Performance of Antinuclear Antibodies for Classifying Systemic Lupus Erythematosus: A Systematic Literature Review and Meta‐Regression of Diagnostic Data

Objective

To review the published literature on the performance of indirect immunofluorescence (IIF)–HEp‐2 antinuclear antibody (ANA) testing for classification of systemic lupus erythematosus (SLE).

Methods

A systematic literature search was conducted in the Medline, Embase, and Cochrane databases for articles published between January 1990 and October 2015. The research question was structured according to Population, Intervention, Comparator, Outcome (PICO) format rules, and Preferred Reporting Items for Systematic Reviews and Meta‐Analyses (PRISMA) recommendations were followed where appropriate. Meta‐regression analysis for diagnostic tests was performed, using the ANA titer as independent variable, while sensitivity and specificity were dependent variables.

Results

Of 4,483 publications screened, 62 matched the eligibility criteria, and another 2 articles were identified through reference analysis. The included studies comprised 13,080 SLE patients in total, of whom 12,542 (95.9%) were reported to be IIF‐ANA positive at various titers. For ANA at titers of 1:40, 1:80, 1:160, and 1:320, meta‐regression gave sensitivity values of 98.4% (95% confidence interval [95% CI] 97.6–99.0%), 97.8% (95% CI 96.8–98.5%), 95.8% (95% CI 94.1–97.1%), and 86.0% (95% CI 77.0–91.9%), respectively. The corresponding specificities were 66.9% (95% CI 57.8–74.9%), 74.7% (95% CI 66.7–81.3%), 86.2% (95% CI 80.4–90.5%), and 96.6% (95% CI 93.9–98.1%), respectively.

Conclusion

The results of this systematic literature review and meta‐regression confirm that IIF‐ANAs have high sensitivity for SLE. ANAs at a titer of 1:80 have sufficiently high sensitivity to be considered as an entry criterion for SLE classification criteria, i.e., formally test other classification criteria for SLE only if ANAs of at least 1:80 have been found.

     

Shear stress-induced up-regulation of the intermediate-conductance Ca(2+)-activated K(+) channel in human endothelium

OBJECTIVE: Wall shear stress associated with blood flow is a major stimuli for generation of endothelial vasodilating and antithrombotic factors and it also regulates endothelial gene expression. Activation of endothelial intermediate-conductance Ca(2+)-activated K(+) channels (IK(Ca)) is important for the control of endothelial function by inducing cell hyperpolarization and thus generation of the endothelium-derived hyperpolarizing factor. In the present study we tested whether the IK(Ca) encoding IKCa1 gene is regulated by laminar shear stress (LSS). METHODS: Human umbilical vein endothelial cells (HUVEC) were subjected to LSS with a magnitude of 0.5-15 dyn/cm(2) and time intervals of 2-24 h in a flow cone apparatus. Expression of the IKCa1 gene and IK(Ca)-functions were determined by using real time RT-PCR and patch-clamp techniques. RESULTS: A short 2-4 h-or long 24 h-exposure to a LSS with a low (venous) magnitude of 0.5 dyn/cm(2) had no effect on IKCa1 expression levels. An exposure for 2 and 4 h to LSS with an intermediate magnitude of 5 dyn/cm(2) was also ineffective, whereas an exposure for 24 h induced a significant threefold up-regulation of IKCa1 expression levels. An exposure to LSS with a higher (arterial) magnitude of 15 dyn/cm(2), resulted in an eightfold up-regulation of IKCa1 expression levels after a 4 h-exposure and a fourfold increase of IKCa1 expression levels at 24 h. The increased IKCa1 expression levels following exposure to high levels of LSS resulted in enhanced IK(Ca) whole-cell currents and in an increased hyperpolarization of the endothelium in response to ATP and the IK(Ca) opener 1-EBIO. Inhibition of the mitogen-activated protein kinase/extracellular-signal-regulated kinase (ERK) kinase 1/2 (MEK/ERK) pathway by PD98059 prevented the LSS-induced up-regulation of IKCa1 expression levels and IK(Ca) whole-cell currents indicating that augmentation of IKCa1 expression levels is mediated by the LSS-induced activation of the MEK/ERK pathway. CONCLUSION: Long term exposure to LSS up-regulates expression and function of endothelial IK(Ca). This increase might represent a new important mechanism in endothelial adaptation to altered hemodynamics.     

Moderation of hemophilia A phenotype by the factor V R506Q mutation

Although many examples of unrelated hemophilia A patients carrying identical point mutations in the factor VIII (FVIII) gene have been reported, the clinical phenotype is not always the same among patients sharing the same molecular defect. Possible explanations for this discrepancy include undetected additional mutations in the FVIII gene or coinheritance of mutations at other genetic loci that modulate FVIII function. We report molecular genetic analysis of potential modifying genes in two sets of unrelated patients carrying common FVIII missense mutations but exhibiting different levels of clinical severity. Both mutations (FVIII R1689C and R2209Q) are associated with severe hemophilia A in some patients and mild/moderate disease in others. The common von Willebrand disease type 2N mutation (R91Q) was excluded as a modifying factor in these groups of patients. However, analysis of the recently described factor V (FV) R506Q mutation (leading to activated protein C resistance) identified a correlation of inheritance of this defect with reduced hemophilia A severity. Two moderately affected hemophilia A patients, each with either of two FVIII gene mutations, were heterozygous for FV R506Q, whereas two severely affected patients and two moderately affected patients were homozygous normal at the FV locus. Our results suggest that coinheritance of the FV R506Q mutation may be an important determinant of clinical phenotype in hemophilia A and that modification of the protein C pathway may offer a new strategy for the treatment of FVIII deficiency.     

Rapid Response to Cyclosporin A and Favorable Renal Outcome in Nongenetic Versus Genetic Steroid–Resistant Nephrotic Syndrome

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.     

From the Cover: Breaking the diffraction limit of light-sheet fluorescence microscopy by RESOLFT

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.     

Distribution of plasma fibronectin (cold-insoluble globulin) and components of the factor VIII complex after heparin-induced precipitation of plasma

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.     

Macrophages retain hematopoietic stem cells in the spleen via VCAM-1

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.     

Concurrent interaction of DCs with CD4+ and CD8+ T cells improves secondary CTL expansion: It takes three to tango

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.     

Influence of cementum on the demineralization and remineralization processes of root surface caries in vitro

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.