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71.
Anja K. Büscher Bodo B. Beck Anette Melk Julia Hoefele Birgitta Kranz Daniel Bamborschke Sabrina Baig B?rbel Lange-Sperandio Theresa Jungraithmayr Lutz T. Weber Markus J. Kemper Burkhard T?nshoff Peter F. Hoyer Martin Konrad Stefanie Weber 《Clinical journal of the American Society of Nephrology》2016,11(2):245-253
Background and objectives
Treatment of congenital nephrotic syndrome (CNS) and steroid–resistant nephrotic syndrome (SRNS) is demanding, and renal prognosis is poor. Numerous causative gene mutations have been identified in SRNS that affect the renal podocyte. In the era of high–throughput sequencing techniques, patients with nongenetic SRNS frequently escape the scientific interest. We here present the long-term data of the German CNS/SRNS Follow-Up Study, focusing on the response to cyclosporin A (CsA) in patients with nongenetic versus genetic disease.Design, setting, participants, & measurements
Cross–sectional and longitudinal clinical data were collected from 231 patients with CNS/SRNS treated at eight university pediatric nephrology units with a median observation time of 113 months (interquartile range, 50–178). Genotyping was performed systematically in all patients.Results
The overall mutation detection rate was high at 57% (97% in CNS and 41% in SRNS); 85% of all mutations were identified by the analysis of three single genes only (NPHS1, NPHS2, and WT1), accounting for 92% of all mutations in patients with CNS and 79% of all mutations in patients with SRNS. Remission of the disease in nongenetic SRNS was observed in 78% of patients after a median treatment period of 2.5 months; 82% of nongenetic patients responded within 6 months of therapy, and 98% of patients with nongenetic SRNS and CsA–induced complete remission (normalbuminemia and no proteinuria) maintained a normal renal function. Genetic SRNS, on the contrary, is associated with a high rate of ESRD in 66% of patients. Only 3% of patients with genetic SRNS experienced a complete remission and 16% of patients with genetic SRNS experienced a partial remission after CsA therapy.Conclusions
The efficacy of CsA is high in nonhereditary SRNS, with an excellent prognosis of renal function in the large majority of patients. CsA should be given for a minimum period of 6 months in these patients with nongenetic SRNS. In genetic SRNS, response to CsA was low and restricted to exceptional patients. 相似文献72.
Patrick Hoyer Gustavo de Medeiros Bálint Balázs Nils Norlin Christina Besir Janina Hanne Hans-Georg Kr?usslich Johann Engelhardt Steffen J. Sahl Stefan W. Hell Lars Hufnagel 《Proceedings of the National Academy of Sciences of the United States of America》2016,113(13):3442-3446
We present a plane-scanning RESOLFT [reversible saturable/switchable optical (fluorescence) transitions] light-sheet (LS) nanoscope, which fundamentally overcomes the diffraction barrier in the axial direction via confinement of the fluorescent molecular state to a sheet of subdiffraction thickness around the focal plane. To this end, reversibly switchable fluorophores located right above and below the focal plane are transferred to a nonfluorescent state at each scanning step. LS-RESOLFT nanoscopy offers wide-field 3D imaging of living biological specimens with low light dose and axial resolution far beyond the diffraction barrier. We demonstrate optical sections that are thinner by 5–12-fold compared with their conventional diffraction-limited LS analogs.Far-field nanoscopy (1, 2) methods discern features within subdiffraction distances by briefly forcing their molecules to two distinguishable states for the time period of detection. Typically, fluorophores are switched between a signaling “on” and a nonsignaling (i.e., dark) “off” state. Depending on the switching and fluorescence registration strategy used, these superresolution techniques can be categorized into coordinate-stochastic and coordinate-targeted approaches (2). The latter group of methods, comprising the so-called RESOLFT [reversible saturable/switchable optical (fluorescence) transitions] (1, 3–7) approaches, have been realized using patterns of switch-off light with one or more zero-intensity points or lines, to single out target point (zero-dimensional) or line (1D) coordinates in space where the fluorophores are allowed to assume the on state. The RESOLFT idea can also be implemented in the inverse mode, by using switch-on light and confining the off state. In any case, probing the presence of molecules in new sets of points or lines at every scanning step produces images.Owing to the nature of the on and off states involved––first excited electronic and ground state––stimulated emission depletion (STED) (3) and saturated structured illumination microscopy (SSIM) (8), which both qualify as variants of the RESOLFT principle, typically apply light intensities in the range of MW/cm2 and above. Especially when imaging sensitive samples where photoinduced changes must be avoided, RESOLFT is preferably realized with fluorophores which lead to the same factor of resolution improvement at much lower intensities of state-switching light. Reversibly switchable fluorescent proteins (RSFPs) are highly suitable for this purpose (4–7, 9), as transitions between their metastable on and off states require 5 orders of magnitude lower threshold intensities than STED/SSIM to guarantee switch-off. Suitable spectral properties, relatively fast millisecond switching kinetics, and high photostability of recently developed yellow-green-emitting RSFPs like rsEGFP (5), rsEGFP2 (7), and rsEGFP(N205S) (10) compared with early RSFPs have indeed enabled RESOLFT nanoscopy in living cells and tissues. To date, RSFP-based RESOLFT has achieved resolution improvements by factors of 4–5 in rsEGFP2-labeled samples (7). To further reduce the imaging time, massive parallelization of scanning has been reported (10). However, the diffraction-limited axial resolution and lack of background suppression restrict applications to thin samples.Imaging applications typically require careful tuning of imaging parameters including speed, contrast, photosensitivity, and spatial resolution, depending on the information that is sought. Light-sheet fluorescence microscopy (LSFM) (11–15) stands out by its ability to balance most of these parameters for 3D imaging of living specimens. Recently reenacted as the selective plane illumination microscope (13), this microscopy mode has sparked increasing interest notably because of its short acquisition times in 3D imaging and low phototoxicity in living specimens. It excites fluorophores only in a thin diffraction-limited slice of the sample, perpendicular to the direction of fluorescence detection. The LS is generated by a cylindrical lens which focuses an expanded laser beam in only one direction onto the specimen or into the back-focal plane of an illumination objective. Alternatively, a single beam is quickly moved as a “virtual” LS (16) across a specimen section.In such conventional LSFM imaging, the lateral resolution is determined by the numerical aperture (N.A.) of the detection objective (17), whereas axial resolution is given by the LS thickness, provided the latter is thinner than the axial extent of the point-spread function describing the imaging process from the focal plane of the detecting lens to the camera. In a previous study, the axial resolution of LSFM was pushed to the diffraction limit by using the full aperture of the illumination objective with Gaussian beams; this was carried out for practically useful combinations of N.A. (e.g., 0.8 for both illumination and detection objectives) permissible in light of the geometrical constraints given by the objective lens dimensions (18). High-N.A. illumination comes with short Rayleigh ranges of Gaussian beams, which inherently limit the field of view (FOV) along the direction of illumination. Scanned Bessel beams for diffraction-limited excitation with a virtual LS (19–21) typically offer larger FOVs (22), but side lobes broaden the scanned LS in the axial direction and contribute to phototoxicity outside of the focal plane of detection (20). A more complex approach has used Bessel-beam excitation in combination with structured illumination to obtain near-isotropic (but still diffraction-limited) resolution as measured on fluorescent beads (20), albeit at the cost of acquisition time and reduced contrast due to fluorescence generated by the side lobes. In different work, axial resolution has also been improved about fourfold by acquiring two complementary orthogonal views of the sample using two alternating LSs, followed by computationally fusing image information with a deconvolution incorporating both views (23). LS approaches have also helped suppress out-of-focus background for single-molecule imaging in biological situations (e.g., in ref. 24), including at superresolution (25–27).Slight axial resolution improvement beyond the diffraction barrier has been demonstrated by overlapping a Gaussian excitation LS with a STED LS featuring a zero-intensity plane (28). Due to scattering and possibly additional aberrations caused by the wavelength difference between excitation and STED light, the maximal achievable resolution in biological specimens was severely limited. This was the case even in fixed samples. A successful application of LS-STED to living cells or organisms has not been reported. The relatively high average STED laser power required for high resolution gains calls for developing a coordinate-targeted superresolution LS approach with low-power operation, meaning a concept that does not solely rely on changing the way the light is directed to––or collected from––the sample, but a concept that harnesses an “on–off” transition for improved feature separation. 相似文献
73.
Factor VIII procoagulant (VIII:C) activity, factor VIII coagulant antigen (VIII:CAg), von Willebrand ristocetin cofactor (VIIIR:RC) activity, factor VIII-related antigen (VIIIR:Ag), and plasma fibronectin (CIg; cold-insoluble globulin) were measured in the heparin precipitable fraction (HPF) and heparin supernatant fraction (HS) of normal human plasma. Following heparin induced precipitation, most measurable VIII:C activity (77% +/- 24%) was recovered in the HS. Although there was little VIII:C activity (less than 1%) in the HPF, 20% +/- 6.5% VIII:CAg was present as well as CIg (81% +/- 5.6%). VIIIR:RC activity (72% +/- 12%), and VIIIR:Ag (34 +/- 5.2%). As assessed by Na dodecyl SO4 glyoxyl agarose electrophoresis, the multimeric forms of plasma VIIIR:Ag could be resolved into a series of bands. Larger multimers tended to precipitate with the HPF whereas the smaller multimers tended to remain supernatant. Plasma from a subject with congenital afibrinogenemia was also studied. Although the afibrinogenemic HPF contained CIg, neither VIIIR:RC activity nor VIIIR:Ag was precipitated. However, both were present in the HPF from afibrinogenemic plasma to which fibrinogen had been added, suggesting that they are incorporated in this precipitate because of an affinity for fibrinogen. The ability of heparin to induce precipitation of CIg while leaving most VIII:C activity in the supernatant plasma may be useful in the preparation of procoagulant-rich plasma subfractions, since VIII:C can subsequently be recovered in good yield by cryoprecipitation. 相似文献
74.
Partha Dutta Friedrich Felix Hoyer Lubov S. Grigoryeva Hendrik B. Sager Florian Leuschner Gabriel Courties Anna Borodovsky Tatiana Novobrantseva Vera M. Ruda Kevin Fitzgerald Yoshiko Iwamoto Gregory Wojtkiewicz Yuan Sun Nicolas Da Silva Peter Libby Daniel G. Anderson Filip K. Swirski Ralph Weissleder Matthias Nahrendorf 《The Journal of experimental medicine》2015,212(4):497-512
Splenic myelopoiesis provides a steady flow of leukocytes to inflamed tissues, and leukocytosis correlates with cardiovascular mortality. Yet regulation of hematopoietic stem cell (HSC) activity in the spleen is incompletely understood. Here, we show that red pulp vascular cell adhesion molecule 1 (VCAM-1)+ macrophages are essential to extramedullary myelopoiesis because these macrophages use the adhesion molecule VCAM-1 to retain HSCs in the spleen. Nanoparticle-enabled in vivo RNAi silencing of the receptor for macrophage colony stimulation factor (M-CSFR) blocked splenic macrophage maturation, reduced splenic VCAM-1 expression and compromised splenic HSC retention. Both, depleting macrophages in CD169 iDTR mice or silencing VCAM-1 in macrophages released HSCs from the spleen. When we silenced either VCAM-1 or M-CSFR in mice with myocardial infarction or in ApoE−/− mice with atherosclerosis, nanoparticle-enabled in vivo RNAi mitigated blood leukocytosis, limited inflammation in the ischemic heart, and reduced myeloid cell numbers in atherosclerotic plaques.Leukocytosis correlates closely with cardiovascular mortality. In the steady state, blood leukocytes derive exclusively from bone marrow hematopoietic stem cells (HSCs). Supporting cells (Sugiyama et al., 2006; Ding et al., 2012; Ding and Morrison, 2013), including macrophages (Winkler et al., 2010; Chow et al., 2011), maintain the bone marrow HSC niche and regulate hematopoietic stem and progenitor cell (HSPC) activity by supplying various cytokines and retention factors. Systemic inflammation can stimulate extramedullary hematopoiesis in adult mice and humans. Splenic myelopoiesis supplies inflammatory monocytes to atherosclerotic plaques (Robbins et al., 2012) and the ischemic myocardium (Leuschner et al., 2012). In ischemic heart disease, HSPCs emigrate from the bone marrow, seed the spleen, and amplify leukocyte production (Dutta et al., 2012). Splenic HSPCs localize in the red pulp near the sinusoids in parafollicular areas (Kiel et al., 2005). Likewise, after adoptive transfer of GFP+ HSPCs, GFP+ colonies populate the splenic red pulp of atherosclerotic ApoE−/− mice (Robbins et al., 2012). During myocardial infarction (MI), proinflammatory monocytes derived from the spleen accelerate atherosclerotic progression (Dutta et al., 2012). Collectively, these data suggest that splenic myelopoiesis has promise as a therapeutic target; however, the components of the splenic hematopoietic niche are incompletely understood, especially compared with the well-studied bone marrow niche. Understanding HSC retention factors and their regulation in the spleen was the purpose of this study.Because the spleen harbors very few HSCs in the steady state, we investigated the splenic hematopoietic niche after injecting the Toll-like receptor ligand LPS to activate extramedullary hematopoiesis. In the bone marrow, macrophages are an integral part of the HSC niche (Winkler et al., 2010; Chow et al., 2011) and differentiation depends on the receptor for macrophage colony-stimulating factor (M-CSFR, CD115; Auffray et al., 2009). We thus hypothesized that splenic hematopoietic niche assembly also requires M-CSFR signaling. In line with knockout studies (Takahashi et al., 1994; Dai et al., 2002), in vivo knockdown of M-CSFR with nanoparticle-encapsulated siRNA reduced splenic macrophage numbers substantially. Interestingly, decreased macrophage numbers were associated with a reduction of splenic HSCs. Depleting macrophages with diphtheria toxin (DT) in CD169 iDTR mice reproduced the findings obtained with M-CSF–directed siRNA treatment, thereby indicating that macrophages have a key role in splenic HSC maintenance. To investigate how splenic macrophages retain HSCs, we measured changes in splenic expression of major bone marrow retention factors after M-CSFR silencing. Silencing M-CSFR selectively reduced splenic VCAM-1, and the adhesion molecule was primarily expressed by macrophages. Inhibiting macrophage expression of VCAM-1 with siRNA targeting this adhesion molecule reduced splenic HSPC numbers. Finally, we found that M-CSFR and macrophage-directed VCAM-1 silencing in mice with atherosclerosis mitigated blood leukocytosis and dampened inflammation in atherosclerotic plaques and the infarcted myocardium. These data reveal the importance of VCAM-1 expression by splenic macrophages for extramedullary hematopoiesis and illustrate the therapeutic potential of RNAi as an antiinflammatory that mutes emergency overproduction and provision of myeloid cells. 相似文献
75.
Concurrent interaction of DCs with CD4+ and CD8+ T cells improves secondary CTL expansion: It takes three to tango 下载免费PDF全文
Stefanie Hoyer Sabrina Prommersberger Isabell A. Pfeiffer Beatrice Schuler‐Thurner Gerold Schuler Jan Dörrie Niels Schaft 《European journal of immunology》2014,44(12):3543-3559
T‐cell help is essential for CTL‐memory formation. Nevertheless, it is unclear whether the continuous presence of CD4+ T‐helper (Th) cells is required during dendritic cell (DC)/CD8+ T‐cell encounters, or whether a DC will remember the helper signal after the Th cell has departed. This question is relevant for the design of therapeutic cancer vaccines. Therefore, we investigated how human DCs need to interact with CD4+ T cells to mediate efficient repetitive CTL expansion in vitro. We established an autologous antigen‐specific in vitro system with monocyte‐derived DCs, as these are primarily used for cancer vaccination. Contrary to common belief, a sequential interaction of licensed DCs with CD8+ T cells barely improved CTL expansion. In sharp contrast, simultaneous encounter of Th cells and CTLs with the same DC during the first in vitro encounter is a prerequisite for optimal subsequent CTL expansion in our in vitro system. These data suggest that, in contrast to DC maturation, the activation of DCs by Th cells, which is necessary for optimal CTL stimulation, is transient. This knowledge has significant implications for the design of new and more effective DC‐based vaccination strategies. Furthermore, our in vitro system could be a valuable tool for preclinical immunotherapeutical studies. 相似文献
76.
Wolfram Dietz Ulrike Kraft Ingrid Hoyer Gunilla Klingberg 《Acta odontologica Scandinavica》2013,71(4):241-247
The influence of the nature of the root surface on the demineralization and remineralization processes within artificial fluoride-treated caries lesions was investigated using microscopic and X-ray microanalytical methods. Traces of fluoride were detected in the outer parts (about 25 µm) of the lesions after the application of fluorides, and a high mineral content was proved for the same region by means of microanalytical calcium estimation. The location of this mineral-rich band in relation to the root surface was deeper into the root depending on the existence and thickness of a cementum layer. However, within the dentine the location and intensity of the mineral content were unaffected by the cementum. Investigation of artificial caries lesions without fluoride treatment showed the following: The degree of mineralization was kept at a higher level near the root surface in the presence of cementum. Consequently, a cementum layer gives some initial caries resistance of the root surface. 相似文献
77.
78.
Jutta Gellermann Lutz Weber Lars Pape Burkhard T?nshoff Peter Hoyer Uwe Querfeld 《Journal of the American Society of Nephrology : JASN》2013,24(10):1689-1697
The severe side effects of long-term corticosteroid or cyclosporin A (CsA) therapy complicate the treatment of children with frequently relapsing steroid-sensitive nephrotic syndrome (FR-SSNS). We conducted a randomized, multicenter, open-label, crossover study comparing the efficacy and safety of a 1-year treatment with mycophenolate mofetil (MMF; target plasma mycophenolic acid trough level of 1.5–2.5 µg/ml) or CsA (target trough level of 80–100 ng/ml) in 60 pediatric patients with FR-SSNS. We assessed the frequency of relapse as the primary endpoint and evaluated pharmacokinetic profiles (area under the curve [AUC]) after 3 and 6 months of treatment. More relapses per patient per year occurred with MMF than with CsA during the first year (P=0.03), but not during the second year (P=0.14). No relapses occurred in 85% of patients during CsA therapy and in 64% of patients during MMF therapy (P=0.06). However, the time without relapse was significantly longer with CsA than with MMF during the first year (P<0.05), but not during the second year (P=0.36). In post hoc analysis, patients with low mycophenolic acid exposure (AUC <50 µg⋅h/ml) experienced 1.4 relapses per year compared with 0.27 relapses per year in those with high exposure (AUC>50 µg⋅h/ml; P<0.05). There were no significant differences between groups with respect to BP, growth, lipid levels, or adverse events. However, cystatin clearance, estimated GFR, and hemoglobin levels increased significantly with MMF compared with CsA. These results indicate that MMF is inferior to CsA in preventing relapses in pediatric patients with FR-SSNS, but may be a less nephrotoxic treatment option.Idiopathic nephrotic syndrome, the most common form of childhood nephrotic syndrome, is most often associated with renal biopsy findings of minimal glomerular and tubulointerstitial changes (minimal change disease). Most patients respond to therapy with corticosteroids,1 but about 70% experience a relapsing course.2 Approximately 30% develop frequently relapsing steroid-sensitive nephrotic syndrome (FR-SSNS), defined as ≥4 relapses per year.3Although corticosteroids are the mainstay of therapy in pediatric patients with minimal change disease, their repeated use in FR-SSNS results in severe side effects, and other therapeutic options are needed to prevent steroid toxicity.4 A 2- to 3-month course of cyclophosphamide or chlorambucil has been shown to produce a longer remission period in many patients5; however, these drugs may have serious side effects and their long-term efficacy is limited.6,7 Levamisole has been shown to reduce the risk of relapse in several small studies, but information is limited regarding long-term efficacy and adverse effects, and the drug is currently not available in most countries. Treatment with cyclosporin A (CsA) is highly effective in maintaining remission in patients with FR-SSNS allowing withdrawal of corticosteroids, but most patients relapse after withdrawal of CsA.8 Importantly, prolonged CsA therapy is accompanied by time- and dose-dependent nephrotoxicity.9Mycophenolate mofetil (MMF), the prodrug of mycophenolic acid (MPA), is a non-nephrotoxic immunosuppressive drug with inhibitory effects on T and B lymphocytes, cell-surface markers, and cytokine gene expression10 and has proven efficacy and tolerability in renal allograft recipients. Several small studies with limited statistical power have shown that MMF has steroid-sparing effects and reduces relapse rates in patients with FR-SSNS, albeit with varying efficacy.11–18We studied efficacy and safety of MMF in patients with FR-SSNS in comparison with CsA in a prospective randomized multicenter open-label crossover trial. 相似文献
79.
Pradeepkumar Charlagorla David Becerra Parth M. Patel Mark Hoyer Robert K. Darragh 《Pediatric cardiology》2016,37(1):125-130
Congenital pulmonary vein stenosis (PVS) is a rare entity with limited outcome literature. Multiple interventional approaches have evolved including surgical and catheterization techniques. Our objective is to report our center experience and to compare short-term and mid-term outcomes among these therapeutic modalities. Retrospective study on 23 patients (n = 23) with PVS that required intervention over the last 13 years (2000–2013). Patients were divided into three groups based on type of initial intervention. Of these, 10 (43.5 %) had balloon angioplasty, 3 (13.0 %) had surgical dilation, and 10 (43.5 %) had surgical marsupialization. Mortality and number of re-interventions were our primary outcomes. Mean age at diagnosis was 10.9 ± 18.4 months. Mean age at initial intervention was 14.5 ± 18.0 months. Mean pre- and post-initial intervention PVS gradients were 9.2 ± 3.4 and 3.4 ± 2.2 mmHg, respectively. Mean survival time and re-intervention-free survival time were 4.8 ± 4.0 and 2.8 ± 3.4 years. No statistical significance was found between the interventions with respect to survival time (p = 0.52) and re-intervention free time (p = 0.78). High initial pre- and post-intervention gradients were significantly associated with re-intervention-free survival (p = 0.01 and p = 0.03, respectively). Patients with bilateral disease have increased mortality (p = 0.01) and decreased 5-year survival (p = 0.009) compared to patients with unilateral disease irrespective of type of intervention. No statistically significant difference in mortality or re-intervention rate was present among these different therapeutic modalities. This study has the longest follow-up so far reported in the current literature (58 months) with overall survival of 78 %. 相似文献
80.
Hoyer J Dreweke A Becker C Göhring I Thiel CT Peippo MM Rauch R Hofbeck M Trautmann U Zweier C Zenker M Hüffmeier U Kraus C Ekici AB Rüschendorf F Nürnberg P Reis A Rauch A 《Journal of medical genetics》2007,44(10):629-636