reported by Berkel et al. 2010 (n = 396 patients and n = 659 controls). We observed a significant enrichment of variants affecting conserved amino acids in 29 of 851 (3.4%) patients and in 16 of 1,090 (1.5%) controls (P = 0.004, OR = 2.37, 95% CI = 1.23-4.70). In neuronal cell cultures, the variants identified in patients were associated with a reduced synaptic density at dendrites compared to the variants only detected in controls (P = 0.0013). Interestingly, the three patients with de novo SHANK2 deletions also carried inherited CNVs at 15q11-q13 previously associated with neuropsychiatric disorders. In two cases, the nicotinic receptor CHRNA7 was duplicated and in one case the synaptic translation repressor CYFIP1 was deleted. These results strengthen the role of synaptic gene dysfunction in ASD but also highlight the presence of putative modifier genes, which is in keeping with the "multiple hit model" for ASD. A better knowledge of these genetic interactions will be necessary to understand the complex inheritance pattern of ASD.
Introduction: Febrile neutropenia is a common and potentially life-threatening complication of treatment for childhood cancer, which has increasingly been subject to targeted treatment based on clinical risk stratification. Our previous meta-analysis demonstrated 16 rules had been described and 2 of them subject to validation in more than one study. We aimed to advance our knowledge of evidence on the discriminatory ability and predictive accuracy of such risk stratification clinical decision rules (CDR) for children and young people with cancer by updating our systematic review.
Methods: The review was conducted in accordance with Centre for Reviews and Dissemination methods, searching multiple electronic databases, using two independent reviewers, formal critical appraisal with QUADAS and meta-analysis with random effects models where appropriate. It was registered with PROSPERO: CRD42011001685.
Results: We found 9 new publications describing a further 7 new CDR, and validations of 7 rules. Six CDR have now been subject to testing across more than two data sets. Most validations demonstrated the rule to be less efficient than when initially proposed; geographical differences appeared to be one explanation for this.
Conclusion: The use of clinical decision rules will require local validation before widespread use. Considerable uncertainty remains over the most effective rule to use in each population, and an ongoing individual-patient-data meta-analysis should develop and test a more reliable CDR to improve stratification and optimise therapy. Despite current challenges, we believe it will be possible to define an internationally effective CDR to harmonise the treatment of children with febrile neutropenia.
Background: Panic disorder is common (5% prevalence) and females are twice as likely to be affected as males. The heritable component of panic disorder is estimated at 48%. Glutamic acid dehydrogenase GAD1, the key enzyme for the synthesis of the inhibitory and anxiolytic neurotransmitter GABA, is supposed to influence various mental disorders, including mood and anxiety disorders. In a recent association study in depression, which is highly comorbid with panic disorder, GAD1 risk allele associations were restricted to females.
Methodology/Principal Findings: Nineteen single nucleotide polymorphisms (SNPs) tagging the common variation in GAD1 were genotyped in two independent gender and age matched case-control samples (discovery sample n = 478; replication sample n = 584). Thirteen SNPs passed quality control and were examined for gender-specific enrichment of risk alleles associated with panic disorder by using logistic regression including a genotype×gender interaction term. The latter was found to be nominally significant for four SNPs (rs1978340, rs3762555, rs3749034, rs2241165) in the discovery sample; of note, the respective minor/risk alleles were associated with panic disorder only in females. These findings were not confirmed in the replication sample; however, the genotype×gender interaction of rs3749034 remained significant in the combined sample. Furthermore, this polymorphism showed a nominally significant association with the Agoraphobic Cognitions Questionnaire sum score.
Conclusions/Significance: The present study represents the first systematic evaluation of gender-specific enrichment of risk alleles of the common SNP variation in the panic disorder candidate gene GAD1. Our tentative results provide a possible explanation for the higher susceptibility of females to panic disorder.
The neurexin genes (NRXN1/2/3) encode two families (α and β) of highly polymorphic presynaptic proteins that are involved in excitatory/inhibitory synaptic balance. Recent studies indicate that neuronal activation and memory formation affect NRXN1/2/3α expression and alternative splicing at splice sites 3 and 4 (SS#3/SS#4). Neurons in the biological clock residing in the suprachiasmatic nuclei of the hypothalamus (SCN) act as self-sustained oscillators, generating rhythms in gene expression and electrical activity, to entrain circadian bodily rhythms to the 24 hours day/night cycles. Cell autonomous oscillations in NRXN1/2/3α expression and SS#3/SS#4 exons splicing and their links to rhythms in excitatory/inhibitory synaptic balance in the circadian clock were explored. NRXN1/2/3α expression and SS#3/SS#4 splicing, levels of neurexin-2α and the synaptic scaffolding proteins PSD-95 and gephyrin (representing excitatory and inhibitory synapses, respectively) were studied in mRNA and protein extracts obtained from SCN of C3H/J mice at different times of the 24 hours day/night cycle. Further studies explored the circadian oscillations in these components and causality relationships in immortalized rat SCN2.2 cells. Diurnal rhythms in mNRXN1α and mNRXN2α transcription, SS#3/SS#4 exon-inclusion and PSD-95 gephyrin and neurexin-2α levels were found in the SCN in vivo. No such rhythms were found with mNRXN3α. SCN2.2 cells also exhibited autonomous circadian rhythms in rNRXN1/2 expression SS#3/SS#4 exon inclusion and PSD-95, gephyrin and neurexin-2α levels. rNRXN3α and rNRXN1/2β were not expressed. Causal relationships were demonstrated, by use of specific siRNAs, between rNRXN2α SS#3 exon included transcripts and gephyrin levels in the SCN2.2 cells. These results show for the first time dynamic, cell autonomous, diurnal rhythms in expression and splicing of NRXN1/2 and subsequent effects on the expression of neurexin-2α and postsynaptic scaffolding proteins in SCN across the 24-h cycle. NRXNs gene transcripts may have a role in coupling the circadian clock to diurnal rhythms in excitatory/inhibitory synaptic balance.
Background: Dopamine plays an important role in orienting, response anticipation and movement evaluation. Thus, we examined the influence of functional variants related to dopamine inactivation in the dopamine transporter (DAT1) and catechol-O-methyltransferase genes (COMT) on the time-course of motor processing in a contingent negative variation (CNV) task.
Methods: 64-channel EEG recordings were obtained from 195 healthy adolescents of a community-based sample during a continuous performance task (A-X version). Early and late CNV as well as motor postimperative negative variation were assessed. Adolescents were genotyped for the COMT Val158Met and two DAT1 polymorphisms (variable number tandem repeats in the 3′-untranslated region and in intron 8).
Results: The results revealed a significant interaction between COMT and DAT1, indicating that COMT exerted stronger effects on lateralized motor post-processing (centro-parietal motor postimperative negative variation) in homozygous carriers of a DAT1 haplotype increasing DAT1 expression. Source analysis showed that the time interval 500–1000 ms after the motor response was specifically affected in contrast to preceding movement anticipation and programming stages, which were not altered.
Conclusions: Motor slow negative waves allow the genomic imaging of dopamine inactivation effects on cortical motor post-processing during response evaluation. This is the first report to point towards epistatic effects in the motor system during response evaluation, i.e. during the post-processing of an already executed movement rather than during movement programming.
Dendritic cells (DCs) and oxLDL play an important role in the atherosclerotic process with DCs accumulating in the plaques during plaque progression. Our aim was to investigate the role of oxLDL in the modulation of the DC homing-receptor CCR7 and endothelial-ligand CCL21. Methods and Results. The expression of the DC homing-receptor CCR7 and its endothelial-ligand CCL21 was examined on atherosclerotic carotic plaques of 47 patients via qRT-PCR and immunofluorescence. In vitro, we studied the expression of CCR7 on DCs and CCL21 on human microvascular endothelial cells (HMECs) in response to oxLDL. CCL21- and CCR7-mRNA levels were significantly downregulated in atherosclerotic plaques versus non-atherosclerotic controls [90% for CCL21 and 81% for CCR7 (P<0.01)]. In vitro, oxLDL reduced CCR7 mRNA levels on DCs by 30% and protein levels by 46%. Furthermore, mRNA expression of CCL21 was significantly reduced by 50% (P<0.05) and protein expression by 24% in HMECs by oxLDL (P<0.05). Conclusions. The accumulation of DCs in atherosclerotic plaques appears to be related to a downregulation of chemokines and their ligands, which are known to regulate DC migration. oxLDL induces an in vitro downregulation of CCR7 and CCL21, which may play a role in the reduction of DC migration from the plaques.
Borrelia burgdorferi evades complement-mediated killing by interacting with complement regulators through distinct complement regulator-acquiring surface proteins (CRASPs). Here, we extend our analyses to the contribution of CRASP-4 in mediating complement resistance of B. burgdorferi and its interaction with human complement regulators. CRASP-4 (also known as ErpC) was immobilized onto magnetic beads and used to capture proteins from human serum. Following Western blotting, factor H (CFH), CFH-related protein 1 (CFHR1), CFHR2, and CFHR5 were identified as ligands of CRASP-4. To analyze the impact of native CRASP-4 on mediating survival of serum-sensitive cells in human serum, a B. garinii strain was generated that ectopically expresses CRASP-4. CRASP-4-producing bacteria bound CFHR1, CFHR2, and CFHR5 but not CFH. In addition, transformed spirochetes deposited significant amounts of lethal complement components on their surface and were susceptible to human serum, thus indicating that CRASP-4 plays a subordinate role in complement resistance of B. burgdorferi.
Background. Leukotriene B4 (LTB4), a proinflammatory lipid mediator correlates well with the acute phase of Acute Respiratory Distress Syndrome (ARDS). Therefore, LTB4-levels were investigated to determine whether they might be a useful clinical marker in predicting pulmonary complications (PC) in multiply traumatized patients. Methods: Plasma levels of LTB4 were determined in 100 patients on admission (ED) and for five consecutive days (daily). Twenty healthy volunteers served as control. LTB4-levels were measured by ELISA. Thirty patients developed PC (pneumonia, respiratory failure, acute lung injury (ALI), ARDS, pulmonary embolism) and 70 had no PC (ØPC). Results. LTB4-levels in the PC-group [127.8 pg/mL, IQR: 104–200pg/ml] were significantly higher compared to the ØPC-group on admission [95.6 pg/mL, IQR: 55–143 pg/mL] or control-group [58.4 pg/mL, IQR: 36–108 pg/mL]. LTB4 continuously declined to basal levels from day 1 to 5 without differences between the groups. The cutoff to predict PC was calculated at 109.6 pg/mL (72% specificity, 67% sensitivity). LTB4 was not influenced by overall or chest injury severity, age, gender or massive transfusion. Patients with PC received mechanical ventilation for a significantly longer period of time, and had prolonged intensive care unit and overall hospital stay. Conclusion. High LTB4-levels indicate risk for PC development in multiply traumatized patients
Treatment of malignant glioma with chemotherapy is limited mostly because of delivery impediment related to the blood-brain tumor barrier (BTB). B1 receptors (B1R), inducible prototypical G-protein coupled receptors (GPCR) can regulate permeability of vessels including possibly that of brain tumors. Here, we determine the extent of BTB permeability induced by the natural and synthetic peptide B1R agonists, LysdesArg9BK (LDBK) and SarLys[DPhe8]desArg9BK (NG29), in syngeneic F98 glioma-implanted Fischer rats. Ten days after tumor inoculation, we detected the presence of B1R on tumor cells and associated vasculature. NG29 infusion increased brain distribution volume and uptake profiles of paramagnetic probes (Magnevist and Gadomer) at tumoral sites (T1-weighted imaging). These effects were blocked by B1R antagonist and non-selective cyclooxygenase inhibitors, but not by B2R antagonist and non-selective nitric oxide synthase inhibitors. Consistent with MRI data, systemic co-administration of NG29 improved brain tumor delivery of Carboplatin chemotherapy (ICP-Mass spectrometry). We also detected elevated B1R expression in clinical samples of high-grade glioma. Our results documented a novel GPCR-signaling mechanism for promoting transient BTB disruption, involving activation of B1R and ensuing production of COX metabolites. They also underlined the potential value of synthetic biostable B1R agonists as selective BTB modulators for local delivery of different sized-therapeutics at (peri)tumoral sites.
The neuroendocrine substance melatonin is a hormone synthesized rhythmically by the pineal gland under the influence of the circadian system and alternating light/dark cycles. Melatonin has been shown to have broad applications, and consequently becoming a molecule of great controversy. Undoubtedly, however, melatonin plays an important role as a time cue for the endogenous circadian system. This review focuses on melatonin as a regulator in the circadian modulation of memory processing. Memory processes (acquisition, consolidation, and retrieval) are modulated by the circadian system. However, the mechanism by which the biological clock is rhythmically influencing cognitive processes remains unknown. We also discuss, how the circadian system by generating cycling melatonin levels can implant information about daytime into memory processing, depicted as day and nighttime differences in acquisition, memory consolidation and/or retrieval.
Oncolytic effects of a novel Influenza A virus expressing Interleukin-15 from the NS reading frame
(2012)
Oncolytic influenza A viruses with deleted NS1 gene (delNS1) replicate selectively in tumour cells with defective interferon response and/or activated Ras/Raf/MEK/ERK signalling pathway. To develop a delNS1 virus with specific immunostimulatory properties, we used an optimised technology to insert the interleukin-15 (IL-15) coding sequence into the viral NS gene segment (delNS1-IL-15). DelNS1 and delNS1-IL-15 exerted similar oncolytic effects. Both viruses replicated and caused caspase-dependent apoptosis in interferon-defective melanoma cells. Virus replication was required for their oncolytic activity. Cisplatin enhanced the oncolytic activity of delNS1 viruses. The cytotoxic drug increased delNS1 replication and delNS1-induced caspase-dependent apoptosis. Interference with MEK/ERK signalling by RNAi-mediated depletion or the MEK inhibitor U0126 did not affect the oncolytic effects of the delNS1 viruses. In oncolysis sensitive melanoma cells, delNS1-IL-15 (but not delNS1) infection resulted in the production of IL-15 levels ranging from 70 to 1140 pg/mL in the cell culture supernatants. The supernatants of delNS1-IL-15-infected (but not of delNS1-infected) melanoma cells induced primary human natural killer cell-mediated lysis of non-infected tumour cells. In conclusion, we constructed a novel oncolytic influenza virus that combines the oncolytic activity of delNS1 viruses with immunostimulatory properties through production of functional IL-15. Moreover, we showed that the oncolytic activity of delNS1 viruses can be enhanced in combination with cytotoxic anti-cancer drugs.