Refine
Year of publication
Document Type
- Article (4914)
- Preprint (172)
- Doctoral Thesis (126)
- Conference Proceeding (81)
- Part of Periodical (12)
- Part of a Book (8)
- Book (7)
- Review (4)
- Working Paper (3)
- Report (1)
Language
- English (5328) (remove)
Has Fulltext
- yes (5328)
Keywords
- inflammation (78)
- COVID-19 (56)
- SARS-CoV-2 (47)
- glioblastoma (38)
- cancer (37)
- apoptosis (36)
- Inflammation (35)
- autophagy (29)
- prostate cancer (29)
- breast cancer (27)
Institute
- Medizin (5328) (remove)
Macrophages show a remarkable functional plasticity, which enables them to change their phenotype in response to environmental signals. They are key players during infection by initiating inflammation through the release of proinflammatory mediators. Furthermore, macrophages contribute to the resolution of inflammation by phagocytosis of apoptotic granulocytes. Phagocytosis of apoptotic cells (AC) induces an anti-inflammatory phenotype in macrophages and protects them against apoptosis. However, mechanistic details provoking these phenotype alterations are incompletely understood. Therefore, the aim of my Ph.D. thesis was to investigate the molecular basis of anti-inflammatory macrophage polarization. In the first part of my studies, I investigated the expression of heme oxygenase (HO)-1 in macrophages following treatment with supernatants from AC. HO-1 catalyzes the first and rate-limiting step of heme degradation and potentially bears anti-inflammatory as well as anti-apoptotic potential. I was able to show biphasic upregulation of HO-1 by AC supernatants. The first phase of HO-1 induction at 6 h required activation of p38 MAPK and was accomplished by the bioactive lipid sphingosine-1-phosphate (S1P) engaging S1P receptor 1 (S1P1). However, the second wave of HO-1 induction at 24 h was attributed to autocrine signaling of vascular endothelial growth factor (VEGF) A, whose expression was facilitated by S1P. The release of VEGFA from macrophages was STAT1-dependent, whereas VEGFA itself acted on the macrophage HO-1 promoter via STAT1/STAT3 heterodimer binding. Knockdown of HO-1 revealed its relevance in promoting enhanced expression of the anti-apoptotic proteins B cell leukemia/lymphoma-2 (Bcl-2) and B cell leukaemia/lymphoma-x long (Bcl-XL), as well as the anti-inflammatory adenosine receptor A2A. MHC II and indoleamine 2,3-dioxygenase expression were also affected by ACsupernanatants, but were not HO-1 dependent. Unexpectedly, S1P1 was also upregulated following treatment with AC supernatants. Thus, I considered whether S1P1 induction could specifically be mediated by alternative macrophage activating factors. The expression of S1P1 was enhanced in the presence of the alternative activation stimuli IL-4 as well as IL-10, whereas it was unchanged following incubations with LPS, interferon-g or S1P. My next aim was to investigate the expression of the different S1P receptor isoforms in macrophages following treatment with supernatants form AC. While the expressions of S1P1 as well as S1P3 were induced by exposure to supernatants from AC, S1P2 expression was unaffected. As S1P1/3 and S1P2 are conflictively involved in the regulation of cell migration, I asked for a correlation between increased S1P receptor expression and enhanced migration rate. Indeed, macrophages showed enhanced motility following treatment with supernatants form AC, which was inhibited in S1P1 knockout macrophages. In summary, my findings indicate that HO-1, which is induced by AC-derived S1P, is critically involved in macrophage polarization towards an alternatively activated macrophage phenotype. S1P1 seems to represent a central checkpoint during macrophage activation. On the one hand, S1P1 is induced by supernatants form AC and promotes migration of macrophages. On the other hand, it mediates the induction of HO-1, which is accompanied by antiinflammatory as well as anti-apoptotic signaling. Furthermore, my studies provide evidence that upregulation of HO-1 and S1P1 in macrophages may contribute to the resolution of inflammation by establishing an anti-inflammatory macrophage phenotype and provoking macrophage migration along the vascular S1P gradient out of an inflammatory environment into the lymph.
Background: Wnt signaling controls the balance between stem cell proliferation and differentiation and body patterning throughout development. Previous data demonstrated that non-canonical Wnts (Wnt5a, Wnt11) increased cardiac gene expression of circulating endothelial progenitor cells (EPC) and bone marrow-derived stem cells cultured in vitro. Since previous studies suggested a contribution of the protein kinase C (PKC) family to the Wnt5a-induced signalling, we investigated which PKC isoforms are activated by non-canonical Wnt5a in human EPC. Methodology/Principal Findings: Immunoblot experiments demonstrated that Wnt5a selectively activated the novel PKC isoform, PKC delta, as evidenced by phosphorylation and translocation. In contrast, the classical Ca2+-dependent PKC isoforms, PKC alpha and beta2, and one of the other novel PKC isoforms, PKC epsilon, were not activated by Wnt5a. The PKC delta inhibitor rottlerin significantly blocked co-culture-induced cardiac differentiation in vitro, whereas inhibitors directed against the classical Ca2+-dependent PKC isoforms or a PKC epsilon-inhibitory peptide did not block cardiac differentiation. In accordance, EPC derived from PKC delta heterozygous mice exhibited a significant reduction of Wnt5a-induced cardiac gene expression compared to wild type mice derived EPC. Conclusions/Significance: These data indicate that Wnt5a enhances cardiac gene expressions of EPC via an activation of PKC delta.
Background: Generalised spike wave (GSW) discharges are the electroencephalographic (EEG) hallmark of absence seizures, clinically characterised by a transitory interruption of ongoing activities and impaired consciousness, occurring during states of reduced awareness. Several theories have been proposed to explain the pathophysiology of GSW discharges and the role of thalamus and cortex as generators. In this work we extend the existing theories by hypothesizing a role for the precuneus, a brain region neglected in previous works on GSW generation but already known to be linked to consciousness and awareness. We analysed fMRI data using dynamic causal modelling (DCM) to investigate the effective connectivity between precuneus, thalamus and prefrontal cortex in patients with GSW discharges. Methodology and Principal Findings: We analysed fMRI data from seven patients affected by Idiopathic Generalized Epilepsy (IGE) with frequent GSW discharges and significant GSW-correlated haemodynamic signal changes in the thalamus, the prefrontal cortex and the precuneus. Using DCM we assessed their effective connectivity, i.e. which region drives another region. Three dynamic causal models were constructed: GSW was modelled as autonomous input to the thalamus (model A), ventromedial prefrontal cortex (model B), and precuneus (model C). Bayesian model comparison revealed Model C (GSW as autonomous input to precuneus), to be the best in 5 patients while model A prevailed in two cases. At the group level model C dominated and at the population-level the p value of model C was ,1. Conclusion: Our results provide strong evidence that activity in the precuneus gates GSW discharges in the thalamo-(fronto) cortical network. This study is the first demonstration of a causal link between haemodynamic changes in the precuneus - an index of awareness - and the occurrence of pathological discharges in epilepsy.
Background: The standard electrode array for the MED-EL MAESTRO cochlear implant system is 31 mm in length which allows an insertion angle of approximately 720°. When fully inserted, this long electrode array is capable of stimulating the most apical region of the cochlea. No investigation has explored Electrically Evoked Compound Action Potential (ECAP) recordings in this region with a large number of subjects using a commercially available cochlear implant system. The aim of this study is to determine if certain properties of ECAP recordings vary, depending on the stimulation site in the cochlea. Methods: Recordings of auditory nerve responses were conducted in 67 subjects to demonstrate the feasibility of ECAP recordings using the Auditory Nerve Response Telemetry (ART™) feature of the MED-EL MAESTRO system software. These recordings were then analyzed based on the site of cochlear stimulation defined as basal, middle and apical to determine if the amplitude, threshold and slope of the amplitude growth function and the refractory time differs depending on the region of stimulation. Results: Findings show significant differences in the ECAP recordings depending on the stimulation site. Comparing the apical with the basal region, on average higher amplitudes, lower thresholds and steeper slopes of the amplitude growth function have been observed. The refractory time shows an overall dependence on cochlear region; however post-hoc tests showed no significant effect between individual regions. Conclusions :Obtaining ECAP recordings is also possible in the most apical region of the cochlea. However, differences can be observed depending on the region of the cochlea stimulated. Specifically, significant higher ECAP amplitude, lower thresholds and steeper amplitude growth function slopes have been observed in the apical region. These differences could be explained by the location of the stimulating electrode with respect to the neural tissue in the cochlea, a higher density, or an increased neural survival rate of neural tissue in the apex. Trial registration: The Clinical Investigation has the Competent Authority registration number DE/CA126/AP4/3332/18/05.
Background: Three genes have been confirmed as major joint susceptibility genes for endocrine autoimmune disease:human leukocyte antigen class II, cytotoxic T-lymphocyte antigen 4 and protein tyrosine phosphatase non-receptor type 22. Recent studies showed that a genetic variation within the interferon induced helicase domain 1 (IFIH1) locus (rs1990760 polymorphism) is an additional risk factor in type 1 diabetes and Graves' disease (GD). Methods: The aim of the present study was to investigate the role of the rs1990760 polymorphism within the IFIH1 gene in German patients with GD (n=258), Hashimoto's thyroiditis (HT,n=106), Addison's disease (AD,n=195) and healthy controls (HC,n=227) as well as in 55 GD families (165 individuals, German) and 100 HT families (300 individuals, Italian). Furthermore, the interaction between rs1990760 polymorphism with human leukocyte antigen (HLA) risk haplotype DQ2(DQA*0501-DQB*0201), the risk haplotypes DQ2/DQ8 (DQA*0301-DQB*0302) and the status of thyroglobulin antibody (TgAb), thyroid peroxidase antibody (TPOAb) and TSH receptor antibody (TRAb) in patients and families were analysed. Results:No significant differences were found between the allele and genotype frequencies for rs1990760 IFIH1 polymorphism in patients with GD, HT, AD and HC. Also no differences were observed when stratifying the IFIH1 rs1990760 polymorphism for gender, presence or absence of thyroid antibodies (GD:TRAb and HT:TPOAb/TgAb) and HLA risk haplotypes (DQ2:for GD and HT, DQ2/DQ8:for AD). Furthermore the transmission analysis in GD and HT families revealed no differences in alleles transmission for rs1990760 IFIH1 from parents with or without HLA risk haplotype DQ2 to the affected offspring. In contrast, by dividing the HT parents according to the presence or absence of thyroid Ab titers, mothers and fathers both positive for TPOAb/TgAb overtransmitted the allele A of IFIH1 rs1990760 to their HT affected offspring (61.8% vs 38.2%;p=0.05;corrected p [pc]=0.1). However, these associations did not remain statistically significant after correction of the p-values. Conclusion: In conclusion, our data suggest, no contribution from IFIH1 rs1990760 polymorphism to the pathogenesis of either Graves' disease, Hashimoto's thyroiditis or Addison's disease in our study populations. However, in order to exclude a possible influence of the studied polymorphism in specified subgroups within patients with autoimmune thyroid disease, further investigations in larger populations are needed.
Background Enhanced activity of histone deacetylases (HDAC) is associated with more aggressive tumour behaviour and tumour progression in various solid tumours. The over-expression of these proteins and their known functions in malignant neoplasms has led to the development of HDAC inhibitors (HDI) as new anti-neoplastic drugs. However, little is known about HDAC expression in renal cell cancer. Methods We investigated the expression of HDAC 1, 2 and 3 in 106 renal cell carcinomas and corresponding normal renal tissue by immunohistochemistry on tissue micro arrays and correlated expression data with clinico-pathological parameters including patient survival. Results Almost 60% of renal cell carcinomas expressed the HDAC isoforms 1 and 2. In contrast, HDAC 3 was only detected in 13% of all renal tumours, with particular low expression rates in the clear cell subtype. HDAC 3 was significantly higher expressed in pT1/2 tumours in comparison to pT3/4 tumours. Expression of class I HDAC isoforms correlated with each other and with the proliferative activity of the tumours. We found no prognostic value of the expression of any of the HDAC isoforms in this tumour entity. Conclusion Class I HDAC isoforms 1 and 2 are highly expressed in renal cell cancer, while HDAC 3 shows low, histology dependent expression rates. These unexpected differences in the expression patterns suggests alternative regulatory mechanisms of class I HDACs in renal cell cancer and should be taken into account when trials with isoform selective HDI are being planned. Whether HDAC expression in renal cancers is predictive of responsiveness for HDI will have to be tested in further studies.
Loss of vascular barrier function causes leak of fluid and proteins into tissues, extensive leak leads to shock and death. Barriers are largely formed by endothelial cell-cell contacts built up by VE-cadherin and are under the control of RhoGTPases. Here we show that a natural plasmin digest product of fibrin, peptide Bß15-42 (also called FX06), significantly reduces vascular leak and mortality in animal models for Dengue shock syndrome. The ability of Bß15-42 to preserve endothelial barriers is confirmed in rats i.v.-injected with LPS. In endothelial cells, Bß15-42 prevents thrombin-induced stress fiber formation, myosin light chain phosphorylation and RhoA activation. The molecular key for the protective effect of Bß15-42 is the src kinase Fyn, which associates with VE-cadherin-containing junctions. Following exposure to Bß15-42 Fyn dissociates from VE-cadherin and associates with p190RhoGAP, a known antagonists of RhoA activation. The role of Fyn in transducing effects of Bß15-42 is confirmed in Fyn -/- mice, where the peptide is unable to reduce LPS-induced lung edema, whereas in wild type littermates the peptide significantly reduces leak. Our results demonstrate a novel function for Bß15-42. Formerly mainly considered as a degradation product occurring after fibrin inactivation, it has now to be considered as a signaling molecule. It stabilizes endothelial barriers and thus could be an attractive adjuvant in the treatment of shock.
Background Bacterial DNA containing motifs of unmethylated CpG dinucleotides (CpG-ODN) initiate an innate immune response mediated by the pattern recognition receptor Toll-like receptor 9 (TLR9). This leads in particular to the expression of proinflammatory mediators such as tumor necrosis factor (TNF-alpha) and interleukin-1beta (IL-1beta). TLR9 is expressed in human and murine pulmonary tissue and induction of proinflammatory mediators has been linked to the development of acute lung injury. Therefore, the hypothesis was tested whether CpG-ODN administration induces an inflammatory response in the lung via TLR9 in vivo. Methods Wild-type (WT) and TLR9-deficient (TLR9-D) mice received CpG-ODN intraperitoneally (1668-Thioat, 1 nmol/g BW) and were observed for up to 6 hrs. Lung tissue and plasma samples were taken and various inflammatory markers were measured. Results In WT mice, CpG-ODN induced a strong activation of pulmonary NFKB as well as a significant increase in pulmonary TNF-alpha and IL-1beta mRNA/protein. In addition, cytokine serum levels were significantly elevated in WT mice. Increased pulmonary content of lung myeloperoxidase (MPO) was documented in WT mice following application of CpG-ODN. Bronchoalveolar lavage (BAL) revealed that CpG-ODN stimulation significantly increased total cell number as well as neutrophil count in WT animals. In contrast, the CpG-ODN-induced inflammatory response was abolished in TLR9-D mice. Conclusion This study suggests that bacterial CpG-ODN causes lung inflammation via TLR9.
Background During gram-negative sepsis, lipopolysaccharide (LPS) induces tissue factor expression on monocytes. The resulting disseminated intravascular coagulation leads to tissue ischemia and worsens the prognosis of septic patients. There are indications, that fever reduces the mortality of sepsis, the effect on tissue factor activity on monocytes is unknown. Therefore, we investigated whether heat shock modulates LPS-induced tissue factor activity in human blood. Methods Whole blood samples and leukocyte suspensions, respectively, from healthy probands (n = 12) were incubated with LPS for 2 hours under heat shock conditions (43°C) or control conditions (37°C), respectively. Subsequent to further 3 hours of incubation at 37°C the clotting time, a measure of tissue factor expression, was determined. Cell integrity was verified by trypan blue exclusion test and FACS analysis. Results Incubation of whole blood samples with LPS for 5 hours at normothermia resulted in a significant shortening of clotting time from 357 ± 108 sec to 82 ± 8 sec compared to samples incubated without LPS (n = 12; p < 0.05). This LPS effect was mediated by tissue factor, as inhibition with active site-inhibited factor VIIa (ASIS) abolished the effect of LPS on clotting time. Blockade of protein synthesis using cycloheximide demonstrated that LPS exerted its procoagulatory effect via an induction of tissue factor expression. Upon heat shock treatment, the LPS effect was blunted: clotting times were 312 ± 66 s in absence of LPS and 277 ± 65 s in presence of LPS (n = 8; p > 0.05). Similarly, heat shock treatment of leukocyte suspensions abolished the LPS-induced tissue factor activity. Clotting time was 73 ± 31 s, when cells were treated with LPS (100 ng/mL) under normothermic conditions, and 301 ± 118 s, when treated with LPS (100 ng/mL) and heat shock (n = 8, p < 0.05). Control experiments excluded cell damage as a potential cause of the observed heat shock effect. Conclusion Heat shock treatment inhibits LPS-induced tissue factor activity in human whole blood samples and isolated leukocytes.
Introduction: Immune paralysis with massive T-cell apoptosis is a central pathogenic event during sepsis and correlates with septic patient mortality. Previous observations implied a crucial role of peroxisome proliferator-activated receptor gamma (PPARγ) during T-cell apoptosis.
Methods: To elucidate mechanisms of PPARγ-induced T-cell depletion, we used an endotoxin model as well as the caecal ligation and puncture sepsis model to imitate septic conditions in wild-type versus conditional PPARγ knockout (KO) mice.
Results: PPARγ KO mice showed a marked survival advantage compared with control mice. Their T cells were substantially protected against sepsis-induced death and showed a significantly higher expression of the pro-survival factor IL-2. Since PPARγ is described to repress nuclear factor of activated T cells (NFAT) transactivation and concomitant IL-2 expression, we propose inhibition of NFAT as the underlying mechanism allowing T-cell apoptosis. Corroborating our hypothesis, we observed up-regulation of the pro-apoptotic protein BIM and downregulation of the anti-apoptotic protein Bcl-2 in control mice, which are downstream effector proteins of IL-2 receptor signaling. Application of a neutralizing anti-IL-2 antibody reversed the pro-survival effect of PPARγ-deficient T cells and confirmed IL-2-dependent apoptosis during sepsis.
Conclusion: Apparently antagonizing PPARγ in T cells might improve their survival during sepsis, which concomitantly enhances defence mechanisms and possibly provokes an increased survival of septic patients.
Background: The c-Cbl-associated protein (CAP), also known as ponsin, localizes to focal adhesions and stress fibers and is involved in signaling events. Phosphorylation has been described for the other two members of the sorbin homology family, vinexin and ArgBP2, but no data exist about the putative phosphorylation of CAP. According to previous findings, CAP binds to tyrosine kinase c-Abl. However, it is not known if CAP is a substrate of c-Abl or other tyrosine kinases or if phosphorylation regulates its localization.
Results: We here show that CAP is Tyr phosphorylated by and interacts with both c-Abl and c-Src. One major phosphorylation site, Tyr360, and two minor contributors Tyr326 and Tyr632 were identified as Abl phosphorylation sites, whereas Src preferentially phosphorylates Tyr326 and Tyr360. Phosphorylation of CAP was not necessary for its localization to focal adhesions and stress fibers, but Tyr326Phe substitution alters the function of CAP during cell spreading.
Conclusion: This is the first demonstration of phosphorylation of CAP by any kinase. Our findings suggest that coordinated action of Src and Abl might regulate the function of CAP and reveal a functional role especially for the Src-mediated Tyr phosphorylation of CAP in cell spreading.
Objectives: We sought to investigate whether statin therapy affects the association between preprocedural C-reactive protein (CRP) levels and the risk for recurrent coronary events in patients undergoing coronary stent implantation.
Background: Low-grade inflammation as detected by elevated CRP levels predicts the risk of recurrent coronary events. The effect of inflammation on coronary risk may be attenuated by statin therapy.
Methods: We investigated a potential interrelation among statin therapy, serum evidence of inflammation, and the risk for recurrent coronary events in 388 consecutive patients undergoing coronary stent implantation. Patients were grouped according to the median CRP level (0.6 mg/dl) and to the presence of statin therapy.
Results: A primary combined end point event occurred significantly more frequently in patients with elevated CRP levels without statin therapy (RR [relative risk] 2.37, 95% CI [confidence interval] [1.3 to 4.2]). Importantly, in the presence of statin therapy, the RR for recurrent events was significantly reduced in the patients with elevated CRP levels (RR 1.27 [0.7 to 2.1]) to about the same degree as in patients with CRP levels below 0.6 mg/dl and who did not receive statin therapy (RR 1.1 [0.8 to 1.3]).
Conclusions: Statin therapy significantly attenuates the increased risk for major adverse cardiac events in patients with elevated CRP levels undergoing coronary stent implantation, suggesting that statin the rapy interferes with the detrimental effects of inflammation on accelerated atherosclerotic disease progression following coronary stenting.
Background Chemoresistance acquisition may influence cancer cell biology. Here, bioinformatics analysis of gene expression data was used to identify chemoresistance-associated changes in neuroblastoma biology. Results Bioinformatics analysis of gene expression data revealed that expression of angiogenesis-associated genes significantly differs between chemosensitive and chemoresistant neuroblastoma cells. A subsequent systematic analysis of a panel of 14 chemosensitive and chemoresistant neuroblastoma cell lines in vitro and in animal experiments indicated a consistent shift to a more pro-angiogenic phenotype in chemoresistant neuroblastoma cells. The molecular mechanims underlying increased pro-angiogenic activity of neuroblastoma cells are individual and differ between the investigated chemoresistant cell lines. Treatment of animals carrying doxorubicin-resistant neuroblastoma xenografts with doxorubicin, a cytotoxic drug known to exert anti-angiogenic activity, resulted in decreased tumour vessel formation and growth indicating chemoresistance-associated enhanced pro-angiogenic activity to be relevant for tumour progression and to represent a potential therapeutic target. Conclusions A bioinformatics approach allowed to identify a relevant chemoresistance-associated shift in neuroblastoma cell biology. The chemoresistance-associated enhanced pro-angiogenic activity observed in neuroblastoma cells is relevant for tumour progression and represents a potential therapeutic target.
TNFAIP3 (A20) is a tumor suppressor gene in Hodgkin lymphoma and primary mediastinal B cell lymphoma
(2009)
Proliferation and survival of Hodgkin and Reed/Sternberg (HRS) cells, the malignant cells of classical Hodgkin lymphoma (cHL), are dependent on constitutive activation of nuclear factor {kappa}B (NF-{kappa}B). NF-{kappa}B activation through various stimuli is negatively regulated by the zinc finger protein A20. To determine whether A20 contributes to the pathogenesis of cHL, we sequenced TNFAIP3, encoding A20, in HL cell lines and laser-microdissected HRS cells from cHL biopsies. We detected somatic mutations in 16 out of 36 cHLs (44%), including missense mutations in 2 out of 16 Epstein-Barr virus–positive (EBV+) cHLs and a missense mutation, nonsense mutations, and frameshift-causing insertions or deletions in 14 out of 20 EBV– cHLs. In most mutated cases, both TNFAIP3 alleles were inactivated, including frequent chromosomal deletions of TNFAIP3. Reconstitution of wild-type TNFAIP3 in A20-deficient cHL cell lines revealed a significant decrease in transcripts of selected NF-{kappa}B target genes and caused cytotoxicity. Extending the mutation analysis to primary mediastinal B cell lymphoma (PMBL), another lymphoma with constitutive NF-{kappa}B activity, revealed destructive mutations in 5 out of 14 PMBLs (36%). This report identifies TNFAIP3 (A20), a key regulator of NF-{kappa}B activity, as a novel tumor suppressor gene in cHL and PMBL. The significantly higher frequency of TNFAIP3 mutations in EBV– than EBV+ cHL suggests complementing functions of TNFAIP3 inactivation and EBV infection in cHL pathogenesis.
Background: In this interdisciplinary project, the biological effects of heavy ions are compared to those of X-rays using tissue slice culture preparations from rodents and humans. Advantages of this biological model are the conservation of an organotypic environment and the independency from genetic immortalization strategies used to generate cell lines. Its open access allows easy treatment and observation via live-imaging microscopy. Materials and methods: Rat brains and human brain tumor tissue are cut into 300 micro m thick tissue slices. These slices are cultivated using a membrane-based culture system and kept in an incubator at 37°C until treatment. The slices are treated with X-rays at the radiation facility of the University Hospital in Frankfurt at doses of up to 40 Gy. The heavy ion irradiations were performed at the UNILAC facility at GSI with different ions of 11.4 A MeV and fluences ranging from 0.5–10 x 106 particles/cm². Using 3D-confocal microscopy, cell-death and immune cell activation of the irradiated slices are analyzed. Planning of the irradiation experiments is done with simulation programs developed at GSI and FIAS. Results: After receiving a single application of either X-rays or heavy ions, slices were kept in culture for up to 9d post irradiation. DNA damage was visualized using gamma H2AXstaining. Here, a dose-dependent increase and time-dependent decrease could clearly be observed for the X-ray irradiation. Slices irradiated with heavy ions showed less gamma H2AX-positive cells distributed evenly throughout the slice, even though particles were calculated to penetrate only 90–100 micro m into the slice. Conclusions: Single irradiations of brain tissue, even at high doses of 40 Gy, will result neither in tissue damage visible on a macroscopic level nor necrosis. This is in line with the view that the brain is highly radio-resistant. However, DNA damage can be detected very well in tissue slices using gamma H2AX-immuno staining. Thus, slice cultures are an excellent tool to study radiation-induced damage and repair mechanisms in living tissues.
The human amygdala is thought to play a pivotal role in the processing of emotionally significant sensory information. The major subdivisions of the human amygdala—the laterobasal group (LB), the superficial group (SF), and the centromedial group (CM)—have been anatomically delineated, but the functional response properties of these amygdala subregions in humans are still unclear. We combined functional MRI with cyto-architectonically defined probabilistic maps to analyze the response characteristics of amygdala subregions in subjects presented with auditory stimuli. We found positive auditory stimulation-related signal changes predominantly in probabilistically defined LB, and negative responses predominantly in SF and CM. In the left amygdala, mean response magnitude in the core area of LB with 90–100% assignment probability was significantly larger than in the core areas of SF and CM. These differences were observed for pleasant and unpleasant stimuli. Our findings reveal that the probabilistically defined anatomical subregions of the human amygdala show distinctive fMRI response patterns. The stronger auditory responses in LB as compared with SF and CM may reflect a predominance of auditory inputs to human LB, similar to many animal species in which the majority of sensory, including auditory, afferents project to this subdivision of the amygdala. Our study indicates that the intrinsic functional differentiation of the human amygdala may be probed using fMRI combined with probabilistic anatomical maps.
Neuronal nAChRs are a diverse family of pentameric ion channels with wide distribution throughout cells of the nervous and immune systems. However, the role of specific subtypes in normal and pathological states remains poorly understood due to the lack of selective probes. Here, we used a binding assay based on acetylcholine-binding protein (AChBP), a homolog of the nicotinic acetylcholine ligand-binding domain, to discover a novel alpha-conotoxin (alpha-TxIA) in the venom of Conus textile. alpha-TxIA bound with high affinity to AChBPs from different species and selectively targeted the alpha3beta2 nAChR subtype. A co-crystal structure of Ac-AChBP with the enhanced potency analog TxIA(A10L), revealed a 20° backbone tilt compared to other AChBP–conotoxin complexes. This reorientation was coordinated by a key salt bridge formed between Arg5 (TxIA) and Asp195 (Ac-AChBP). Mutagenesis studies, biochemical assays and electrophysiological recordings directly correlated the interactions observed in the co-crystal structure to binding affinity at AChBP and different nAChR subtypes. Together, these results establish a new pharmacophore for the design of novel subtype-selective ligands with therapeutic potential in nAChR-related diseases.
Only a few Methyl-[11C]-l-methionine (MET) positron emission tomography (PET) studies have focused on children and young adults with brain neoplasm. Due to radiation exposure, long scan acquisition time, and the need for sedation in young children MET-PET studies should be restricted to this group of patients when a decision for further therapy is not possible from routine diagnostic procedures alone, e.g., structural imaging. We investigated the diagnostic accuracy of MET-PET for the differentiation between tumorous and non-tumorous lesions in this group of patients. Forty eight MET-PET scans from 39 patients aged from 2 to 21 years (mean 15 ± 5.0 years) were analyzed. The MET tumor-uptake relative to a corresponding control region was calculated. A receiver operating characteristic (ROC) was performed to determine the MET-uptake value that best distinguishes tumorous from non-tumorous brain lesions. A differentiation between tumorous (n = 39) and non-tumorous brain lesions (n = 9) was possible at a threshold of 1.48 of relative MET-uptake with a sensitivity of 83% and a specificity of 92%, respectively. A differentiation between high grade malignant lesions (mean MET-uptake = 2.00 ± 0.46) and low grade tumors (mean MET-uptake = 1.84 ± 0.31) was not possible. There was a significant difference in MET-uptake between the histologically homogeneous subgroups of astrocytoma WHO grade II and anaplastic astrocytoma WHO grade III (P = 0.02). MET-PET might be a useful tool to differentiate tumorous from non-tumorous lesions in children and young adults when a decision for further therapy is difficult or impossible from routine structural imaging procedures alone. Keywords Brain tumor - Children - PET - Methionine - Molecular imaging
L1-CAM (L1 cell-adhesion molecule), or more simply L1, plays an important role in the progression of human carcinoma. Overexpression promotes tumour-cell invasion and motility, growth in nude mice and tumour metastasis. It is feasible that L1-dependent signalling contributes to these effects. However, little is known about its mechanism in tumour cells. We reported previously that L1 is cleaved by ADAM (a disintegrin and metalloprotease) and that the cytoplasmic part is essential for L1 function. Here we analysed more closely the role of proteolytic cleavage in L1-mediated nuclear signalling. Using OVMz carcinoma cells and L1-transfected cells as a model, we found that ADAM10-mediated cleavage of L1 proceeds in lipid raft and non-raft domains. The cleavage product, L1-32, is further processed by PS (presenilin)/gamma-secretase to release L1-ICD, an L1 intracellular domain of 28 kDa. Overexpression of dominantnegative PS1 or use of a specific gamma-secretase inhibitor leads to an accumulation of L1-32. Fluorescence and biochemical analysis revealed a nuclear localization for L1-ICD. Moreover, inhibition of ADAM10 and/or gamma-secretase blocks nuclear translocation of L1-ICD and L1-dependent gene regulation. Overexpression of recombinant L1-ICD mediates gene regulation in a similar manner to full-length L1. Our results establish for the first time that regulated proteolytic processing by ADAM10 and PS/gamma-secretase is essential for the nuclear signalling of L1 in human carcinoma cell lines. Key words: a disintegrin and metalloprotease 10 (ADAM10), L1 cell-adhesion molecule (L1-CAM), nuclear translocation, presenilin (PS)/gamma-secretase activity, raft, signalling.
In the European Union (EU), health policy and the institutional reform of health systems have been treated primarily as national affairs, and health care systems within the EU thus differ considerably. However, the health policy field is undergoing a dynamic process of Europeanization. This process is stimulated by the orientation towards a more competitive economy, recently inaugurated and known as the Lisbon Strategy, while the regulatory requirements of the European Economic and Monetary Union are stimulating the Europeanization of health policy. In addition, the so-called open method of coordination, representing a new mode of regulation within the European multi-level system, is applied increasingly to the health policy area. Diverse trends are thus emerging. While the Lisbon Strategy goes along with a strategic upgrading of health policy more generally, health policy is increasingly used to strengthen economic competitiveness. Pressure on Member States is expected to increase to contain costs and promote market-based health care provision.
Introduction: Reliable predictive and prognostic markers for routine diagnostic purposes are needed for breast cancer patients treated with neoadjuvant chemotherapy. We evaluated protein biomarkers in a cohort of 116 participants of the GeparDuo study on anthracycline/taxane-based neoadjuvant chemotherapy for operable breast cancer to test for associations with pathological complete response (pCR) and disease-free survival (DFS). Particularly, we evaluated if interactions between hormone receptor (HR) and human epidermal growth factor receptor 2 (HER2) expression might lead to a different clinical behavior of HR+/HER2+ coexpressing and HR+/HER2- tumors and whether subgroups of triple negative tumors might be identified by the help of Ki67 labeling index, cytokeratin 5/6 (CK5/6), as well as cyclooxygenase-2 (COX-2), and Y-box binding protein 1 (YB-1) expression. Methods: Expression analysis was performed using immunohistochemistry and silver-enhanced in situ hybridization on tissue microarrays (TMAs) of pretherapeutic core biopsies. Results: pCR rates were significantly different between the biology-based tumor types (P = 0.044) with HR+/HER2+ and HR-/HER2- tumors having higher pCR rates than HR+/HER2-tumors. Ki67 labeling index, confirmed as significant predictor of pCR in the whole cohort (P = 0.001), identified HR-/HER- (triple negative) carcinomas with a higher chance for a pCR (P = 0.006). Biology-based tumor type (P = 0.046 for HR+/HER2+vs. HR+/HER2-), Ki67 labeling index (P = 0.028), and treatment arm (P = 0.036) were independent predictors of pCR in a multivariate model. DFS was different in the biology-based tumor types (P < 0.0001) with HR+/HER2- and HR+/HER2+ tumors having the best prognosis and HR-/HER2+ tumors showing the worst outcome. Biology-based tumor type was an independent prognostic factor for DFS in multivariate analysis (P < 0.001). Conclusions: Our data demonstrate that a biology-based breast cancer classification using estrogen receptor (ER), progesterone receptor (PgR), and HER2 bears independent predictive and prognostic potential. The HR+/HER2+ coexpressing carcinomas emerged as a group of tumors with a good response rate to neoadjuvant chemotherapy and a favorable prognosis. HR+/HER2- tumors had a good prognosis irrespective of a pCR, whereas patients with HR-/HER- and HR-/HER+ tumors, especially if they had not achieved a pCR, had an unfavorable prognosis and are in need of additional treatment options. Trial registration ClinicalTrials.gov identifier: NCT00793377
In 1911 Eugen Bleuler (Bleuler, 1911) postulated that schizophrenia was a disorder resulting from inability to properly integrate mental processes. Around the same time, Carl Wernicke (Wernicke, 1894) proposed that psychosis might result from disruption of white matter tracts. Both of these statements can be considered early cornerstones of modern connectivity hypotheses developed towards the end of the twentieth century by such researchers as Karl Friston (1998) and Nancy Andreansen (1998). In the current work, the hypothesis that schizophrenia, rather than being a disorder or either anatomical or functional connectivity, is a disorder where both of these processes interact and influence the clinical presentation of patients, is examined. This is achieved through a detailed examination of a sample of chronic schizophrenia patients using a combination of functional and anatomical Magnetic Resonance Imaging techniques. The relationship of these measures to clinical symptoms is also explored. In the first study, anatomical connectivity at the whole-brain level is examined using Diffusion Tensor Imaging. The results of the study contribute to the previous literature on auditory hallucinations in schizophrenia and provide the first direct correlation between increased anatomical connectivity and increased severity of psychotic symptoms. The second study provides a thorough examination of the interhemispheric connectivity. This is achieved through a detailed examination of the corpus callosum using a combination of diffusivity and volumetric values. This is the first study to date where several anatomical methods are used in one sample. The results illustrate the importance of using different techniques to accurately characterize anatomical abnormalities observed in schizophrenia. In addition, contrary to previous research reports, the results of the current study imply that only specific sub-sections of the corpus callosum are affected by anatomical abnormalities. The pattern of these changes may influence clinical presentation of patients. Finally, functional connectivity at the whole-brain level is examined during resting-state using Independent Component Analysis. Similarly to the results of the anatomical examinations, it provides further supporting evidence that the pattern of disturbances observed in the current sample of schizophrenia patients examined herein reflects a combination of hypo- and hyperconnectivity. Moreover, the study further validates resting-state functional Magnetic Resonance Imaging as a reliable tool for examining functional abnormalities in schizophrenia.
Background: Nitric oxide (NO) is an essential vasodilator. In vascular diseases, oxidative stress attenuates NO signaling by both chemical scavenging of free NO and oxidation and down-regulation of its major intracellular receptor, the alpha/beta heterodimeric heme-containing soluble guanylate cyclase (sGC). Oxidation can also induce loss of sGC's heme and responsiveness to NO.
Results: sGC activators such as BAY 58-2667 bind to oxidized/heme-free sGC and reactivate the enzyme to exert disease-specific vasodilation. Here we show that oxidation-induced down-regulation of sGC protein extends to isolated blood vessels. Mechanistically, degradation was triggered through sGC ubiquitination and proteasomal degradation. The heme-binding site ligand, BAY 58-2667, prevented sGC ubiquitination and stabilized both alpha and beta subunits.
Conclusion: Collectively, our data establish oxidation-ubiquitination of sGC as a modulator of NO/cGMP signaling and point to a new mechanism of action for sGC activating vasodilators by stabilizing their receptor, oxidized/heme-free sGC.
Poster presentation: Self-organized critical (SOC) systems are complex dynamical systems that may express cascades of events, called avalanches [1]. The SOC state was proposed to govern brain function, because of its activity fluctuations over many orders of magnitude, its sensitivity to small input and its long term stability [2,3]. In addition, the critical state is optimal for information storage and processing [4]. Both hallmark features of SOC systems, a power law distribution f(s) for the avalanche size s and a branching parameter (bp) of unity, were found for neuronal avalanches recorded in vitro [5]. However, recordings in vivo yielded contradictory results [6]. Electrophysiological recordings in vivo only cover a small fraction of the brain, while criticality analysis assumes that the complete system is sampled. We hypothesized that spatial subsampling might influence the observed avalanche statistics. In addition, SOC models can have different connectivity, but always show a power law for f(s) and bp = 1 when fully sampled. This may not be the case under subsampling, however. Here, we wanted to know whether a state change from awake to asleep could be modeled by changing the connectivity of a SOC model without leaving the critical state. We simulated a SOC model [1] and calculated f(s) and bp obtained from sampling only the activity of a set of 4 × 4 sites, representing the electrode positions in the cortex. We compared these results with results obtained from multielectrode recordings of local field potentials (LFP) in the cortex of behaving monkeys. We calculated f(s) and bp for the LFP activity recorded while the monkey was either awake or asleep and compared these results to results obtained from two subsampled SOC model with different connectivity. f(s) and bp were very similar for both the experiments and the subsampled SOC model, but in contrast to the fully sampled model, f(s) did not show a power law and bp was smaller than unity. With increasing the distance between the sampling sites, f(s) changed from "apparently supercritical" to "apparently subcritical" distributions in both the model and the LFP data. f(s) and bp calculated from LFP recorded during awake and asleep differed. These changes could be explained by altering the connectivity in the SOC model. Our results show that subsampling can prevent the observation of the characteristic power law and bp in SOC systems, and misclassifications of critical systems as sub- or supercritical are possible. In addition, a change in f(s) and bp for different states (awake/asleep) does not necessarily imply a change from criticality to sub- or supercriticality, but can also be explained by a change in the effective connectivity of the network without leaving the critical state.
Poster presentation: The analysis of neuronal processes distributed across multiple cortical areas aims at the identification of interactions between signals recorded at different sites. Such interactions can be described by measuring the stability of phase angles in the case of oscillatory signals or other forms of signal dependencies for less regular signals. Before, however, any form of interaction can be analyzed at a given time and frequency, it is necessary to assess whether all potentially contributing signals are present. We have developed a new statistical procedure for the detection of coincident power in multiple simultaneously recorded analog signals, allowing the classification of events as 'non-accidental co-activation'. This method can effectively operate on single trials, each lasting only for a few seconds. Signals need to be transformed into time-frequency space, e.g. by applying a short-time Fourier transformation using a Gaussian window. The discrete wavelet transform (DWT) is used in order to weight the resulting power patterns according to their frequency. Subsequently, the weighted power patterns are binarized via applying a threshold. At this final stage, significant power coincidence is determined across all subgroups of channel combinations for individual frequencies by selecting the maximum ratio between observed and expected duration of co-activation as test statistic. The null hypothesis that the activity in each channel is independent from the activity in every other channel is simulated by independent, random rotation of the respective activity patterns. We applied this procedure to single trials of multiple simultaneously sampled local field potentials (LFPs) obtained from occipital, parietal, central and precentral areas of three macaque monkeys. Since their task was to use visual cues to perform a precise arm movement, co-activation of numerous cortical sites was expected. In a data set with 17 channels analyzed, up to 13 sites expressed simultaneous power in the range between 5 and 240 Hz. On average, more than 50% of active channels participated at least once in a significant power co-activation pattern (PCP). Because the significance of such PCPs can be evaluated at the level of single trials, we are confident that this procedure is useful to study single trial variability with sufficient accuracy that much of the behavioral variability can be explained by the dynamics of the underlying distributed neuronal processes.
Poster presentation: Introduction The ability of neurons to emit different firing patterns is considered relevant for neuronal information processing. In dopaminergic neurons, prominent patterns include highly regular pacemakers with separate spikes and stereotyped intervals, processes with repetitive bursts and partial regularity, and irregular spike trains with nonstationary properties. In order to model and quantify these processes and the variability of their patterns with respect to pharmacological and cellular properties, we aim to describe the two dimensions of burstiness and regularity in a single model framework. Methods We present a stochastic spike train model in which the degree of burstiness and the regularity of the oscillation are described independently and with two simple parameters. In this model, a background oscillation with independent and normally distributed intervals gives rise to Poissonian spike packets with a Gaussian firing intensity. The variability of inter-burst intervals and the average number of spikes in each burst indicate regularity and burstiness, respectively. These parameters can be estimated by fitting the model to the autocorrelograms. This allows to assign every spike train a position in the two-dimensional space described by regularity and burstiness and thus, to investigate the dependence of the firing patterns on different experimental conditions. Finally, burst detection in single spike trains is possible within the model because the parameter estimates determine the appropriate bandwidth that should be used for burst identification. Results and Discussion We applied the model to a sample data set obtained from dopaminergic substantia nigra and ventral tegmental area neurons recorded extracellularly in vivo and studied differences between the firing activity of dopaminergic neurons in wildtype and K-ATP channel knock-out mice. The model is able to represent a variety of discharge patterns and to describe changes induced pharmacologically. It provides a simple and objective classification scheme for the observed spike trains into pacemaker, irregular and bursty processes. In addition to the simple classification, changes in the parameters can be studied quantitatively, also including the properties related to bursting behavior. Interestingly, the proposed algorithm for burst detection may be applicable also to spike trains with nonstationary firing rates if the remaining parameters are unaffected. Thus, the proposed model and its burst detection algorithm can be useful for the description and investigation of neuronal firing patterns and their variability with cellular and experimental conditions.
Poster presentation: Introduction Adequate anesthesia is crucial to the success of surgical interventions and subsequent recovery. Neuroscientists, surgeons, and engineers have sought to understand the impact of anesthetics on the information processing in the brain and to properly assess the level of anesthesia in an non-invasive manner. Studies have indicated a more reliable depth of anesthesia (DOA) detection if multiple parameters are employed. Indeed, commercial DOA monitors (BIS, Narcotrend, M-Entropy and A-line ARX) use more than one feature extraction method. Here, we propose TESPAR (Time Encoded Signal Processing And Recognition) a time domain signal processing technique novel to EEG DOA assessment that could enhance existing monitoring devices. ...
Poster presentation: Functional connectivity of the brain describes the network of correlated activities of different brain areas. However, correlation does not imply causality and most synchronization measures do not distinguish causal and non-causal interactions among remote brain areas, i.e. determine the effective connectivity [1]. Identification of causal interactions in brain networks is fundamental to understanding the processing of information. Attempts at unveiling signs of functional or effective connectivity from non-invasive Magneto-/Electroencephalographic (M/EEG) recordings at the sensor level are hampered by volume conduction leading to correlated sensor signals without the presence of effective connectivity. Here, we make use of the transfer entropy (TE) concept to establish effective connectivity. The formalism of TE has been proposed as a rigorous quantification of the information flow among systems in interaction and is a natural generalization of mutual information [2]. In contrast to Granger causality, TE is a non-linear measure and not influenced by volume conduction. ...
Poster presentation: Introduction Dopaminergic neurons in the midbrain show a variety of firing patterns, ranging from very regular firing pacemaker cells to bursty and irregular neurons. The effects of different experimental conditions (like pharmacological treatment or genetical manipulations) on these neuronal discharge patterns may be subtle. Applying a stochastic model is a quantitative approach to reveal these changes. ...
Poster presentation: Introduction The brain is a highly interconnected network of constantly interacting units. Understanding the collective behavior of these units requires a multi-dimensional approach. The results of such analyses are hard to visualize and interpret. Hence tools capable of dealing with such tasks become imperative. ....
Poster presentation: NO-sensitive guanylyl cyclases (sGCs) are cytosolic receptors for nitric oxide (NO) catalyzing the conversion of GTP to cGMP. sGCs are obligate heterodimers composed of one alpha and beta subunit each. The allosteric mechanism of sGC activation via NO is well understood, however, our knowledge about alternative mechanisms such as protein-protein interactions regulating activity, availability, translocation and expression of sGC is rather limited. In a search by the yeast two-hybrid system using the catalytic domain of the alpha1 subunit as the bait, we have identified two structurally related proteins AGAP1 [1] and MRIP2 as novel sGC interacting proteins. MRIP2 is a multi-domain protein of 75 kDa comprising a single PH and ArfGAP domain each and two ankyrin repeats. Co-immunoprecipitation experiments using COS1 cells overexpressing both proteins demonstrated the interaction of MRIP2 with both subunits of the sGC alpha1beta1. Confocal microscopical analysis showed a prominent plasma membrane staining of MRIP2. This membrane association is mediated through an N-terminal myristoylation site and through binding of its PH domain to phospholipids such as phosphatidylinositol-3,5-bisphosphate (PI(3,5)P2). We hypothesize that MRIP2 may represent an acceptor protein for sGC that mediates recruitment of cytosolic sGC to the plasma membrane or other subcellular compartments.
Poster presentation: NO-sensitive guanylyl cyclases (GC) are the principal receptors for nitric oxide (NO) and convert GTP into the second messenger cGMP. We showed that GC is prone to tyrosine phosphorylation in COS1 cells overexpressing the human holoenzyme. Similar results were obtained in PC12 cells and in rat aortic tissue slices. The major phosphorylation site was mapped to position 192 in the regulatory domain of the beta1 subunit. Tyrosine phosphorylation of GC was reduced in the presence of the inhibitors PP1 and PP2 indicating that Src-like kinases are critically involved in phosphorylation. Moreover, co-immunoprecipitation experiments revealed an interaction between Src and GC. To further analyse the relevance of this posttranslational modification we generated a phospho-specific antibody raised against pTyr192. This antibody clearly distinguishes between phosphorylated and non-phosphorylated GC and may be a powerful tool to analyse the subcellular localisation of the phosphorylated enzyme.
Poster presentation NO-sensitive guanylyl cyclases (soluble guanylyl cyclase, sGC) are among the key regulators of intracellular cGMP concentration. The mechanisms underlying NO-mediated activation of sGC are quite well understood, however, little is known about the fine-tuning of sGC activity through alternative mechanisms such as protein phosphorylation. Several reports have demonstrated the reversible phosphorylation of sGC on serine/threonine residues, and it has been speculated, though not experimentally proven, that sGC might also be phosphorylated on tyrosine residues. Using broad-spectrum phosphatase inhibitors we were able to demonstrate tyrosine phosphorylation at Tyr192 of the beta 1 subunit of human sGC in COS1 cells. This residue forms part of a sequence segment (YEDL) representing a preferential binding site for SH2 domains of Src-like kinases. Pull-down assays and co-immunoprecipitation experiments showed that Src can indeed bind via its SH2 domain to pTyr192 of beta 1 indicating that tyrosine phosphorylation of sGC may be followed by recruitment of Src-like kinases to the phosphorylated beta 1 subunit. In support of this hypothesis, immunofluorescence studies showed a colocalization of overexpressed sGC and Src at the plasma membrane of COS1 and Hela cells. Together, our results point to an unexpected crosstalk between tyrosine kinase pathway(s) and the NO/cGMP signalling cascade which may result in translocation of the predominantly cytosolic sGC to the cytosolic face of the plasma membrane.
Poster presentation: Purpose of the study First-line HAART with tenofovir DF (TDF) and FTC in pivotal trials has been associated with high efficacy and good tolerability. However, real-life clinical practice often differs from clinical trials due to co-morbidities, co-infections, and less intensive clinical monitoring. To evaluate efficacy and safety of first-line HAART in a day-to-day setting, this Gilead-sponsored non-interventional cohort was established. Methods Between July 2005 and August 2006, 533 HIV-1 infected antiretroviral-naïve patients from 50 German centres enrolled in this non-interventional cohort. All patients were followed every 3 months for 3 years to monitor efficacy (viral load [VL], CD4), tolerability, renal safety, regimen changes and resistance profile. All patients received TDF+FTC as a single tablet (Truvada, TVD) in combination with either an NNRTI or PI/r as their first antiretroviral regimen. Summary of results As of June 2008, 2 years of therapy have been documented for 330/533 (62%) patients. At treatment initiation, 81% were male; median age was 39 years; clinical AIDS diagnosis was documented in 22%; 47% started therapy with CD4 <200 cells/mm3. TVD was combined with an NNRTI (43%) or a PI/r (57%). After 24 months, in an As-Treated (AT) analysis, 85% patients achieved a VL <50 copies/ml (VL <500 copies/ml: 97%), median CD4 count increased from 217 at baseline to 450 cells/mm3 (IQR: 325–608). Truvada showed a good safety profile; 76 adverse events (AEs) of any grade were reported in 66/533 patients (12%); six of these were judged serious. Fourteen (2.6%) patients discontinued TVD due to AEs. Renal abnormalities of any grade were reported in 10 patients (1.9%). Virological failure was documented in nine patients, of which eight were genotyped; M184V/I was detected in three, K65R in two patients. Conclusion During 2 years of follow-up, the overall safety of TVD was good; renal AEs of any grade were reported in 1.9% of patients. K65R was detected in two patients. First-line HAART with TVD plus an NNRTI or PI/r in clinical practice showed comparable efficacy to that observed in controlled clinical trials.
Purpose of the study: There is a clinical need for antiretroviral therapy (ART) regimens that simplify dosing and make adherence easier for specific patient groups such as former intravenous drug users (IVDU) receiving opiate substitution. Availability of tenofovir DF (TDF) and other once-daily (OD) agents could offer a viable OD regimen. The 3OD study was designed to evaluate the use of OD HAART in IVDU patients.
Methods: 3OD was a single-arm, multicentre, 48-weeks trial to assess efficacy, tolerability and adherence to a OD TDF-containing HAART regimen in former IVDU patients receiving opiate substitution. Of 67 patients enrolled, 27 were antiretroviral treatment naïve, 10 were virologically suppressed (<400 copies/mL), and 30 were re-starting HAART without prior virological failure. Opiate substitution was adjusted according to subject symptoms of opiate overdosing or withdrawal. Various methods were used to assess adherence: besides pill count, patients were asked to fill in a MASRI (Medication Adherence Self-Report Inventory) questionnaire and an electronic log pad diary. Calculation of adherence by pill count assumed that unreturned pills had been taken by the subjects.
Summary of results: Overall, 55% (n = 37, ITT, M = F) of patients had viral load <400 copies/mL at week 48. Using an ITT, M = E analysis, 90% (37/41) of patients reached undetectable VL (<400 copies/mL), 56% (23/41 patients) had plasma HIV-1 RNA concentrations <50 copies/mL at week 48. Only 30 patients (45%) completed the full study and the follow-up period. In 51% of patients, TDF adherence was >100% using pill count. MASRI showed adherence rates of 80–100% in 83–85% of patients; however, 15 patients never entered any data. Diary data were entered by 57 patients; diary data were entered for fewer days than patients received treatment (mean difference 113 days, calculated from treatment start and stop dates).
Conclusion: TDF in combination with other OD antiretrovirals in former IVDU patients showed comparable efficacy to that seen in the average HIV-1 infected population. However, measurement of adherence to self-administered HAART via pill count, MASRI or diary may be misleading in this population.
Poster presentation: Background Maraviroc is a new drug used to treat HIV infection from the new class of drugs called CCR5 entry inhibitors. As the active principle of these drugs is to block the CCR5-receptor on the surface of the target cells, it has to be known if the virus in the patient is using only CCR5 as co-receptor or if there are populations that can also use CXCR4. Therefore, an assay to determine the tropism has to be performed before starting a therapy. Besides phenotypic assays like the TROFILE® assay by Monogram, used in the approval studies, there exist several genotyping systems like geno2pheno-coreceptor, Wetcat (providing five different genotypic tropism schemes) and WebPSSM. ...
Background: In Germany, 17% of 59,000 persons living with HIV/AIDS are female. Accordingly, the research focus in clinical studies as well as in cohort analyses has been almost exclusively on HIV-positive men. As a consequence, there is an urgent need to characterize and evaluate the outcome of HAART in HIV-positive women and to identify special requirements of this particular patient population.
Methods: Cross-sectional multicentre (n = 31 centres) evaluation to observe characteristics of 1,557 HIV-positive women receiving medical care in Germany between June 2007 and March 2008. Data acquisition was performed using standardized questionnaires.
Summary of results: Of 1,557 HIV-positive women studied, 1,191 (77%) received HAART. Mean age was 40 years and average time of known HIV-infection was 9 years. Risk of HIV transmission was: 40% heterosexual intercourse in Germany, 36% heterosexual intercourse in a high prevalence country; 17% IDU; 7% other reasons for transmission. 46% of the women had a migration background. Mean time on antiretroviral treatment was 7 years. 53% of the female participants had been treated with >2 HAART-regimens. 47% of the study subjects received a PI-based regimen, 33% a NNRTI-based regimen; 20% were on other combinations. The most commonly used PI and NNRTI were lopinavir/r and nevirapine, respectively. Only 48% of all women under HAART achieved a viral load <40 copies/ml. There was a significant difference between the PI-treated group with 44% patients <40 copies/ml and the NNRTI-treated group with 56% <40 copies/ml (p = 0.003).
Conclusion: We found that HIV-positive women depicted an inferior virological response to HAART compared to those previously published in German cohort analyses dominated by men (response rates >75%). Possible differences in adherence or drug resistance may have impacted these results and are currently being evaluated in ongoing sub-analyses. Of note, the lack of a study arm with male patients is a limitation of this investigation. However, this is partly off-set by the fact that there are good comparative data in the male population found in other cohorts. We conclude that our results are in discordance to the popular assumption that there are no gender specific differences in virological treatment outcome of HAART.
Background: The combination of stavudine (d4T), 3TC and NVP was the WHO recommended first-line regimen for the treatment of naïve HIV-1 infected patients in resource-limited settings. But peripheral polyneuropathy, lipoatrophy and symptomatic hyperlactatemia are frequent and treatment-limiting adverse events associated with stavudine, especially when combined with antituberculous drugs. Tenofovir combined with lamivudine and efavirenz has proven excellent efficacy, but there is little experience when given with NVP.
Methods: Retrospective analysis of all patients receiving TDF, NVP and 3TC or FTC as first-line treatment in the Frankfurt HIV cohort.
Summary of results: 70 patients (15 female) with a median CD4 cell count of 210/μl (47–949) and HIV-RNA PCR of 140,000 copies/ml (2,500–2,000,000) at baseline received TDF, NVP and 3TC/FTC, and were treated for a median of 68 weeks (16–278). CD4 cells rose up to cells/μl 322 (119–1075) and 75% of the patients remained on treatment. All patients on treatment at week 48 were <50 c/ml, even those starting with CD4 cells of <200 cells/μl or a HIV-RNA PCR >100,000 c/m. Reasons for discontinuation (24%) were mainly adverse events (13%), with rash (7%) and liver toxicity (6%) being the two most common, whereas virologic failure, drug interaction and non-adherence were all relatively rare (each 3%).
Conclusion: The combination of NVP, TDF and 3TC or FTC is effective and well tolerated in previously naïve HIV-1 infected patients even when started with low CD4 cell counts (<200/ml) and high viral loads (>100,000 c/ml). In the latest amendment of the WHO guidelines TDF, instead of d4T, is the recommended first-line treatment in resource-limited settings.
Poster presentation: Purpose of the study To compare the virological, immunological and clinical response to three boosted double protease inhibitor (PI) regimens of saquinavir and ritonavir in combination with lopinavir (LOPSAQ), atazanavir (ATSAQ) or fosamprenavir (FOSAQ) without reverse transcriptase inhibitors (RTI) in HIV-positive patients with limited RTI treatment options. ...
Poster presentation: Background In the past years, once-daily (QD) dosing of antiretroviral combination therapy has become an increasingly available treatment option for HIV-1+ patients. Methods Open label study in which HIV-1+ patients treated with SAQ/RTV (1000/100 mg BID) and two NRTIs with HIV-RNA-PCR < 50 copies/ml were switched to SAQ/RTV(2000/100 mg QD) with unchanged NRTI-backbone. CD4-cells, HIV-RNA-PCR, SAQ and RTV drug-levels and metabolic parameters were compared. Summary of results 17 patients (15 male, 42 years), median CD4 456 ± 139/micro l were included so far. The median follow-up time is 4 months. The HIV-RNA-PCR remained <50 copies/ml for all patients. Fasting metabolic parameters remained unchanged. The SAQ AUC 0–12 h were significantly higher when given QD vs. BID (median 29,400 vs. 18,500 ng*h/ml; p = 0.009), whereas the Cmin, Cmax and AUC was lower for RTV when given QD vs. BID (7,400 vs. 11,700 ng*h/ml; p = 0.02). Conclusion In this ongoing study SAQ/RTV (2000/100 mg QD) was well tolerated and demonstrated higher SAQ and lower RTV drug levels as compared to the BID dosing schedule. (Table 1 and Figure 1.)
Poster presentation: Purpose of the study To compare the lipometabolic profiles of three double-boosted protease inhibitor (PI) regimens at standard dose, containing saquinavir and ritonavir in combination with lopinavir (LOPSAQ), atazanavir (ATSAQ) or fosamprenavir (FOSAQ) in HIV-positive patients, treated without reverse transcriptase inhibitors (RTI). ...
Poster presentation: Purpose of the study The aim of the Rainbow Cohort is to assess the tolerability and efficacy of initiating treatment with, or switching treatment to saquinavir (SQV) 500 mg film-coated tablet formulation. We present the final 48-week subgroup analysis of antiretroviral therapy (ART)-naïve patients. ...
Poster presentation: Purpose of the study The aim of the Rainbow Cohort is to assess the tolerability and efficacy of initiating treatment with, or switching treatment to the saquinavir (SQV) 500 mg film-coated tablet formulation. We present the final 48-week subgroup analysis of PI-experienced, but SQV-naïve patients. ...
Background: Microarray analysis still remains a powerful tool to identify new components of the transcriptosome and it has helped to increase the knowledge of targets triggered by stress conditions such as hypoxia and nitric oxide. However, analysis of transcriptional regulatory events remain elusive due to the contribution of altered mRNA stability to gene expression patterns, as well as changes in the half-life of mRNAs, which influence mRNA expression levels and their turn over rates. To circumvent these problems, we have focused on the analysis of newly transcribed (nascent) mRNAs by nuclear run on (NRO), followed by microarray analysis. Result: We identified 188 genes that were significantly regulated by hypoxia, 81 genes were affected by nitric oxide, and 292 genes were induced by the co-treatment of macrophages with both NO and hypoxia. Fourteen genes (Bnip3, Ddit4, Vegfa, Trib3, Atf3, Cdkn1a, Scd1, D4Ertd765e, Sesn2, Son, Nnt, Lst1, Hps6 and Fxyd5) were common to hypoxia and/or nitric oxide treatments, but with different levels of expression. We observed that 166 transcripts were regulated only when cells were co-treated with hypoxia and NO but not with either treatment alone, pointing to the importance of a crosstalk between hypoxia and NO. In addition, both array and proteomics data supported a consistent repression of hypoxia regulated targets by NO. Conclusion: By eliminating the interference of steady state mRNA in gene expression profiling, we increased the sensitivity of mRNA analysis and identified previously unknown hypoxia-induced targets. Gene analysis profiling corroborated the interplay between NO- and hypoxia-induced signalling.
Background and objectives: Constrictive pericarditis (CP) is the result of a spectrum of primary cardiac and non-cardiac conditions. Little data exists on the cause-specific survival after pericardiectomy in the modern era. The impact of pericardial calcification (CA) on survival is unclear. We sought to determine the association of etiology of CP, CA and other clinical variables with long-term survival after pericardiectomy. Methods: We analyzed the records of 163 patients who underwent pericardiectomy for CP over a 24-year period at a single center. The diagnosis of CP was established by surgical report. Vital status was obtained by the Social Security Death Index. The Kaplan Meier method was used to estimate overall survival and survival by etiology group. Cox proportional hazards regression analysis was performed to assess the effect of various causes for CP on longterm survival while adjusting for age. Results: The etiology of CP was idiopathic in 75 patients (46%), prior cardiac surgery in 60 (37%), radiation treatment in 15 (9%) and miscellaneous in 13 patients (8%). Vital status was obtained in 160 patients (98%). Median follow-up was 6.9 years (range: 0.8 to 24.5 years). Perioperative mortality for all patients was 6%. Idiopathic CP had the best prognosis (7 year survival: 88%, 95% confidence interval [CI] 76% to 94%) followed by postsurgical (66%, 95% CI 52% to 78%) and postradiation CP (27%, 95% CI 9% to 58%). In bootstrap-validated proportional hazards analyses, predictors of poor survival were prior radiation, worse renal function, higher pulmonary artery pressure (PAP), abnormal left ventricular (LV) systolic function, lower serum sodium level, and older age. Pericardial calcification had no impact on survival. Conclusion: Long-term survival after pericardiectomy for CP is determined by the underlying etiology of constriction, LV systolic function, renal function, serum sodium, and PAP. Patients with postsurgical as well as postradiation CP have a survival inferior to patients with idiopathic CP. Perioperative Mortality is low. The relatively good survival after pericardiectomy in patients with idiopathic CP emphasizes the safety of pericardiectomy in this group.
We report the case of a 62-year-old man with chronic pancreatitis who presented with increasing abdominal pain. Sonography, magnetic resonance imaging, contrast-enhanced computed tomography, and ultimately catheter angiography demonstrated a pancreatic pseudocyst that had eroded into the splenoportal venous confluence, mimicking an arterial aneurysm. The diagnostic was confirmed at the time of surgical treatment. This case demonstrates the use of imaging to diagnose complications of pancreatitis, and the difficulty of distinguishing an eroding pseudocyst from an arterial aneurysm.
The pathogenesis of nodular lymphocyte–predominant Hodgkin lymphoma (NLPHL) and its relationship to other lymphomas are largely unknown. This is partly because of the technical challenge of analyzing its rare neoplastic lymphocytic and histiocytic (L&H) cells, which are dispersed in an abundant nonneoplastic cellular microenvironment. We performed a genome-wide expression study of microdissected L&H lymphoma cells in comparison to normal and other malignant B cells that indicated a relationship of L&H cells to and/or that they originate from germinal center B cells at the transition to memory B cells. L&H cells show a surprisingly high similarity to the tumor cells of T cell–rich B cell lymphoma and classical Hodgkin lymphoma, a partial loss of their B cell phenotype, and deregulation of many apoptosis regulators and putative oncogenes. Importantly, L&H cells are characterized by constitutive nuclear factor {kappa}B activity and aberrant extracellular signal-regulated kinase signaling. Thus, these findings shed new light on the nature of L&H cells, reveal several novel pathogenetic mechanisms in NLPHL, and may help in differential diagnosis and lead to novel therapeutic strategies.
Anaphylactic shock is a severe allergic reaction involving multiple organs including the bronchial and cardiovascular system. Most anaphylactic mediators, like platelet-activating factor (PAF), histamine, and others, act through G protein – coupled receptors, which are linked to the heterotrimeric G proteins Gq /G 11 , G12/G13 , and Gi . The role of downstream signaling pathways activated by anaphylactic mediators in defi ned organs during anaphylactic reactions is largely unknown. Using genetic mouse models that allow for the conditional abrogation of G q /G 11 - and G 12 /G 13 -mediated signaling pathways by inducible Cre/loxP-mediated mutagenesis in endothelial cells (ECs), we show that Gq /G11 -mediated signaling in ECs is required for the opening of the endothelial barrier and the stimulation of nitric oxide formation by various infl ammatory mediators as well as by local anaphylaxis. The systemic effects of anaphylactic mediators like histamine and PAF, but not of bacterial lipopolysaccharide (LPS), are blunted in mice with endothelial G alpha q/G alpha 11 deficiency. Mice with endothelium-specific G alpha q /G alpha 11 deficiency, but not with G alpha 12/G alpha 13 deficiency, are protected against the fatal consequences of passive and active systemic anaphylaxis. This identifies endothelial Gq/G11 -mediated signaling as a critical mediator of fatal systemic anaphylaxis and, hence, as a potential new target to prevent or treat anaphylactic reactions.
Antibodies to citrulline-modifi ed proteins have a high diagnostic value in rheumatoid arthritis (RA). However, their biological role in disease development is still unclear. To obtain insight into this question, a panel of mouse monoclonal antibodies was generated against a major triple helical collagen type II (CII) epitope (position 359 – 369; ARGLTGRPGDA) with or without arginines modifi ed by citrullination. These antibodies bind cartilage and synovial tissue, and mediate arthritis in mice. Detection of citrullinated CII from RA patients ’ synovial fl uid demonstrates that cartilage-derived CII is indeed citrullinated in vivo. The structure determination of a Fab fragment of one of these antibodies in complex with a citrullinated peptide showed a surprising beta -turn conformation of the peptide and provided information on citrulline recognition. Based on these findings, we propose that autoimmunity to CII, leading to the production of antibodies specific for both native and citrullinated CII, is an important pathogenic factor in the development of RA.
Background Multimorbidity is a highly frequent condition in older people, but well designed longitudinal studies on the impact of multimorbidity on patients and the health care system have been remarkably scarce in numbers until today. Little is known about the long term impact of multimorbidity on the patients' life expectancy, functional status and quality of life as well as health care utilization over time. As a consequence, there is little help for GPs in adjusting care for these patients, even though studies suggest that adhering to present clinical practice guidelines in the care of patients with multimorbidity may have adverse effects. Methods The study is designed as a multicentre prospective, observational cohort study of 3.050 patients aged 65 to 85 at baseline with at least three different diagnoses out of a list of 29 illnesses and syndromes. The patients will be recruited in approx. 120 to 150 GP surgeries in 8 study centres distributed across Germany. Information about the patients' morbidity will be collected mainly in GP interviews and from chart reviews. Functional status, resources/risk factors, health care utilization and additional morbidity data will be assessed in patient interviews, in which a multitude of well established standardized questionnaires and tests will be performed. Discussion The main aim of the cohort study is to monitor the course of the illness process and to analyse for which reasons medical conditions are stable, deteriorating or only temporarily present. First, clusters of combinations of diseases/disorders (multimorbidity patterns) with a comparable impact (e.g. on quality of life and/or functional status) will be identified. Then the development of these clusters over time will be analysed, especially with regard to prognostic variables and the somatic, psychological and social consequences as well as the utilization of health care resources. The results will allow the development of an instrument for prediction of the deterioration of the illness process and point at possibilities of prevention. The practical consequences of the study results for primary care will be analysed in expert focus groups in order to develop strategies for the inclusion of the aspects of multimorbidity in primary care guidelines.
The use of neutralizing antibodies is one of the most successful methods to interfere with receptor–ligand interactions in vivo. In particular blockade of soluble inflammatory mediators or their corresponding cellular receptors was proven an effective way to regulate inflammation and/or prevent its negative consequences. However, one problem that comes along with an effective neutralization of inflammatory mediators is the general systemic immunomodulatory effect. It is, therefore, important to design a treatment regimen in a way to strike at the right place and at the right time in order to achieve maximal effects with minimal duration of immunosuppression or hyperactivation. In this review, we reflect on two examples of how short time administration of such neutralizing antibodies can block two distinct inflammatory consequences of viral infection. First, we review recent findings that blockade of IL-10/IL-10R interaction can resolve chronic viral infection and second, we reflect on how neutralization of the chemokine CXCL10 can abrogate virus-induced type 1 diabetes.
Breaking tolerance to the natural human liver autoantigen cytochrome P450 2D6 by virus infection
(2008)
Autoimmune liver diseases, such as autoimmune hepatitis (AIH) and primary biliary cirrhosis, often have severe consequences for the patient. Because of a lack of appropriate animal models, not much is known about their potential viral etiology. Infection by liver-tropic viruses is one possibility for the breakdown of self-tolerance. Therefore, we infected mice with adenovirus Ad5 expressing human cytochrome P450 2D6 (Ad-2D6). Ad-2D6–infected mice developed persistent autoimmune liver disease, apparent by cellular infiltration, hepatic fibrosis, “fused” liver lobules, and necrosis. Similar to type 2 AIH patients, Ad-2D6–infected mice generated type 1 liver kidney microsomal–like antibodies recognizing the immunodominant epitope WDPAQPPRD of cytochrome P450 2D6 (CYP2D6). Interestingly, Ad-2D6–infected wild-type FVB/N mice displayed exacerbated liver damage when compared with transgenic mice expressing the identical human CYP2D6 protein in the liver, indicating the presence of a stronger immunological tolerance in CYP2D6 mice. We demonstrate for the first time that infection with a virus expressing a natural human autoantigen breaks tolerance, resulting in a chronic form of severe, autoimmune liver damage. Our novel model system should be instrumental for studying mechanisms involved in the initiation, propagation, and precipitation of virus-induced autoimmune liver diseases.
Background Production of interferon (IFN)-gamma is key to efficient anti-tumor immunity. The present study was set out to investigate effects of IFNgamma on the release of the potent pro-angiogenic mediator IL-8 by human A549 lung carcinoma cells.
Methods A549 cells were cultured and stimulated with interleukin (IL)-1beta alone or in combination with IFNgamma. IL-8 production by these cells was analyzed with enzyme linked immuno sorbent assay (ELISA). mRNA-expression was analyzed by real-time PCR and RNase protection assay (RPA), respectively. Expression of inhibitor-kappaBalpha, cellular IL-8, and cyclooxygenase-2 was analyzed by Western blot analysis.
Results Here we demonstrate that IFNgamma efficiently reduced IL-8 secretion under the influence of IL-1beta. Surprisingly, real-time PCR analysis and RPA revealed that the inhibitory effect of IFNgamma on IL-8 was not associated with significant changes in mRNA levels. These observations concurred with lack of a modulatory activity of IFNgamma on IL-1beta-induced NF-kappaB activation as assessed by cellular IkappaB levels. Moreover, analysis of intracellular IL-8 suggests that IFNgamma modulated IL-8 secretion by action on the posttranslational level. In contrast to IL-8, IL-1beta-induced cyclooxygenase-2 expression and release of IL-6 were not affected by IFNgamma indicating that modulation of IL-1beta action by this cytokine displays specificity.
Conclusions Data presented herein agree with an angiostatic role of IFNgamma as seen in rodent models of solid tumors and suggest that increasing T helper type 1 (Th1)-like functions in lung cancer patients e.g. by local delivery of IFNgamma may mediate therapeutic benefit via mechanisms that potentially include modulation of pro-angiogenic IL-8.
Introduction Loss of intestinal integrity has been implicated as an important contributor to the development of excessive inflammation following severe trauma. Thus far, clinical data concerning the occurrence and significance of intestinal damage after trauma remain scarce. This study investigates whether early intestinal epithelial cell damage occurs in trauma patients and, if present, whether such cell injury is related to shock, injury severity and the subsequent inflammatory response. Methods Prospective observational cohort study in 96 adult trauma patients. Upon arrival at the emergency room (ER) plasma levels of intestinal fatty acid binding protein (i-FABP), a specific marker for damage of differentiated enterocytes, were measured. Factors that potentially influence the development of intestinal cell damage after trauma were determined, including the presence of shock and the extent of abdominal trauma and general injury severity. Furthermore, early plasma levels of i-FABP were related to inflammatory markers interleukin-6 (IL-6), procalcitonin (PCT) and C-reactive protein (CRP). Results Upon arrival at the ER, plasma i-FABP levels were increased compared with healthy volunteers, especially in the presence of shock (P < 0.01). The elevation of i-FABP was related to the extent of abdominal trauma as well as general injury severity (P < 0.05). Circulatory i-FABP concentrations at ER correlated positively with IL-6 and PCT levels at the first day (r2 = 0.19; P < 0.01 and r2 = 0.36; P < 0.001 respectively) and CRP concentrations at the second day after trauma (r2 = 0.25; P < 0.01). Conclusions This study reveals early presence of intestinal epithelial cell damage in trauma patients. The extent of intestinal damage is associated with the presence of shock and injury severity. Early intestinal damage precedes and is related to the subsequent developing inflammatory response.
Abstract: Bcl-2 family proteins including the pro-apoptotic BH3-only proteins are central regulators of apoptotic cell death. Here we show by a focused siRNA miniscreen that the synergistic action of the BH3-only proteins Bim and Bmf is required for apoptosis induced by infection with Neisseria gonorrhoeae (Ngo). While Bim and Bmf were associated with the cytoskeleton of healthy cells, they both were released upon Ngo infection. Loss of Bim and Bmf from the cytoskeleton fraction required the activation of Jun-N-terminal kinase-1 (JNK-1), which in turn depended on Rac-1. Depletion and inhibition of Rac-1, JNK-1, Bim, or Bmf prevented the activation of Bak and Bax and the subsequent activation of caspases. Apoptosis could be reconstituted in Bim-depleted and Bmf-depleted cells by additional silencing of antiapoptotic Mcl-1 and Bcl-XL, respectively. Our data indicate a synergistic role for both cytoskeletal-associated BH3-only proteins, Bim, and Bmf, in an apoptotic pathway leading to the clearance of Ngo-infected cells. Author Summary: A variety of physiological death signals, as well as pathological insults, trigger apoptosis, a genetically programmed form of cell death. Pathogens often induce host cell apoptosis to establish a successful infection. Neisseria gonorrhoeae (Ngo), the etiological agent of the sexually transmitted disease gonorrhoea, is a highly adapted obligate human-specific pathogen and has been shown to induce apoptosis in infected cells. Here we unveil the molecular mechanisms leading to apoptosis of infected cells. We show that Ngo-mediated apoptosis requires a special subset of proapoptotic proteins from the group of BH3-only proteins. BH3-only proteins act as stress sensors to translate toxic environmental signals to the initiation of apoptosis. In a siRNA-based miniscreen, we found Bim and Bmf, BH3-only proteins associated with the cytoskeleton, necessary to induce host cell apoptosis upon infection. Bim and Bmf inactivated different inhibitors of apoptosis and thereby induced cell death in response to infection. Our data unveil a novel pathway of infection-induced apoptosis that enhances our understanding of the mechanism by which BH3-only proteins control apoptotic cell death.
CD8 T cells are recognized key players in control of persistent virus infections, but increasing evidence suggests that assistance from other immune mediators is also needed. Here, we investigated whether specific antibody responses contribute to control of lymphocytic choriomeningitis virus (LCMV), a prototypic mouse model of systemic persistent infection. Mice expressing transgenic B cell receptors of LCMV-unrelated specificity, and mice unable to produce soluble immunoglobulin M (IgM) exhibited protracted viremia or failed to resolve LCMV. Virus control depended on immunoglobulin class switch, but neither on complement cascades nor on Fc receptor gamma chain or Fc gamma receptor IIB. Cessation of viremia concurred with the emergence of viral envelope-specific antibodies, rather than with neutralizing serum activity, and even early nonneutralizing IgM impeded viral persistence. This important role for virus-specific antibodies may be similarly underappreciated in other primarily T cell–controlled infections such as HIV and hepatitis C virus, and we suggest this contribution of antibodies be given consideration in future strategies for vaccination and immunotherapy.
Background Endothelium-derived nitric oxide plays an important role for the bone marrow microenvironment. Since several important effects of nitric oxide are mediated by cGMP-dependent pathways, we investigated the role of the cGMP downstream effector cGMP-dependent protein kinase I (cGKI) on postnatal neovascularization. Methodology/Principal Findings In a disc neovascularization model, cGKI -/- mice showed an impaired neovascularization as compared to their wild-type (WT) littermates. Infusion of WT, but not cGKI -/- bone marrow progenitors rescued the impaired ingrowth of new vessels in cGKI-deficient mice. Bone marrow progenitors from cGKI -/- mice showed reduced proliferation and survival rates. In addition, we used cGKI alpha leucine zipper mutant (LZM) mice as model for cGKI deficiency. LZM mice harbor a mutation in the cGKI alpha leucine zipper that prevents interaction with downstream signaling molecules. Consistently, LZM mice exhibited reduced numbers of vasculogenic progenitors and impaired neovascularization following hindlimb ischemia compared to WT mice. Conclusions/Significance Our findings demonstrate that the cGMP-cGKI pathway is critical for postnatal neovascularization and establish a new role for cGKI in vasculogenesis, which is mediated by bone marrow-derived progenitors.
Background Parkinson's disease (PD) is an adult-onset movement disorder of largely unknown etiology. We have previously shown that loss-of-function mutations of the mitochondrial protein kinase PINK1 (PTEN induced putative kinase 1) cause the recessive PARK6 variant of PD. Methodology/Principal Findings Now we generated a PINK1 deficient mouse and observed several novel phenotypes: A progressive reduction of weight and of locomotor activity selectively for spontaneous movements occurred at old age. As in PD, abnormal dopamine levels in the aged nigrostriatal projection accompanied the reduced movements. Possibly in line with the PARK6 syndrome but in contrast to sporadic PD, a reduced lifespan, dysfunction of brainstem and sympathetic nerves, visible aggregates of alpha-synuclein within Lewy bodies or nigrostriatal neurodegeneration were not present in aged PINK1-deficient mice. However, we demonstrate PINK1 mutant mice to exhibit a progressive reduction in mitochondrial preprotein import correlating with defects of core mitochondrial functions like ATP-generation and respiration. In contrast to the strong effect of PINK1 on mitochondrial dynamics in Drosophila melanogaster and in spite of reduced expression of fission factor Mtp18, we show reduced fission and increased aggregation of mitochondria only under stress in PINK1-deficient mouse neurons. Conclusion Thus, aging Pink1 -/- mice show increasing mitochondrial dysfunction resulting in impaired neural activity similar to PD, in absence of overt neuronal death.
Background Transplantation of vasculogenic progenitor cells (VPC) improves neovascularization after ischemia. However, patients with type 2 diabetes mellitus show a reduced VPC number and impaired functional activity. Previously, we demonstrated that p38 kinase inhibition prevents the negative effects of glucose on VPC number by increasing proliferation and differentiation towards the endothelial lineage in vitro. Moreover, the functional capacity of progenitor cells is reduced in a mouse model of metabolic syndrome including type 2 diabetes (Leprdb) in vivo. Findings The aim of this study was to elucidate the underlying signalling mechanisms in vitro and in vivo. Therefore, we performed DNA-protein binding arrays in the bone marrow of mice with metabolic syndrome, in blood-derived progenitor cells of diabetic patients as well as in VPC ex vivo treated with high levels of glucose. The transcriptional activation of ETS transcription factors was increased in all samples analyzed. Downregulation of ETS1 expression by siRNA abrogated the reduction of VPC number induced by high-glucose treatment. In addition, we observed a concomitant suppression of the non-endothelial ETS-target genes matrix metalloproteinase 9 (MMP9) and CD115 upon short term lentiviral delivery of ETS-specific shRNAs. Long term inhibition of ETS expression by lentiviral infection increased the number of cells with the endothelial markers CD144 and CD105. Conclusion These data demonstrate that diabetes leads to dysregulated activation of ETS, which blocks the functional activity of progenitor cells and their commitment towards the endothelial cell lineage.
Blood oxygen level-dependent (BOLD) responses were measured in parts of primary visual cortex that represented unstimulated visual field regions at different distances from a stimulated central target location. The composition of the visual scene varied by the presence or absence of additional peripheral distracter stimuli. Bottom-up effects were assessed by comparing peripheral activity during central stimulation vs. no stimulation. Top-down effects were assessed by comparing active vs. passive conditions. In passive conditions subjects simply watched the central letter stimuli and in active conditions they had to report occurrence of pre-defined targets in a rapid serial letter stream. Onset of the central letter stream enhanced activity in V1 representations of the stimulated region. Within representations of the periphery activation decreased and finally turned into deactivation with increasing distance from the stimulated location. This pattern was most pronounced in the active conditions and during the presence of peripheral stimuli. Active search for a target did not lead to additional enhancement at areas representing the attentional focus but to a stronger deactivation in the vicinity. Suppressed neuronal activity was also found in the non distracter condition suggesting a top-down attention driven effect. Our observations suggest that BOLD signal decreases in primary visual cortex are modulated by bottom-up sensory-driven factors such as the presence of distracters in the visual field as well as by top-down attentional processes.
Introduction Chemokines and their receptors control immune cell migration during infections as well as in autoimmune responses. A 32 bp deletion in the gene of the chemokine receptor CCR5 confers protection against HIV infection, but has also been reported to decrease susceptibility to rheumatoid arthritis (RA). The influence of this deletion variant on the clinical course of this autoimmune disease was investigated. Methods Genotyping for CCR5d32 was performed by PCR and subsequent electrophoretic fragment length determination. For the clinical analysis, the following extra-articular manifestations of RA were documented by the rheumatologist following the patient: presence of rheumatoid nodules, major organ vasculitis, pulmonary fibrosis, serositis or a Raynaud's syndrome. All documented CRP levels were analyzed retrospectively, and the last available hand and feet radiographs were analyzed with regards to the presence or absence of erosive disease. Results Analysis of the CCR5 polymorphism in 503 RA patients and in 459 age-matched healthy controls revealed a significantly decreased disease susceptibility for carriers of the CCR5d32 deletion (Odds ratio 0.67, P = 0.0437). Within the RA patient cohort, CCR5d32 was significantly less frequent in patients with extra-articular manifestations compared with those with limited, articular disease (13.2% versus 22.8%, P = 0.0374). In addition, the deletion was associated with significantly lower average CRP levels over time (median 8.85 vs. median 14.1, P = 0.0041) and had a protective effect against the development of erosive disease (OR = 0.40, P = 0.0047). Intriguingly, homozygosity for the RA associated DNASE2 -1066 G allele had an additive effect on the disease susceptibility conferred by the wt allele of CCR5 (OR = 2.24, P = 0.0051 for carrier of both RA associated alleles) Conclusions The presence of CCR5d32 significantly influenced disease susceptibility to and clinical course of RA in a German study population. The protective effect of this deletion, which has been described to lead to a decreased receptor expression in heterozygous patients, underlines the importance of chemokines in the pathogenesis of RA.
Objective. A study supported by the EULAR and the ACR being conducted to establish classification criteria for polymyalgia rheumatica (PMR) will include ultrasound examination of the shoulders and hips. Ultrasound (US) depicts glenohumeral joint effusion, biceps tenosynovitis, subdeltoid bursitis, hip joint synovitis, and trochanteric bursitis in PMR. These findings may aid in distinguishing PMR from other diseases. The purpose of this study was to assess standards and US interreader agreement of participants in the PMR classification criteria study. Methods. Sixteen physicians in four groups examined shoulders and hips of 4 patients and 4 healthy adults with ultrasound. Overall agreement and interobserver agreement were calculated. Results. The overall agreement (OA) between groups was 87%. The OA for healthy shoulders was 88.8%, for healthy hips 100%, for shoulders with pathology 85.2%, and 74.3% for hips with pathology, respectively. Conclusion. There was a high degree of agreement found for the examination of healthy shoulders and pathologic hips. Agreement was moderate for pathologic shoulders and perfect for healthy hips. US of shoulder and hips performed by different examiners is a reliable and feasible tool for assessment of PMR related disease pathology and can be incorporated into a classification criteria study.
The blood–brain barrier (BBB) is confined to the endothelium of brain capillaries and is indispensable for fluid homeostasis and neuronal function. In this study, we show that endothelial Wnt/beta-catenin (beta-cat) signaling regulates induction and maintenance of BBB characteristics during embryonic and postnatal development. Endothelial specific stabilization of beta-cat in vivo enhances barrier maturation, whereas inactivation of beta-cat causes significant down-regulation of claudin3 (Cldn3), up-regulation of plamalemma vesicle-associated protein, and BBB breakdown. Stabilization of beta-cat in primary brain endothelial cells (ECs) in vitro by N-terminal truncation or Wnt3a treatment increases Cldn3 expression, BBB-type tight junction formation, and a BBB characteristic gene signature. Loss of beta-cat or inhibition of its signaling abrogates this effect. Furthermore, stabilization of beta-cat also increased Cldn3 and barrier properties in nonbrain-derived ECs. These findings may open new therapeutic avenues to modulate endothelial barrier function and to limit the devastating effects of BBB breakdown.
It has been shown that certain chemokine receptor polymorphisms may correspond to certain complications after organ transplantation. Ischemic-type biliary lesion (ITBL) encounters for major morbidity and mortality in liver transplant recipients. So far, the exact cause for ITBL remains unclear. Certain risk factors for the development of ITBL like donor age and cold ischemic time are well described. In a previous study, a 32-nucleotide deletion of the chemokine receptor-5Delta32 (CCR-5Delta32) was strongly associated with the incidence of ITBL in adult liver transplantation. This study re-evaluates the association of CCR-5Delta32 gene polymorphism and the incidence of ITBL. 169 patients were included into this retrospective analysis. 134 patients were homozygous for wild-type CCR-5, 33 patients heterozygous, and 2 patients were homozygous for CCR-5Delta32 mutation. There were no major differences in donor or recipients demographics. No association was found between CCR-5Delta32 mutation and the development of ITBL.We conclude that CCR-5Delta32 is no risk factor for the development of ITBL in our patient cohort.
U1-snRNA is an integral part of the U1 ribonucleoprotein pivotal for pre-mRNA splicing. Toll-like receptor (TLR) signaling has recently been associated with immunoregulatory capacities of U1-snRNA. Using lung A549 epithelial/carcinoma cells, we report for the first time on interferon regulatory factor (IRF)-3 activation initiated by endosomally delivered U1-snRNA. This was associated with expression of the IRF3-inducible genes interferon-b (IFN-b), CXCL10/IP-10 and indoleamine 2,3-dioxygenase. Mutational analysis of the U1-snRNA-activated IFN-b promoter confirmed the crucial role of the PRDIII element, previously proven pivotal for promoter activation by IRF3. Notably, expression of these parameters was suppressed by bafilomycin A1, an inhibitor of endosomal acidification, implicating endosomal TLR activation. Since resiquimod, an agonist of TLR7/8, failed to stimulate A549 cells, data suggest TLR3 to be of prime relevance for cellular activation. To assess the overall regulatory potential of U1-snRNA-activated epithelial cells on cytokine production, co-cultivation with peripheral blood mononuclear cells (PBMC) was performed. Interestingly, A549 cells activated by U1-snRNA reinforced phytohemagglutinin-induced interleukin-10 release by PBMC but suppressed that of tumor necrosis factor-a, indicating an antiinflammatory potential of U1-snRNA. Since U1-snRNA is enriched in apoptotic bodies and epithelial cells are capable of performing efferocytosis, the present data in particular connect to immunobiological aspects of apoptosis at host/environment interfaces.
Background: To facilitate in the identification of gene products important in regulating renal glomerular structure and function, we have produced an annotated transcriptome database for normal human glomeruli using the SAGE approach. Description: The database contains 22,907 unique SAGE tag sequences, with a total tag count of 48,905. For each SAGE tag, the ratio of its frequency in glomeruli relative to that in 115 non-glomerular tissues or cells, a measure of transcript enrichment in glomeruli, was calculated. A total of 133 SAGE tags representing well-characterized transcripts were enriched 10-fold or more in glomeruli compared to other tissues. Comparison of data from this study with a previous human glomerular Sau3A-anchored SAGE library reveals that 47 of the highly enriched transcripts are common to both libraries. Among these are the SAGE tags representing many podocyte-predominant transcripts like WT-1, podocin and synaptopodin. Enrichment of podocyte transcript tags SAGE library indicates that other SAGE tags observed at much higher frequencies in this glomerular compared to non-glomerular SAGE libraries are likely to be glomerulus-predominant. A higher level of mRNA expression for 19 transcripts represented by glomerulus-enriched SAGE tags was verified by RT-PCR comparing glomeruli to lung, liver and spleen. Conclusions: The database can be retrieved from, or interrogated online at http://cgap.nci.nih.gov/SAGE. The annotated database is also provided as an additional file with gene identification for 9,022, and matches to the human genome or transcript homologs in other species for 1,433 tags. It should be a useful tool for in silico mining of glomerular gene expression.
Introduction Complex psychopathological and behavioral symptoms, such as delusions and aggression against care providers, are often the primary cause of acute hospital admissions of elderly patients to emergency units and psychiatric departments. This issue resembles an interdisciplinary clinically highly relevant diagnostic and therapeutic challenge across many medical subjects and general practice. At least 50% of the dramatically growing number of patients with dementia exerts aggressive and agitated symptoms during the course of clinical progression, particularly at moderate clinical severity. Methods Commonly used rating scales for agitation and aggression are reviewed and discussed. Furthermore, we focus in this article on benefits and limitations of all available data of anticonvulsants published in this specific indication, such as valproate, carbamazepine, oxcarbazepine, lamotrigine, gabapentin and topiramate. Results To date, most positive and robust data are available for carbamazepine, however, pharmacokinetic interactions with secondary enzyme induction limit its use. Controlled data of valproate do not seem to support the use in this population. For oxcarbazepine only one controlled but negative trial is available. Positive small series and case reports have been reported for lamotrigine, gabapentin and topiramate. Conclusions So far, data of anticonvulsants in demented patients with behavioral disturbances are not convincing. Controlled clinical trials using specific, valid and psychometrically sound instruments of newer anticonvulsants with a better tolerability profile are mandatory to verify whether they can contribute as treatment option in this indication.
The role of microglial cells in the pathogenesis of Alzheimer’s disease (AD) neurodegeneration is unknown. Although several works suggest that chronic neuroinflammation caused by activated microglia contributes to neurofibrillary degeneration, anti-inflammatory drugs do not prevent or reverse neuronal tau pathology. This raises the question if indeed microglial activation occurs in the human brain at sites of neurofibrillary degeneration. In view of the recent work demonstrating presence of dystrophic (senescent) microglia in aged human brain, the purpose of this study was to investigate microglial cells in situ and at high resolution in the immediate vicinity of tau-positive structures in order to determine conclusively whether degenerating neuronal structures are associated with activated or with dystrophic microglia. We used a newly optimized immunohistochemical method for visualizing microglial cells in human archival brain together with Braak staging of neurofibrillary pathology to ascertain the morphology of microglia in the vicinity of tau-positive structures. We now report histopathological findings from 19 humans covering the spectrum from none to severe AD pathology, including patients with Down’s syndrome, showing that degenerating neuronal structures positive for tau (neuropil threads, neurofibrillary tangles, neuritic plaques) are invariably colocalized with severely dystrophic (fragmented) rather than with activated microglial cells. Using Braak staging of Alzheimer neuropathology we demonstrate that microglial dystrophy precedes the spread of tau pathology. Deposits of amyloid-beta protein (A beta) devoid of tau-positive structures were found to be colocalized with non-activated, ramified microglia, suggesting that A beta does not trigger microglial activation. Our findings also indicate that when microglial activation does occur in the absence of an identifiable acute central nervous system insult, it is likely to be the result of systemic infectious disease. The findings reported here strongly argue against the hypothesis that neuroinflammatory changes contribute to AD dementia. Instead, they offer an alternative hypothesis of AD pathogenesis that takes into consideration: (1) the notion that microglia are neuron-supporting cells and neuroprotective; (2) the fact that development of non-familial, sporadic AD is inextricably linked to aging. They support the idea that progressive, aging-related microglial degeneration and loss of microglial neuroprotection rather than induction of microglial activation contributes to the onset of sporadic Alzheimer’s disease. The results have far-reaching implications in terms of reevaluating current treatment approaches towards AD.
Owing to higher performance on the Raven’s Progressive Matrices (RPM) than on the Wechsler Intelligence Scales (WIS), it has recently been argued that intelligence is underestimated in autism. This study examined RPM and WIS IQs in 48 individuals with autism, a mixed clinical (n = 28) and a neurotypical (n = 25) control group. Average RPM IQ was higher than WIS IQ only in the autism group, albeit to a much lesser degree than previously reported and only for individuals with WIS IQs <85. Consequently, and given the importance of reliable multidimensional IQ estimates in autism, the WIS are recommended as first choice IQ measure in high functioning individuals. Additional testing with the RPM might be required in the lower end of the spectrum.
Background The role of the Fcgamma receptor IIa (FcgammaRIIa), a receptor for C-reactive protein (CRP), the classical acute phase protein, in atherosclerosis is not yet clear. We sought to investigate the association of FcgammaRIIa genotype with risk of coronary heart disease (CHD) in two large population-based samples. Methods FcgammaRIIa-R/H131 polymorphisms were determined in a population of 527 patients with a history of myocardial infarction and 527 age and gender matched controls drawn from a population-based MONICA- Augsburg survey. In the LURIC population, 2227 patients with angiographically proven CHD, defined as having at least one stenosis [greater than or equal to]50%, were compared with 1032 individuals with stenosis <50%. Results In both populations genotype frequencies of the FcgammaRIIa gene did not show a significant departure from the Hardy-Weinberg equilibrium. FcgammaRIIa R(-131)->H genotype was not independently associated with lower risk of CHD after multivariable adjustments, neither in the MONICA population (odds ratio (OR) 1.08; 95% confidence interval (CI) 0.81 to 1.44), nor in LURIC (OR 0.96; 95% CI 0.81 to 1.14). Conclusion Our results do not confirm an independent relationship between FcgammaRIIa genotypes and risk of CHD in these populations.
Background Treatment options for metastatic renal cell carcinoma (RCC) are limited due to resistance to chemo- and radiotherapy. The development of small-molecule multikinase inhibitors have now opened novel treatment options. The influence of the receptor tyrosine kinase inhibitor AEE788, applied alone or combined with the mammalian target of rapamycin (mTOR) inhibitor RAD001, on RCC cell adhesion and proliferation in vitro has been evaluated. Methods RCC cell lines Caki-1, KTC-26 or A498 were treated with various concentrations of RAD001 or AEE788 and tumor cell proliferation, tumor cell adhesion to vascular endothelial cells or to immobilized extracellular matrix proteins (laminin, collagen, fibronectin) evaluated. The anti-tumoral potential of RAD001 combined with AEE788 was also investigated. Both, asynchronous and synchronized cell cultures were used to subsequently analyze drug induced cell cycle manipulation. Analysis of cell cycle regulating proteins was done by western blotting. Results RAD001 or AEE788 reduced adhesion of RCC cell lines to vascular endothelium and diminished RCC cell binding to immobilized laminin or collagen. Both drugs blocked RCC cell growth, impaired cell cycle progression and altered the expression level of the cell cycle regulating proteins cdk2, cdk4, cyclin D1, cyclin E and p27. The combination of AEE788 and RAD001 resulted in more pronounced RCC growth inhibition, greater rates of G0/G1 cells and lower rates of S-phase cells than either agent alone. Cell cycle proteins were much more strongly altered when both drugs were used in combination than with single drug application. The synergistic effects were observed in an asynchronous cell culture model, but were more pronounced in synchronous RCC cell cultures. Conclusions Potent anti-tumoral activitites of the multikinase inhibitors AEE788 or RAD001 have been demonstrated. Most importantly, the simultaneous use of both AEE788 and RAD001 offered a distinct combinatorial benefit and thus may provide a therapeutic advantage over either agent employed as a monotherapy for RCC treatment.
Background Many systems in nature are characterized by complex behaviour where large cascades of events, or avalanches, unpredictably alternate with periods of little activity. Snow avalanches are an example. Often the size distribution f(s) of a system's avalanches follows a power law, and the branching parameter sigma, the average number of events triggered by a single preceding event, is unity. A power law for f(s), and sigma=1, are hallmark features of self-organized critical (SOC) systems, and both have been found for neuronal activity in vitro. Therefore, and since SOC systems and neuronal activity both show large variability, long-term stability and memory capabilities, SOC has been proposed to govern neuronal dynamics in vivo. Testing this hypothesis is difficult because neuronal activity is spatially or temporally subsampled, while theories of SOC systems assume full sampling. To close this gap, we investigated how subsampling affects f(s) and sigma by imposing subsampling on three different SOC models. We then compared f(s) and sigma of the subsampled models with those of multielectrode local field potential (LFP) activity recorded in three macaque monkeys performing a short term memory task. Results Neither the LFP nor the subsampled SOC models showed a power law for f(s). Both, f(s) and sigma, depended sensitively on the subsampling geometry and the dynamics of the model. Only one of the SOC models, the Abelian Sandpile Model, exhibited f(s) and sigma similar to those calculated from LFP activity. Conclusions Since subsampling can prevent the observation of the characteristic power law and sigma in SOC systems, misclassifications of critical systems as sub- or supercritical are possible. Nevertheless, the system specific scaling of f(s) and sigma under subsampling conditions may prove useful to select physiologically motivated models of brain function. Models that better reproduce f(s) and sigma calculated from the physiological recordings may be selected over alternatives.
Background Evidence-based guidelines potentially improve healthcare. However, their de-novo-development requires substantial resources - especially for complex conditions, and adaptation may be biased by contextually influenced recommendations in source guidelines. In this paper we describe a new approach to guideline development - the systematic guideline review method (SGR), and its application in the development of an evidence-based guideline for family physicians on chronic heart failure (CHF). Methods A systematic search for guidelines was carried out. Evidence-based guidelines on CHF management in adults in ambulatory care published in English or German between the years 2000 and 2004 were included. Guidelines on acute or right heart failure were excluded. Eligibility was assessed by two reviewers, methodological quality of selected guidelines was appraised using the AGREE-instrument, and a framework of relevant clinical questions for diagnostics and treatment was derived. Data were extracted into evidence tables, systematically compared by means of a consistency analysis and synthesized in a preliminary draft. Most relevant primary sources were re-assessed to verify the cited evidence. Evidence and recommendations were summarized in a draft guideline. Results Of 16 included guidelines five were of good quality. A total of 35 recommendations were systematically compared: 25/35 were consistent, 9/35 inconsistent, and 1/35 unratable (derived from a single guideline). Of the 25 consistencies, 14 based on consensus, seven on evidence and four differed in grading. Major inconsistencies were found in 3/9 of the inconsistent recommendations. We re-evaluated the evidence for 17 recommendations (evidence-based, differing evidence levels and minor inconsistencies) the majority was congruent. Incongruencies were found, where the stated evidence could not be verified in the cited primary sources, or where the evaluation in the source guidelines focused on treatment benefits and underestimated the risks. The draft guideline was completed in 8.5 man-months. The main limitation to this study was the lack of a second reviewer. Conclusions The systematic guideline review including framework development, consistency analysis and validation is an effective, valid, and resource saving-approach to the development of evidence-based guidelines.
Mesenchymal stem cells (MSC), also referred to as marrow stromal cells, maintain the capacity to differentiate into multiple mesenchymal lineages such as osteoblasts, chondrocytes, adipocytes, myoblasts, stromal, neural and endothelial cells. The use of autologous MSC has generated widespread interest due to their developing application in regenerative medicine and tissue engineering in orthopedic surgery. They have become an indispensable cell source for successful implementation in many bone reconstruction procedures. In addition to their multipotency and selfrenewal capacity, they are easily harvested, have demonstrated a homing mechanism and can be efficiently expanded in vitro, thus providing a safe and costefficient tissue replacement for patients with skeletal injury or disease. Little information is currently available concerning donor characteristics for tissue engineering growth of osseous tissue. This study examines the influences of such donor characteristics, including injury pattern, gender, age, and site of harvest on the quantity, quality and osteogenic differentiation of MSC. The goal is to evaluate whether certain patient groups are practically suitable for an ex vivo expansion and therapeutic reimplantation of MSC. The effect of injury pattern on the reservoir and proliferative capacity of MSC in human bone marrow is clearly demonstrated in this analysis. Age and gender were also shown to influence MSC number and proliferation, as in previous studies. A total of 53 participants (46 patients and 7 healthy volunteers ranging from 18 to 64 years of age), who were scheduled to undergo operative procedures on the pelvis, vertebrae, tibia or hip as well as cancellous bone autografts for reconstruction of various bone defects, were included in the study. Participants were divided into 4 groups for each gender: single fracture, multiple trauma, atrophic nonunion and healthy volunteers. A minimum of 6 ml bone marrow samples were aspirated intraoperatively and processed immediately according to protocol. Following cultivation and expansion for 14 days, the cells were then stained for the colony forming unit-fibroblast (CFU-F) assay and each culture flask was photographed, digitized and converted to an 8 bit grey level TIF-format. Using the digitized CFU-F assay, the mean colony number, mean colony area and mean cell number per microscopic field of view (cell density) could be determined. In addition, confirmation of MSC phenotype was established using fluorescent activated cell sorting (FACS). MSC potential for osteogenic differentiation was quantified by von Kossa, alkaline phosphatase and alizarin staining. Furthermore, serum from a total of 39 randomly chosen participants was collected and tested for hormone levels of 17β-estradiol, testosterone and prolactin as well as the cytokine interleukin-6. These analyses demonstrate several significant trauma-related modifications in MSC reservoir and proliferation, in both male and female patients. In multiple trauma patients, the highest MSC frequency was found, independent of gender and age. Proliferative capacity was also highest in male multiple trauma patients. In the case of atrophic nonunion, the lowest MSC reservoir was detected, independent of gender. Furthermore, MSC frequency in male patients was significantly higher than in female, although analyses of hormone and interleukin-6 levels provided no correlation. Agerelated changes in MSC reservoir could also be observed, whereas the proliferative capacity produced only a tendency toward decreasing values with increasing age. Concerning the site of cell harvest, MSC isolated from the proximal extremity of the tibia, greater trochanter and vertebral body did not proliferate sufficiently enough to be included in statistical analysis, supporting the use of the iliac crest for efficient expansion of MSC. This data suggests the interaction of yet to be identified processes in bone marrow in multiple trauma situations which stimulate the activation and mobilization of MSC. Moreover, in the case of atrophic nonunion, the concentration in bone marrow is depleted and the absence of systemic stimulation present in multiple trauma results in reduced activation of proliferative capacity. Such patients, with severe injury or atrophic nonunion, represent a group of patients with an especially acute necessity for effective and successful bone reconstruction. This data can be used to determine the applicability of MSC from various patient groups for osseous tissue replacement procedures. Especially in such medically challenging situations, further research is essential not only to delineate the factors involved in MSC regulation but also to develop methods to stimulate MSC expansion and proliferation.
Forgotten features of head zones and their relation to diagnostically relevant acupuncture points
(2011)
In the 1890s Sir Henry Head discovered certain areas of the skin that develop tenderness (allodynia) in the course of visceral disease. These areas were later termed ‘Head zones’. In addition, he also emphasized the existence of specific points within these zones, that he called ‘maximum points’, a finding that seems to be almost forgotten today. We hypothesized that two important groups of acupuncture points, the diagnostically relevant Mu and Shu points, spatially and functionally coincide with these maximum points to a large extent. A comparison of Head's papers with the Huang Di Neijing (Yellow Thearch's Inner Classic) and the Zhen Jiu Jia Yi Jing (Systematic Classic of Acupuncture and Moxibustion), two of the oldest still extant Chinese sources on acupuncture, revealed astonishing parallels between the two concepts regarding both point locations and functional aspects. These findings suggest that the Chinese discovery of viscerocutaneous reflexes preceded the discovery in the West by more than 2000 years. Furthermore, the fact that Chinese medicine uses Mu and Shu points not only diagnostically but also therapeutically may give us new insights into the underlying mechanisms of acupuncture.
Oligonucleotides suppress PKB/Akt and act as superinductors of apoptosis in human keratinocytes
(2009)
DNA oligonucleotides (ODN) applied to an organism are known to modulate the innate and adaptive immune system. Previous studies showed that a CpG-containing ODN (CpG-1-PTO) and interestingly, also a non-CpG-containing ODN (nCpG- 5-PTO) suppress inflammatory markers in skin. In the present study it was investigated whether these molecules also influence cell apoptosis. Here we show that CpG-1-PTO, nCpG-5-PTO, and also natural DNA suppress the phosphorylation of PKB/Akt in a cell-type-specific manner. Interestingly, only epithelial cells of the skin (normal human keratinocytes, HaCaT and A-431) show a suppression of PKB/Akt. This suppressive effect depends from ODN lengths, sequence and backbone. Moreover, it was found that TGFa-induced levels of PKB/Akt and EGFR were suppressed by the ODN tested. We hypothesize that this suppression might facilitate programmed cell death. By testing this hypothesis we found an increase of apoptosis markers (caspase 3/7, 8, 9, cytosolic cytochrome c, histone associated DNA fragments, apoptotic bodies) when cells were treated with ODN in combination with low doses of staurosporin, a wellknown pro-apoptotic stimulus. In summary the present data demonstrate DNA as a modulator of apoptosis which specifically targets skin epithelial cells.
Several regions in human temporal and frontal cortex are known to integrate visual and auditory object features. The processing of audio–visual (AV) associations in these regions has been found to be modulated by object familiarity. The aim of the present study was to explore training-induced plasticity in human cortical AV integration. We used functional magnetic resonance imaging to analyze the neural correlates of AV integration for unfamiliar artificial object sounds and images in naïve subjects (PRE training) and after a behavioral training session in which subjects acquired associations between some of these sounds and images (POST-training). In the PRE-training session, unfamiliar artificial object sounds and images were mainly integrated in right inferior frontal cortex (IFC). The POST-training results showed extended integration-related IFC activations bilaterally, and a recruitment of additional regions in bilateral superior temporal gyrus/sulcus and intraparietal sulcus. Furthermore, training-induced differential response patterns to mismatching compared with matching (i.e., associated) artificial AV stimuli were most pronounced in left IFC. These effects were accompanied by complementary training-induced congruency effects in right posterior middle temporal gyrus and fusiform gyrus. Together, these findings demonstrate that short-term cross-modal association learning was sufficient to induce plastic changes of both AV integration of object stimuli and mechanisms of AV congruency processing.
Background The to date evidence for a dose-response relationship between physical workload and the development of lumbar disc diseases is limited. We therefore investigated the possible etiologic relevance of cumulative occupational lumbar load to lumbar disc diseases in a multi-center case-control study. Methods In four study regions in Germany (Frankfurt/Main, Freiburg, Halle/Saale, Regensburg), patients seeking medical care for pain associated with clinically and radiologically verified lumbar disc herniation (286 males, 278 females) or symptomatic lumbar disc narrowing (145 males, 206 females) were prospectively recruited. Population control subjects (453 males and 448 females) were drawn from the regional population registers. Cases and control subjects were between 25 and 70 years of age. In a structured personal interview, a complete occupational history was elicited to identify subjects with certain minimum workloads. On the basis of job task-specific supplementary surveys performed by technical experts, the situational lumbar load represented by the compressive force at the lumbosacral disc was determined via biomechanical model calculations for any working situation with object handling and load-intensive postures during the total working life. For this analysis, all manual handling of objects of about 5 kilograms or more and postures with trunk inclination of 20 degrees or more are included in the calculation of cumulative lumbar load. Confounder selection was based on biologic plausibility and on the change-in-estimate criterion. Odds ratios (OR) and 95% confidence intervals (CI) were calculated separately for men and women using unconditional logistic regression analysis, adjusted for age, region, and unemployment as major life event (in males) or psychosocial strain at work (in females), respectively. To further elucidate the contribution of past physical workload to the development of lumbar disc diseases, we performed lag-time analyses. Results We found a positive dose-response relationship between cumulative occupational lumbar load and lumbar disc herniation as well as lumbar disc narrowing among men and women. Even past lumbar load seems to contribute to the risk of lumbar disc disease. Conclusions According to our study, cumulative physical workload is related to lumbar disc diseases among men and women.
Background Since June 2002, revised regulations in Germany have required "Emergency Medical Care" as an interdisciplinary subject, and state that emergency treatment should be of increasing importance within the curriculum. A survey of the current status of undergraduate medical education in emergency medical care establishes the basis for further committee work. Methods Using a standardized questionnaire, all medical faculties in Germany were asked to answer questions concerning the structure of their curriculum, representation of disciplines, instructors' qualifications, teaching and assessment methods, as well as evaluation procedures. Results Data from 35 of the 38 medical schools in Germany were analysed. In 32 of 35 medical faculties, the local Department of Anaesthesiology is responsible for the teaching of emergency medical care; in two faculties, emergency medicine is taught mainly by the Department of Surgery and in another by Internal Medicine. Lectures, seminars and practical training units are scheduled in varying composition at 97% of the locations. Simulation technology is integrated at 60% (n=21); problem-based learning at 29% (n=10), e-learning at 3% (n=1), and internship in ambulance service is mandatory at 11% (n=4). In terms of assessment methods, multiple-choice exams (15 to 70 questions) are favoured (89%, n=31), partially supplemented by open questions (31%, n=11). Some faculties also perform single practical tests (43%, n=15), objective structured clinical examination (OSCE; 29%, n=10) or oral examinations (17%, n=6). Conclusion Emergency Medical Care in undergraduate medical education in Germany has a practical orientation, but is very inconsistently structured. The innovative options of simulation technology or state-of-the-art assessment methods are not consistently utilized. Therefore, an exchange of experiences and concepts between faculties and disciplines should be promoted to guarantee a standard level of education in emergency medical care.
Background Brain-computer interface methodology based on self-regulation of slow-cortical potentials (SCPs) of the EEG (electroencephalogram) was used to assess conditional associative learning in one severely paralyzed, late-stage ALS patient. After having been taught arbitrary stimulus relations, he was evaluated for formation of equivalence classes among the trained stimuli. Methods A monitor presented visual information in two targets. The method of teaching was matching to sample. Three types of stimuli were presented: signs (A), colored disks (B), and geometrical shapes (C). The sample was one type, and the choice was between two stimuli from another type. The patient used his SCP to steer a cursor to one of the targets. A smiley was presented as a reward when he hit the correct target. The patient was taught A-B and B-C (sample – comparison) matching with three stimuli of each type. Tests for stimulus equivalence involved the untaught B-A, C-B, A-C, and C-A relations. An additional test was discrimination between all three stimuli of one equivalence class presented together versus three unrelated stimuli. The patient also had sessions with identity matching using the same stimuli. Results The patient showed high accuracy, close to 100%, on identity matching and could therefore discriminate the stimuli and control the cursor correctly. Acquisition of A-B matching took 11 sessions (of 70 trials each) and had to be broken into simpler units before he could learn it. Acquisition of B-C matching took two sessions. The patient passed all equivalence class tests at 90% or higher. Conclusion The patient may have had a deficit in acquisition of the first conditional association of signs and colored disks. In contrast, the patient showed clear evidence that A-B and B-C training had resulted in formation of equivalence classes. The brain-computer interface technology combined with the matching to sample method is a useful way to assess various cognitive abilities of severely paralyzed patients, who are without reliable motor control.
Background The purpose of the trial was to evaluate the safety and performance of the new Protégé stent in the treatment of common and/or internal carotid artery stenoses. Methods The Protégé® GPS stent is a self-expanding Nitinol stent system. It is mounted on a 6 Fr 0.018” (6-9mm stent) or 7 Fr, 0.035” (10mm stent) over-the-wire-delivery system and includes a new stent release system which allows exact placement of the stent. Seventyseven patients were enrolled in the trial. Study patient assessments were conducted clinically and by duplex scan at baseline, peri-procedure, discharge, one and six months post procedure. Results Seventyseven lesions were treated. Thirtyone lesions were symptomatic, 46 lesions were asymptomatic. The procedure was technically successful in 76 (99%) lesions. The percentage of stenosis was reduced from 86 ± 7 % to 16 ± 8 %. One procedure failed because the embolic protection device could not be retrieved and the patient was sent to surgery. Within 30 days there were 4 (5.2%) Major Adverse Neurological Events (MANEs). Three of the MANEs were major strokes (3.9%), one a minor stroke. The fifth MANE occurred prior to the six month follow-up visit; this patient had a major stroke 75 days after the procedure and died 36 days later. One additional death occurred due to urosepsis. Conclusions The trial shows that the Protégé stent satisfies safety and performance criteria for the treatment of carotid artery stenosis. The complication rate was comparable to the incidence of these events in other recent carotid stent and endarterectomy studies.
Background Titanium and titanium alloys are widely used for fabrication of dental implants. Since the material composition and the surface topography of a biomaterial play a fundamental role in osseointegration, various chemical and physical surface modifications have been developed to improve osseous healing. Zirconia-based implants were introduced into dental implantology as an altenative to titanium implants. Zirconia seems to be a suitable implant material because of its tooth-like colour, its mechanical properties and its biocompatibility. As the osseointegration of zirconia implants has not been extensively investigated, the aim of this study was to compare the osseous healing of zirconia implants with titanium implants which have a roughened surface but otherwise similar implant geometries. Methods Forty-eight zirconia and titanium implants were introduced into the tibia of 12 minipigs. After 1, 4 or 12 weeks, animals were sacrificed and specimens containing the implants were examined in terms of histological and ultrastructural techniques. Results Histological results showed direct bone contact on the zirconia and titanium surfaces. Bone implant contact as measured by histomorphometry was slightly better on titanium than on zirconia surfaces. However, a statistically significant difference between the two groups was not observed. Conclusion The results demonstrated that zirconia implants with modified surfaces result in an osseointegration which is comparable with that of titanium implants.
Background Osseointegration is crucial for the long-term success of dental implants and depends on the tissue reaction at the tissue-implant interface. Mechanical properties and biocompatibility make zirconia a suitable material for dental implants, although surface processings are still problematic. The aim of the present study was to compare osteoblast behavior on structured zirconia and titanium surfaces under standardized conditions. Methods The surface characteristics were determined by scanning electron microscopy (SEM). In primary bovine osteoblasts attachment kinetics, proliferation rate and synthesis of bone-associated proteins were tested on different surfaces. Results The results demonstrated that the proliferation rate of cells was significantly higher on zirconia surfaces than on titanium surfaces (p < 0.05; Student's t-test). In contrast, attachment and adhesion strength of the primary cells was significant higher on titanium surfaces (p < 0.05; U test). No significant differences were found in the synthesis of bone-specific proteins. Ultrastructural analysis revealed phenotypic features of osteoblast-like cells on both zirconia and titanium surfaces. Conclusion The study demonstrates distinct effects of the surface composition on osteoblasts in culture. Zirconia improves cell proliferation significantly during the first days of culture, but it does not improve attachment and adhesion strength. Both materials do not differ with respect to protein synthesis or ultrastructural appearance of osteoblasts. Zirconium oxide may therefore be a suitable material for dental implants.
Background Heme oxygenase-1 is an inducible cytoprotective enzyme which handles oxidative stress by generating anti-oxidant bilirubin and vasodilating carbon monoxide. A (GT)n dinucleotide repeat and a -413A>T single nucleotide polymorphism have been reported in the promoter region of HMOX1 to both influence the occurrence of coronary artery disease and myocardial infarction. We sought to validate these observations in persons scheduled for coronary angiography. Methods We included 3219 subjects in the current analysis, 2526 with CAD including a subgroup of CAD and MI (n = 1339) and 693 controls. Coronary status was determined by coronary angiography. Risk factors and biochemical parameters (bilirubin, iron, LDL-C, HDL-C, and triglycerides) were determined by standard procedures. The dinucleotide repeat was analysed by PCR and subsequent sizing by capillary electrophoresis, the -413A>T polymorphism by PCR and RFLP. Results In the LURIC study the allele frequency for the -413A>T polymorphism is A = 0,589 and T = 0,411. The (GT)n repeats spread between 14 and 39 repeats with 22 (19.9%) and 29 (47.1%) as the two most common alleles. We found neither an association of the genotypes or allelic frequencies with any of the biochemical parameters nor with CAD or previous MI. Conclusion Although an association of these polymorphisms with the appearance of CAD and MI have been published before, our results strongly argue against a relevant role of the (GT)n repeat or the -413A>T SNP in the HMOX1 promoter in CAD or MI.
Introduction Impaired renal function and/or pre-existing atherosclerosis in the deceased donor increase the risk of delayed graft function and impaired long-term renal function in kidney transplant recipients. Case presentation We report delayed graft function occurring simultaneously in two kidney transplant recipients, aged 57-years-old and 39-years-old, who received renal allografts from the same deceased donor. The 62-year-old donor died of cardiac arrest during an asthmatic state. Renal-allograft biopsies performed in both kidney recipients because of delayed graft function revealed cholesterol-crystal embolism. An empiric statin therapy in addition to low-dose acetylsalicylic acid was initiated. After 10 and 6 hemodialysis sessions every 48 hours, respectively, both renal allografts started to function. Glomerular filtration rates at discharge were 26 ml/min/1.73 m2 and 23.9 ml/min/1.73 m2, and remained stable in follow-up examinations. Possible donor and surgical procedure-dependent causes for cholesterol-crystal embolism are discussed. Conclusion Cholesterol-crystal embolism should be considered as a cause for delayed graft function and long-term impaired renal allograft function, especially in the older donor population.
Visual information is processed hierarchically in the human visual system. Early during processing basic features are analysed separately while at later stages of processing, they are integrated into a unified percept. By investigating a basic visual feature and following its integration at different levels of processing one can identify specific patterns. In certain visual impairments, these patterns can function defectively and their detailed study can clarify the cause of the visual deficit. Here we investigate orientation as a basic feature and use a property of the visual system called adaptation. Adaptation occurs as a decrease in the level of neural activity during repetitive presentation of the same stimulus. Psychophysical studies have shown that adaptation transfers interocularly, meaning that if only one eye is adapted the other eye shows also adaptation effects. Our aim was to investigate interocular transfer by means of functional magnetic resonance imaging (fMRI). Even though adaptation was demonstrated in the fMRI environment, the interocular transfer was never investigated in such a setup. First, we developed a method to measure interocular transfer of adaptation to gratings with fMRI. We then went further to test it in various groups of subjects. In normally sighted humans interocular transfer was present both in early (striate) as well as later visual areas (extrastriate). In subjects with impaired stereovision (with or without normal visual acuity) interocular transfer was absent in the investigated regions. Detailed analysis of the recorded differences between subjects with and subjects without stereovision was performed. The results of this analysis are presented in detail in this book. These results suggest that the neuronal mechanisms involved in the interocular transfer of pattern adaptation share, at least in part, the neural circuitry underlying binocular functions and stereopsis. We conclude that fMRI adaptation can be used for the assessment of cortical binocularity in humans with normal and impaired stereopsis. Further investigations are needed to address more subtle aspects of the lack of interocular transfer. Towards this purpose, through a fourth experiment we propose further directions that might shed more light on the issue of stereovision and its clinical implications. We show that carefully tuned variations in our experimental procedure might reveal other aspects of binocularity in the human visual system. We believe that the method we developed, apart from the interesting results shown here, has a high potential to be further used for other research questions. Following the above summarized ideas, the thesis comprises of three parts (chapters). The first chapter provides the main theoretical backgrounds of the visual system and of the MRI imaging technique, chapter two describes the experimental procedures while the results and their detailed discussion are detailed in chapter three.
Background The treatment of Herpes-simplex-virus-encephalitis (HSVE) remains a major unsolved problem in Neurology. Current gold standard for therapy is acyclovir, a drug that inhibits viral replication. Despite antiviral treatment, mortality remains up to 15%, less than 20% of patients are able to go back to work, and the majority of patients suffer from severe disability. This is a discouraging, unsatisfactory situation for treating physicians, the disabled patients and their families, and constitutes an enormous burden to the public health services. The information obtained from experimental animal research and from recent retrospective clinical observations, indicates that a substantial benefit in outcome can be expected in patients with HSVE who are treated with adjuvant dexamethasone. But currently there is no available evidence to support the routine use of adjuvant corticosteroid treatment in HSVE. A randomized multicenter trial is the only useful instrument to address this question. Design GACHE is a multicenter, randomized, double-blind, placebo-controlled, parallel group clinical trial of treatment with acyclovir and adjuvant dexamethasone, as compared with acyclovir and placebo in adults with HSVE. The statistical design will be that of a 3-stage-group sequential trial with potential sample size adaptation in the last stage. Conclusion 372 patients with proven HSVE (positive HSV-DNA-PCR), aged 18 up to 85 years; with focal neurological signs no longer than 5 days prior to admission, and who give informed consent will be recruited from Departments of Neurology of academic medical centers in Germany, Austria and The Netherlands. Sample size will potentially be extended after the second interim analysis up to a maximum of 450 patients. Trial Registration Current Controlled Trials ISRCTN45122933
Aims: The purpose of this study was to evaluate the feasibility and short-term efficacy of transcatheter paravalvular leak closure using different occlusion devices. Methods and Results: Twenty one patients underwent transcatheter closure of either aortic or mitral paravalvular leak from June 2002 to February 2006 using the Amplatzer PDA, ASD or VSD occluder. All patients had symptoms and signs of haemolysis and/or cardiac decompensation with dyspnoea. Implantation of a device was technically successful in twenty patients (95 %). Immediate residual leak was found in seventeen patients (85 %). Significant shunting persisted in nine cases during follow up (45 %). Permanent leaflet obstruction was observed in one patient. Severe complications during follow up led to early death in one patient and surgical intervention in three. A successful second catheter treatment was performed in another three patients. The event-free survival from reoperation, death and stroke at the end of the observation period was 80 %. Conclusion: Transcatheter closure of paravalvular leaks is a technically feasible, but demanding procedure. Residual leaks are common and may worsen pre-existing haemolysis. Due to the significant ongoing morbidity in this group of patients and the complexity of follow up individual patient results differ considerably. Nevertheless, it is possible to achieve some symptomatic relief, thus an interventional approach should be discussed as a potential treatment option for those patients with a limited defect and who are not deemed suitable for another operation.
Background Cyclin B1, the regulatory subunit of cyclin-dependent kinase 1 (Cdk1), is essential for the transition from G2 phase to mitosis. Cyclin B1 is very often found to be overexpressed in primary breast and cervical cancer cells as well as in cancer cell lines. Its expression is correlated with the malignancy of gynecological cancers. Methods In order to explore cyclin B1 as a potential target for gynecological cancer therapy, we studied the effect of small interfering RNA (siRNA) on different gynecological cancer cell lines by monitoring their proliferation rate, cell cycle profile, protein expression and activity, apoptosis induction and colony formation. Tumor formation in vivo was examined using mouse xenograft models. Results Downregulation of cyclin B1 inhibited proliferation of several breast and cervical cancer cell lines including MCF-7, BT-474, SK-BR-3, MDA-MB-231 and HeLa. After combining cyclin B1 siRNA with taxol, we observed an increased apoptotic rate accompanied by an enhanced antiproliferative effect in breast cancer cells. Furthermore, control HeLa cells were progressively growing, whereas the tumor growth of HeLa cells pre-treated with cyclin B1 siRNA was strongly inhibited in nude mice, indicating that cyclin B1 is indispensable for tumor growth in vivo. Conclusion Our data support the notion of cyclin B1 being essential for survival and proliferation of gynecological cancer cells. Concordantly, knockdown of cyclin B1 inhibits proliferation in vitro as well as in vivo. Moreover, targeting cyclin B1 sensitizes breast cancer cells to taxol, suggesting that specific cyclin B1 targeting is an attractive strategy for the combination with conventionally used agents in gynecological cancer therapy.
Biventricular pacing has been suggested in end-stage heart failure. We present a 59-year-old patient undergoing second re-do CABG (coronary artery bypass graft) and carotid artery endarterectomy. Ejection fraction was 15%, QRS-width 175 ms. Following the carotid and CABG procedure, an implanted single-chamber ICD (implantable cardioverter defibrillator) was upgraded to permanent biventricular DDD pacing by implantation of one epicardial left ventricular and one epicardial atrial electrode. At follow-up two months postoperatively ejection fraction had significantly improved to 45%, the patient underwent stress test with adequate load and reported a good quality of life.
Background Hepatitis C virus (HCV) is a leading cause of chronic liver disease, end-stage cirrhosis, and liver cancer, but little is known about the burden of disease caused by the virus. We summarised burden of disease data presently available for Europe, compared the data to current expert estimates, and identified areas in which better data are needed. Methods Literature and international health databases were systematically searched for HCV-specific burden of disease data, including incidence, prevalence, mortality, disability-adjusted life-years (DALYs), and liver transplantation. Data were collected for the WHO European region with emphasis on 22 countries. If HCV-specific data were unavailable, these were calculated via HCV-attributable fractions. Results HCV-specific burden of disease data for Europe are scarce. Incidence data provided by national surveillance are not fully comparable and need to be standardised. HCV prevalence data are often inconclusive. According to available data, an estimated 7.3–8.8 million people (1.1–1.3%) are infected in our 22 focus countries. HCV-specific mortality, DALY, and transplantation data are unavailable. Estimations via HCV-attributable fractions indicate that HCV caused more than 86000 deaths and 1.2 million DALYs in the WHO European region in 2002. Most of the DALYs (95%) were accumulated by patients in preventable disease stages. About one-quarter of the liver transplants performed in 25 European countries in 2004 were attributable to HCV. Conclusion Our results indicate that hepatitis C is a major health problem and highlight the importance of timely antiviral treatment. However, data on the burden of disease of hepatitis C in Europe are scarce, outdated or inconclusive, which indicates that hepatitis C is still a neglected disease in many countries. What is needed are public awareness, co-ordinated action plans, and better data. European physicians should be aware that many infections are still undetected, provide timely testing and antiviral treatment, and avoid iatrogenic transmission.
Background Public health systems are confronted with constantly rising costs. Furthermore, diagnostic as well as treatment services become more and more specialized. These are the reasons for an interdisciplinary project on the one hand aiming at simplification of planning and scheduling patient appointments, on the other hand at fulfilling all requirements of efficiency and treatment quality. Methods As to understanding procedure and problem solving activities, the responsible project group strictly proceeded with four methodical steps: actual state analysis, analysis of causes, correcting measures, and examination of effectiveness. Various methods of quality management, as for instance opinion polls, data collections, and several procedures of problem identification as well as of solution proposals were applied. All activities were realized according to the requirements of the clinic's ISO 9001:2000 certified quality management system. The development of this project is described step by step from planning phase to inauguration into the daily routine of the clinic and subsequent control of effectiveness. Results Five significant problem fields could be identified. After an analysis of causes the major remedial measures were: installation of a patient telephone hotline, standardization of appointment arrangements for all patients, modification of the appointments book considering the reason for coming in planning defined working periods for certain symptoms and treatments, improvement of telephonic counselling, and transition to flexible time planning by daily updates of the appointments book. After implementation of these changes into the clinic's routine success could be demonstrated by significantly reduced waiting times and resulting increased patient satisfaction. Conclusion Systematic scrutiny of the existing organizational structures of the outpatients' department of our clinic by means of actual state analysis and analysis of causes revealed the necessity of improvement. According to rules of quality management correcting measures and subsequent examination of effectiveness were performed. These changes resulted in higher satisfaction of patients, referring colleagues and clinic staff the like. Additionally the clinic is able to cope with an increasing demand for appointments in outpatients' departments, and the clinic's human resources are employed more effectively.
Background Ongoing changes in cancer care cause an increase in the complexity of cases which is characterized by modern treatment techniques and a higher demand for patient information about the underlying disease and therapeutic options. At the same time, the restructuring of health services and reduced funding have led to the downsizing of hospital care services. These trends strongly influence the workplace environment and are a potential source of stress and burnout among professionals working in radiotherapy. Methods and patients A postal survey was sent to members of the workgroup "Quality of Life" which is part of DEGRO (German Society for Radiooncology). Thus far, 11 departments have answered the survey. 406 (76.1%) out of 534 cancer care workers (23% physicians, 35% radiographers, 31% nurses, 11% physicists) from 8 university hospitals and 3 general hospitals completed the FBAS form (Stress Questionnaire of Physicians and Nurses; 42 items, 7 scales), and a self-designed questionnaire regarding work situation and one question on global job satisfaction. Furthermore, the participants could make voluntary suggestions about how to improve their situation. Results Nurses and physicians showed the highest level of job stress (total score 2.2 and 2.1). The greatest source of job stress (physicians, nurses and radiographers) stemmed from structural conditions (e.g. underpayment, ringing of the telephone) a "stress by compassion" (e.g. "long suffering of patients", "patients will be kept alive using all available resources against the conviction of staff"). In multivariate analyses professional group (p < 0.001), working night shifts (p = 0.001), age group (p = 0.012) and free time compensation (p = 0.024) gained significance for total FBAS score. Global job satisfaction was 4.1 on a 9-point scale (from 1 – very satisfied to 9 – not satisfied). Comparing the total stress scores of the hospitals and job groups we found significant differences in nurses (p = 0.005) and physicists (p = 0.042) and a borderline significance in physicians (p = 0.052). In multivariate analyses "professional group" (p = 0.006) and "vocational experience" (p = 0.036) were associated with job satisfaction (cancer care workers with < 2 years of vocational experience having a higher global job satisfaction). The total FBAS score correlated with job satisfaction (Spearman-Rho = 0.40; p < 0.001). Conclusion Current workplace environments have a negative impact on stress levels and the satisfaction of radiotherapy staff. Identification and removal of the above-mentioned critical points requires various changes which should lead to the reduction of stress.
Mitochondria are essential for respiration and oxidative phosphorylation. Mitochondrial dysfunction due to aging processes is involved in pathologies and pathogenesis of a series of cardiovascular disorders. New results accumulate showing that the enzyme telomerase with its catalytic subunit telomerase reverse transcriptase (TERT) has a beneficial effect on heart functions. The benefit of short-term running of mice for heart function is dependent on TERT expression. TERT can translocate into the mitochondria and mitochondrial TERT (mtTERT) is protective against stress induced stimuli and binds to mitochondrial DNA (mtDNA). Because mtDNA is highly susceptible to damage produced by reactive oxygen species (ROS) which are generated in close proximity to the respiratory chain, the aim of this study was to determine the functions of mtTERT in vivo and in vitro. Therefore, mitochondria from hearts of adult, 2nd generation TERT-deficient mice (TERT -/-) and wt littermates were isolated and state 3 respiration was measured. Strikingly mitochondria from TERT -/- revealed a significantly lower state 3 respiration (TERTwt: 987 +/- 72 pmol/s*mg vs. TERT-/-: 774 +/- 38 pmol/s*mg, p < 0.05, n = 5). These results demonstrated that TERT -/- mice have a so far undiscovered heart phenotype. In contrast mitochondria isolated from liver tissues did not show any differences. To get further insights in the molecular mechanisms, we reduced endogenous TERT levels by shRNA and measured mitochondrial reactive oxygen species (mtROS). mtROS were increased after ablation of TERT (scrambled: 4.98 +/- 1.1% gated vs. shTERT: 2.03 +/- 0.7% gated, p < 0.05, n = 4). We next determined mtDNA deletions, which are caused by mtROS. Semiquantitative realtime PCR of mtDNA deletions revealed that mtTERT protects mtDNA from oxidative damage. To analyze whether mitochondrial integrity is required to protect from apoptosis, vectors with mitochondrially targeted TERT (mitoTERT) and wildtype TERT (wtTERT) were transfected and apoptosis was measured. mitoTERT showed the most prominent protective effect on H2O2 induced apoptosis. In conclusion, mtTERT has a protective role in mitochondria by importantly contributing to mtDNA integrity and thereby enhancing respiration capacity of the heart.
Introduction: Lymphocyte infiltration (LI) is often seen in breast cancer but its importance remains controversial. A positive correlation of human epidermal growth factor receptor 2 (HER2) amplification and LI has been described, which was associated with a more favorable outcome. However, specific lymphocytes might also promote tumor progression by shifting the cytokine milieu in the tumor.
Methods: Affymetrix HG-U133A microarray data of 1,781 primary breast cancer samples from 12 datasets were included. The correlation of immune system-related metagenes with different immune cells, clinical parameters, and survival was analyzed.
Results: A large cluster of nearly 600 genes with functions in immune cells was consistently obtained in all datasets. Seven robust metagenes from this cluster can act as surrogate markers for the amount of different immune cell types in the breast cancer sample. An IgG metagene as a marker for B cells had no significant prognostic value. In contrast, a strong positive prognostic value for the T-cell surrogate marker (lymphocyte-specific kinase (LCK) metagene) was observed among all estrogen receptor (ER)-negative tumors and those ER-positive tumors with a HER2 overexpression. Moreover ER-negative tumors with high expression of both IgG and LCK metagenes seem to respond better to neoadjuvant chemotherapy.
Conclusions: Precise definitions of the specific subtypes of immune cells in the tumor can be accomplished from microarray data. These surrogate markers define subgroups of tumors with different prognosis. Importantly, all known prognostic gene signatures uniformly assign poor prognosis to all ER-negative tumors. In contrast, the LCK metagene actually separates the ER-negative group into better or worse prognosis.
Background Imatinib mesylate, a selective inhibitor of Abl tyrosine kinase, is efficacious in treating chronic myeloid leukaemia (CML) and Ph+ acute lymphoblastic leukaemia (ALL). However, most advanced-phase CML and Ph+ ALL patients relapse on Imatinib therapy. Several mechanisms of refractoriness have been reported, including the activation of the Src-family kinases (SFK). Here, we investigated the biological effect of the new specific dual Src/Abl kinase inhibitor AZD0530 on Ph+ leukaemic cells. Methods Cell lines used included BV173 (CML in myeloid blast crisis), SEM t(4;11), Ba/F3 (IL-3 dependent murine pro B), p185Bcr-Abl infected Ba/F3 cells, p185Bcr-Abl mutant infected Ba/F3 cells, SupB15 (Ph+ ALL) and Imatinib resistant SupB15 (RTSupB15) (Ph+ ALL) cells. Cells were exposed to AZD0530 and Imatinib. Cell proliferation, apoptosis, survival and signalling pathways were assessed by dye exclusion, flow cytometry and Western blotting respectively. Results AZD0530 specifically inhibited the growth of, and induced apoptosis in CML and Ph+ ALL cells in a dose dependent manner, but showed only marginal effects on Ph- ALL cells. Resistance to Imatinib due to the mutation Y253F in p185Bcr-Abl was overcome by AZD0530. Combination of AZD0530 and Imatinib showed an additive inhibitory effect on the proliferation of CML BV173 cells but not on Ph+ ALL SupB15 cells. An ongoing transphosphorylation was demonstrated between SFKs and Bcr-Abl. AZD0530 significantly down-regulated the activation of survival signalling pathways in Ph+ cells, resistant or sensitive to Imatinib, with the exception of the RTSupB15. Conclusion Our results indicate that AZD0530 targets both Src and Bcr-Abl kinase activity and reduces the leukaemic maintenance by Bcr-Abl.
Background The effect of additional treatment strategies with antineoplastic agents on intraperitoneal tumor stimulating interleukin levels are unclear. Taurolidine and Povidone-iodine have been mainly used for abdominal lavage in Germany and Europe. Methods In the settings of a multicentre (three University Hospitals) prospective randomized controlled trial 120 patients were randomly allocated to receive either 0.5% taurolidine/2,500 IU heparin (TRD) or 0.25% povidone-iodine (control) intraperitoneally for resectable colorectal, gastric or pancreatic cancers. Due to the fact that IL-1beta (produced by macrophages) is preoperatively indifferent in various gastrointestinal cancer types our major outcome criterion was the perioperative (overall) level of IL-1beta in peritoneal fluid. Results Cytokine values were significantly lower after TRD lavage for IL-1beta, IL-6, and IL-10. Perioperative complications did not differ. The median follow-up was 50.0 months. The overall mortality rate (28 vs. 25, p = 0.36), the cancer-related death rate (17 vs. 19, p = .2), the local recurrence rate (7 vs. 12, p = .16), the distant metastasis rate (13 vs. 18, p = 0.2) as well as the time to relapse were not statistically significant different. Conclusion Reduced cytokine levels might explain a short term antitumorigenic intraperitoneal effect of TRD. But, this study analyzed different types of cancer. Therefore, we set up a multicentre randomized trial in patients undergoing curative colorectal cancer resection. Trial registration : ISRCTN66478538
Background The differential diagnosis between follicular thyroid adenoma and minimal invasive follicular thyroid carcinoma is often difficult for several reasons. One major aspect is the lack of typical cytological criteria in well differentiated specimens. New marker molecules, shown by poly- or monoclonal antibodies proved helpful. Methods We performed global gene expression analysis of 12 follicular thyroid tumours (4 follicular adenomas, 4 minimal invasive follicular carcinomas and 4 widely invasive follicular carcinomas), followed by immunohistochemical staining of 149 cases. The specificity of the antibody was validated by western blot analysis Results In gene expression analysis QPRT was detected as differently expressed between follicular thyroid adenoma and follicular thyroid carcinoma. QPRT protein could be detected by immunohistochemistry in 65% of follicular thyroid carcinomas including minimal invasive variant and only 22% of follicular adenomas. Conclusion Consequently, QPRT is a potential new marker for the immunohistochemical screening of follicular thyroid nodules.
Although the concept that dendritic cells (DCs) recognize pathogens through the engagement of Toll-like receptors is widely accepted, we recently suggested that immature DCs might sense kinin-releasing strains of Trypanosoma cruzi through the triggering of G-protein-coupled bradykinin B2 receptors (B2R). Here we report that C57BL/6.B2R-/- mice infected intraperitoneally with T. cruzi display higher parasitemia and mortality rates as compared to B2R+/+ mice. qRT-PCR revealed a 5-fold increase in T. cruzi DNA (14 d post-infection [p.i.]) in B2R-/- heart, while spleen parasitism was negligible in both mice strains. Analysis of recall responses (14 d p.i.) showed high and comparable frequencies of IFN-gamma-producing CD4+ and CD8+ T cells in the spleen of B2R-/- and wild-type mice. However, production of IFN-gamma by effector T cells isolated from B2R-/- heart was significantly reduced as compared with wild-type mice. As the infection continued, wild-type mice presented IFN-gamma-producing (CD4+CD44+ and CD8+CD44+) T cells both in the spleen and heart while B2R-/- mice showed negligible frequencies of such activated T cells. Furthermore, the collapse of type-1 immune responses in B2R-/- mice was linked to upregulated secretion of IL-17 and TNF-alpha by antigen-responsive CD4+ T cells. In vitro analysis of tissue culture trypomastigote interaction with splenic CD11c+ DCs indicated that DC maturation (IL-12, CD40, and CD86) is controlled by the kinin/B2R pathway. Further, systemic injection of trypomastigotes induced IL-12 production by CD11c+ DCs isolated from B2R+/+ spleen, but not by DCs from B2R-/- mice. Notably, adoptive transfer of B2R+/+ CD11c+ DCs (intravenously) into B2R-/- mice rendered them resistant to acute challenge, rescued development of type-1 immunity, and repressed TH17 responses. Collectively, our results demonstrate that activation of B2R, a DC sensor of endogenous maturation signals, is critically required for development of acquired resistance to T. cruzi infection. Author Summary: Antibodies and IFN-gamma-producing effector T cells are essential for the immune control of infection by Trypanosoma cruzi, the intracellular protozoa that causes human Chagas disease. Despite the potency of anti-parasite immunity, the parasites are not cleared from their intracellular niches. Instead, a low grade chronic infection prevails, provoking severe immunopathology in the myocardium. Although it is well established that innate sentinel cells sense T. cruzi through receptors for microbial structures, such as Toll-like receptors, it remained unclear whether endogenous inflammatory signals also contribute to the development of adaptive immunity. The present study was motivated by awareness that T. cruzi trypomastigotes (extracellular infective forms) are equipped with proteases that liberate the pro-inflammatory bradykinin peptide from an internal segment of kininogens. Here we demonstrate that splenic dendritic cells (DCs), the antigen-presenting cells that coordinate the adaptive branch of immunity in lymphoid tissues, are potently activated via G-protein-coupled bradykinin B2 receptors (B2R). Analysis of the outcome of infection in B2R-knockout mice revealed that the mutant mice developed a typical susceptible phenotype, owing to impaired development of IFN-gamma-producing effector T cells. Notably, the immune dysfunction of B2R-knockout mice was corrected upon cell transfer of wild-type DCs, thus linking development of protective T cells to DCs' sensing of endogenous danger signals (kinins) released by trypomastigotes.
The timing of feedback to early visual cortex in the perception of long-range apparent motion
(2008)
When 2 visual stimuli are presented one after another in different locations, they are often perceived as one, but moving object. Feedback from area human motion complex hMT/V5+ to V1 has been hypothesized to play an important role in this illusory perception of motion. We measured event-related responses to illusory motion stimuli of varying apparent motion (AM) content and retinal location using Electroencephalography. Detectable cortical stimulus processing started around 60-ms poststimulus in area V1. This component was insensitive to AM content and sequential stimulus presentation. Sensitivity to AM content was observed starting around 90 ms post the second stimulus of a sequence and most likely originated in area hMT/V5+. This AM sensitive response was insensitive to retinal stimulus position. The stimulus sequence related response started to be sensitive to retinal stimulus position at a longer latency of 110 ms. We interpret our findings as evidence for feedback from area hMT/V5+ or a related motion processing area to early visual cortices (V1, V2, V3).