Expression and function of microRNA-188-5p in activated rheumatoid arthritis synovial fibroblasts

Activated synovial fibroblasts in rheumatoid arthritis (RASF) play a critical role in the pathology of rheumatoid arthritis (RA). Recent studies suggested that deregulation of microRNAs (miRs) affects the development and progression of RA. Therefore, we aimed to identify de-regulated miRs in RASF and to identify target genes that may contribute to the aggressive phenotype of RASF. Quantitative real-time PCR revealed a marked downregulation of miR-188-5p in synovial tissue samples of RA patients as well as in RASF. Exposure to the cytokine interleukine-1β lead to a further downregulation of miR-188-5p expression levels compared to control cells. Re-expression of miR-188-5p in RASF by transient transfection significantly inhibited cell migration. However, miR-188-5p re-expression had no effects on glycosaminoglycan degradation or expression of repellent factors, which have been previously shown to affect the invasive behavior of RASF. In search for target genes of miR-188-5p in RASF we performed gene expression profiling in RASF and found a strong regulatory effect of miR-188-5p on the hyaluronan binding protein KIAA1199 as well as collagens COL1A1 and COL12A1, which was confirmed by qRT-PCR. In silico analysis revealed that KIAA1199 carries a 3’UTR binding site for miR-188-5p. COL1A1 and COL12A1 showed no binding site in the mRNA region, suggesting an indirect regulation of these two genes by miR-188-5p. In summary, our study showed that miR-188-5p is down-regulated in RA in vitro and in vivo, most likely triggered by an inflammatory environment. MiR-188-5p expression is correlated to the activation state of RASF and inhibits migration of these cells. Furthermore, miR-188-5p is directly and indirectly regulating the expression of genes, which may play a role in extracellular matrix formation and destruction in RA. Herewith, this study identified potential novel therapeutic targets to inhibit the development and progression of RA.     

Eating disorders and biochemical composition of saliva: a retrospective matched case–control study

This study aimed to compare the biochemical composition of saliva from patients with eating disorders (EDs) with saliva from control subjects with no ED. All patients who initiated outpatient treatment in an ED clinic during a 12‐month period were invited to participate. Of the 65 patients who started treatment during the period, 54 (50 female patients/four male patients; mean age: 21.5 yr) agreed to participate. The controls were 54 sex‐ and age‐matched patients from a dental health clinic. All participants completed a questionnaire and underwent dental clinical examinations, including laboratory analyses of saliva. The proportion of subjects with unstimulated salivary hyposalivation was lower in the ED group and not correlated with intake of xerogenic drugs. Significant differences in the biochemical composition of saliva were found almost exclusively in the unstimulated state, with albumin, inorganic phosphate, aspartate aminotransferase (ASAT), chloride, magnesium, and total protein all being significantly higher in the ED group. Conditional logistic regression showed that higher ASAT and total protein concentrations were relatively good predictors of ED, with sensitivity and specificity of 65% and 67%, respectively. In conclusion, elevated salivary concentrations of ASAT and total protein may serve as indicators of ED as well as of disease severity. Future studies are needed to corroborate these initial findings.     

Resistance‐associated substitutions in patients with chronic hepatitis C virus genotype 4 infection

Data on the prevalence of resistance‐associated substitutions (RASs) and their implications for treatment with direct‐acting antivirals (DAAs) are sparse in European patients with HCV genotype 4. This study investigated RASs before and after DAA failure in different genotype 4 subtypes and evaluated retreatment efficacies. Samples of 195 genotype 4‐infected patients were collected in the European Resistance Database and investigated for NS3, NS5A and NS5B RASs. Retreatment efficacies in DAA failure patients were analysed retrospectively. After NS5A inhibitor (NS5Ai) failure, subtype 4r was frequent (30%) compared to DAA‐naïve patients (5%) and the number of NS5A RASs was significantly higher in subtype 4r compared to 4a or 4d (median three RASs vs no or one RAS, respectively, P < .0001). RASsL28V, L30R and M31L pre‐existed in subtype 4r and were maintained after NS5Ai failure. Typical subtype 4r RASs were located in subdomain 1a of NS5A, close to membrane interaction and protein‐protein interaction sites that are responsible for multimerization and hence viral replication. Retreatment of 37 DAA failure patients was highly effective with 100% SVR in prior SOF/RBV, PI/SOF and PI/NS5Ai failures. Secondary virologic failures were rare (n = 2; subtype 4d and 4r) and only observed in prior NS5Ai/SOF failures (SVR 90%). In conclusion, subtype 4r harboured considerably more RASs compared to other subtypes. A resistance‐tailored retreatment using first‐ and second‐generation DAAs was highly effective with SVR rates ≥90% across all subtypes and first‐line treatment regimens.     

Assessment of Viral Targeted Sequence Capture Using Nanopore Sequencing Directly from Clinical Samples

Shotgun metagenomic sequencing (SMg) enables the simultaneous detection and characterization of viruses in human, animal and environmental samples. However, lack of sensitivity still poses a challenge and may lead to poor detection and data acquisition for detailed analysis. To improve sensitivity, we assessed a broad scope targeted sequence capture (TSC) panel (ViroCap) in both human and animal samples. Moreover, we adjusted TSC for the Oxford Nanopore MinION and compared the performance to an SMg approach. TSC on the Illumina NextSeq served as the gold standard. Overall, TSC increased the viral read count significantly in challenging human samples, with the highest genome coverage achieved using the TSC on the MinION. TSC also improved the genome coverage and sequencing depth in clinically relevant viruses in the animal samples, such as influenza A virus. However, SMg was shown to be adequate for characterizing a highly diverse animal virome. TSC on the MinION was comparable to the NextSeq and can provide a valuable alternative, offering longer reads, portability and lower initial cost. Developing new viral enrichment approaches to detect and characterize significant human and animal viruses is essential for the One Health Initiative.     

Heterogeneity of stromal cells in the human splenic white pulp. Fibroblastic reticulum cells,follicular dendritic cells and a third superficial stromal cell type

At least three phenotypically and morphologically distinguishable types of branched stromal cells are revealed in the human splenic white pulp by subtractive immunohistological double-staining. CD271 is expressed in fibroblastic reticulum cells of T-cell zones and in follicular dendritic cells of follicles. In addition, there is a third CD271⁻ and CD271^+/− stromal cell population surrounding T-cell zones and follicles. At the surface of follicles the third population consists of individually variable partially overlapping shells of stromal cells exhibiting CD90 (Thy-1), MAdCAM-1, CD105 (endoglin), CD141 (thrombomodulin) and smooth muscle α-actin (SMA) with expression of CD90 characterizing the broadest shell and SMA the smallest. In addition, CXCL12, CXCL13 and CCL21 are also present in third-population stromal cells and/or along fibres. Not only CD27⁺ and switched B lymphocytes, but also scattered IgD⁺⁺ B lymphocytes and variable numbers of CD4⁺ T lymphocytes often occur close to the third stromal cell population or one of its subpopulations at the surface of the follicles. In contrast to human lymph nodes, neither podoplanin nor RANKL (CD254) were detected in adult human splenic white pulp stromal cells. The superficial stromal cells of the human splenic white pulp belong to a widespread cell type, which is also found at the surface of red pulp arterioles surrounded by a mixed T-cell/B-cell population. Superficial white pulp stromal cells differ from fibroblastic reticulum cells and follicular dendritic cells not only in humans, but apparently also in mice and perhaps in rats. However, the phenotype of white pulp stromal cells is species-specific and more heterogeneous than described so far.     

Synthesis and Characterization of Polymerized Ionic Liquids: Mechanical and Thermal Properties of a Novel Type of Hydrogels

An easy and efficient route to synthesize gel materials based on polymeric ionic liquids (PILs) is presented. The radical polymerization of imidazolium (Im)‐based ionic liquids (ILs) bearing a vinyl group ([VEIm][Br], [VEIm][Ac], [VBIm][Br], [VBIm][Cl]) with crosslinker (CL) N,N′‐methylenebisacrylamide (Bis) in water results in polyionic liquid hydrogels. Thermal and mechanical properties (tensile and compression tests) are investigated and compared with two different types of hydrogels. One is a polyacrylamide (PAAm) hydrogel having covalent‐type crosslinking. The other is an alginate‐based hydrogel having ionic‐type crosslinking. Prepared IL‐hydrogel materials provide favorable flexibility, adjustable by varying the CL ratio and water content. The higher the CL ratio is, the higher the fragility of the gel matrix. The gelation time of the hydrogels depends on the alkyl chain length, as well as the size of the anion.

     

Diffusion and Interface Effects during Preparation of All-Solid Microstructured Fibers

All-solid microstructured optical fibers (MOF) allow the realization of very flexible optical waveguide designs. They are prepared by stacking of doped silica rods or canes in complex arrangements. Typical dopants in silica matrices are germanium and phosphorus to increase the refractive index (RI), or boron and fluorine to decrease the RI. However, the direct interface contact of stacking elements often causes interrelated chemical reactions or evaporation during thermal processing. The obtained fiber structures after the final drawing step thus tend to deviate from the targeted structure risking degrading their favored optical functionality. Dopant profiles and design parameters (e.g., the RI homogeneity of the cladding) are controlled by the combination of diffusion and equilibrium conditions of evaporation reactions. We show simulation results of diffusion and thermal dissociation in germanium and fluorine doped silica rod arrangements according to the monitored geometrical disturbances in stretched canes or drawn fibers. The paper indicates geometrical limits of dopant structures in sub-µm-level depending on the dopant concentration and the thermal conditions during the drawing process. The presented results thus enable an optimized planning of the preform parameters avoiding unwanted alterations in dopant concentration profiles or in design parameters encountered during the drawing process.     

Roles of cGMP-dependent protein kinase I (cGKI) and PDE5 in the regulation of Ang II-induced cardiac hypertrophy and fibrosis

Conflicting results have been reported for the roles of cGMP and cGMP-dependent protein kinase I (cGKI) in various pathological conditions leading to cardiac hypertrophy and fibrosis. A cardioprotective effect of cGMP/cGKI has been reported in whole animals and isolated cardiomyocytes, but recent evidence from a mouse model expressing cGKIβ only in smooth muscle (βRM) but not in cardiomyocytes, endothelial cells, or fibroblasts has forced a reevaluation of the requirement for cGKI activity in the cardiomyocyte antihypertrophic effects of cGMP. In particular, βRM mice developed the same hypertrophy as WT controls when subjected to thoracic aortic constriction or isoproterenol infusion. Here, we challenged βRM and WT (Ctr) littermate control mice with angiotensin II (AII) infusion (7 d; 2 mg⋅kg⁻¹⋅d⁻¹) to induce hypertrophy. Both genotypes developed cardiac hypertrophy, which was more pronounced in Ctr animals. Cardiomyocyte size and interstitial fibrosis were increased equally in both genotypes. Addition of sildenafil, a phosphodiesterase 5 (PDE5) inhibitor, in the drinking water had a small effect in reducing myocyte hypertrophy in WT mice and no effect in βRM mice. However, sildenafil substantially blocked the increase in collagen I, fibronectin 1, TGFβ, and CTGF mRNA in Ctr but not in βRM hearts. These data indicate that, for the initial phase of AII-induced cardiac hypertrophy, lack of cardiomyocyte cGKI activity does not worsen hypertrophic growth. However, expression of cGKI in one or more cell types other than smooth muscle is necessary to allow the antifibrotic effect of sildenafil.Cyclic GMP-dependent protein kinase I (cGKI) is expressed in a wide variety of cells including cardiomyocytes (CMs), cardiac myofibroblasts (CMFs), cardiac fibroblasts (CFs), endothelial cells (ECs), and smooth muscle cells (SMCs) (1). Increased cGKI activity has been reported to protect against cardiac hypertrophy induced by pressure overload (2, 3). For example, mice that carry a mutated form of cGKI unable to interact with downstream targets, develop increased pathologic hypertrophy, accelerated mortality, and congestive heart failure when subjected to thoracic aorta constriction (TAC) (4).Hypertrophy induced by angiotensin II (AII) is thought to be attenuated by cGKI, because AII signaling through G_q/11 is abrogated by the regulator of G-protein signaling protein (RGS), a known substrate of cGKI (5). However, selective deletion of the AII receptor 1 (AT-R1) in the kidney ameliorated AII-induced cardiac hypertrophy, suggesting that the cardiac AT-R1 is dispensable for the induction of this response (6), whereas transgenic mice that overexpress the human AT-R1 specifically in CMs develop hypertrophy, fibrosis, dysfunctions, and early death (7). Furthermore, activation of TRPC channels followed by elevated [Ca²⁺]_i may contribute to the induction of the cardiac hypertrophy gene response (8–11). Again, TRPC3 and TRPC6 channels are negatively regulated by cGKI (12–14).It also has been reported that particulate guanylyl cyclase A, the major receptor for atrial natriuretic peptide (ANP) in the heart, couples to and directly activates the TRPC3/C6 channels in chronic cardiac hypertrophy, thus bypassing cGMP and cGKI (15). It was reported that cardiac cGMP can affects cardiac properties by modulating cAMP levels, bypassing again cGKI (16, 17). Thus, the site(s) of action of cGMP/cGKI are not entirely clear (18) and interpretation of cGMP/cGKI effects on cardiac hypertrophy may be quite complicated.Sildenafil, a phosphodiesterase 5 (PDE5) inhibitor, elevates cardiac cGMP at high doses (100 mg⋅kg⁻¹⋅d⁻¹), increases cGKI activity, and has been reported to reverse TAC-induced cardiac hypertrophy (2). This effect is postulated to be caused by an increased activity of RGS2 (19), and in part by the direct regulation of TRPC3/6 conductance by cGKI (11, 14) mentioned above. However, two clinical studies did not report positive therapeutic results after prolonged treatment of diastolic dysfunction with sildenafil (20, 21).We (22) and others (23) have tested the hypothesis that CM-cGKI attenuates cardiac hypertrophy by using the cGKIβ rescue mouse line (βRM). These animals express the cGKIβ isozyme under the SM22α promoter, which is active principally in SMCs and activated CMFs, but do not express cGKIβ in other cell types (24). Chronic infusion of isoproterenol or TAC induced in βRM mice a cardiac hypertrophy that was identical to that of WT littermate controls (22), a result that argues against the hypothesis that CM, EC, or CF cGKI is responsible for the antihypertrophic effects of cGMP in the heart. To examine the role(s) of sildenafil to inhibit cardiac hypertrophy and fibrosis, we now extend these experiments using AII-induced hypertrophy and high concentrations of sildenafil (0.6 mM) in the drinking water. Once again, the lack of cGKI in CM and CF did not cause an increased hypertrophic response as predicted (2). Moreover, little or no antihypertrophic effect of sildenafil was observed in the WT mice and no effect was seen in the βRM mice. However, a large effect of sildenafil was observed on fibrosis in WT but not the βRM mice, suggesting that cGKI present in cardiac SMCs or activated CMFs is an important regulator of cardiac fibrosis.     

CXCR4 blockade and Sphingosine‐1‐phosphate activation facilitate engraftment of haematopoietic stem and progenitor cells in a non‐myeloablative transplant model

Both immunosuppressive and cytoreductive effects of γ‐irradiation contribute to engraftment of allogeneic haematopoietic stem and progenitor cells. We hypothesized that a release of host stem and progenitor cells from the niche prior to conditioning would permit engraftment after less intensive conditioning. Administration of AMD3100 and SEW2871 on days ?4 to ?2 followed by irradiation on day ?1 in a non‐myeloablative zebrafish transplant model resulted in a reduced radiation minimum dose of 10 Gy from 15 Gy being sufficient for engraftment. Targeting the SDF‐1 (CXCL12)/CXCR4‐ and S1P/S1P₁‐axis increased the efficacy of allografting in an experimental transplant model.