Laquinimod is an orally administered compound that is under investigation in relapsing-remitting multiple sclerosis. To understand the mechanism by which laquinimod exerts its clinical effects, we ...have performed human and murine studies assessing its immunomodulatory properties. In experimental autoimmune encephalomyelitis, the therapeutic administration of laquinimod beginning during the recovery of SJL mice, prevented further relapses as expected and strongly reduced infiltration of CD4+ and CD8+ T cells in the central nervous system. We hypothesized that this beneficial effect was mediated by dendritic cells, since we and others found a modulation of different dendritic cell subsets under treatment. According to the findings on antigen-presenting cells in the murine system, we found a reduced capacity of human monocyte-derived dendritic cells treated with therapeutic concentrations of laquinimod, upon maturation with lipopolysaccharide, to induce CD4+ T cell proliferation and secretion of pro-inflammatory cytokines. Furthermore, laquinimod treatment of mature dendritic cells resulted in a decreased chemokine production by both murine and human dendritic cells, associated with a decreased monocyte chemo-attraction. In laquinimod-treated patients with multiple sclerosis we consistently found reduced chemokine and cytokine secretion by conventional CD1c+ dendritic cells upon lipopolysaccharide stimulation. Similarly to the animal model of relapsing-remitting multiple sclerosis, dendritic cell subsets were altered in patients upon laquinimod treatment, as the number of conventional CD1c+ and plasmacytoid CD303+ dendritic cells were decreased within peripheral blood mononuclear cells. Moreover, laquinimod treatment in patients with multiple sclerosis and mice modified the maturation of dendritic cells demonstrated by an upregulation of CD86 expression in vivo. Our data suggest that inhibition of the NF-κB pathway is responsible for the changes observed in dendritic cell maturation and functions. These findings indicate that laquinimod exhibits its disease-modulating activity in multiple sclerosis by downregulating immunogenicity of dendritic cell responses. We suggest that monitoring dendritic cell properties in multiple sclerosis should be implemented in future therapeutic trials.
Cladribine is a purine nucleoside analog developed to treat lymphoid malignancies. Reported therapeutic benefits for the autoimmune disease multiple sclerosis indicate additional immunomodulatory ...effects beyond the well-characterized cytotoxic activity causing lymphopenia. Here, we demonstrate that cladribine reduces the secretion of inflammatory cytokines and chemokines by murine and human dendritic cells, the most potent antigen-presenting cells. This compound also modulates the expression of the activation markers CD86 and MHC II. Furthermore, cladribine affects the T cell priming capacity of dendritic cells, resulting in reduced induction of interferon-γ- and tumor necrosis factor-α-producing T cells and increased induction of interleukin-10-producing T cells. These effects, observed at cladribine concentrations in the therapeutically relevant range of serum steady-state concentrations for leukemia and multiple sclerosis, confirm the immunomodulatory activity of cladribine.
•Cladribine reduces chemokine production by DC.•Cladribine modulates T cell polarizing capacity of DC.•Cladribine alters DC to a semi-mature differentiation-locked tolerogenic phenotype.•Cladribine shows immunomodulatory effects at therapeutic doses.
To assess the impact of laquinimod treatment on monocytes and to investigate the underlying immunomodulatory mechanisms in MS.
In this cross-sectional study, we performed in vivo and in vitro ...analyses of cluster of differentiation (CD14
) monocytes isolated from healthy donors (n = 15), untreated (n = 13), and laquinimod-treated patients with MS (n = 14). Their frequency and the expression of surface activation markers were assessed by flow cytometry and the viability by calcein staining. Cytokine concentrations in the supernatants of lipopolysaccharide (LPS)-stimulated monocytes were determined by flow cytometry. The messenger ribonucleic acid (mRNA) expression level of genes involved in cytokine expression was measured by quantitative PCR. The LPS-mediated nuclear factor kappa-light-chain-enhancer of activated B-cell (NF-κB) activation was determined by the quantification of the phosphorylation level of the p65 subunit. Laquinimod-treated monocytes were cocultured with CD4
T cells, and the resulting cytokine production was analyzed by flow cytometry after intracellular cytokine staining. The interleukin (IL)-17A concentration of the supernatant was assessed by ELISA.
Laquinimod did not alter the frequency or viability of circulating monocytes, but led to an upregulation of CD86 expression. LPS-stimulated monocytes of laquinimod-treated patients with MS secreted less IL-1β following a downregulation of IL-1β gene expression. Phosphorylation levels of the NF-κB p65 subunit were reduced after laquinimod treatment, indicating a laquinimod-associated inhibition of the NF-κB pathway. T cells primed with laquinimod-treated monocytes differentiated significantly less into IL-17A-producing T helper (Th)-17 cells.
Our findings suggest that inhibited NF-κB signaling and downregulation of IL-1β expression in monocytes contributes to the immunomodulatory effects of laquinimod and that the impairment of Th17 polarization might mediate its disease-modifying activity in MS.
In addition to the pathogenetic CAG repeat expansion other genetic factors play a significant role in determining age at onset (AO) in Huntington disease (HD), e.g. variations in the NR2A and NR2B ...glutamate receptor subunit genes (GRIN2A, GRIN2B). In order to expand these findings we fine-mapped a larger HD patient panel (n = 250) using densely spaced markers flanking the originally associated SNPs in GRIN2A and GRIN2B. In GRIN2A association fine-mapping based on eight additional SNPs confirmed intron 2 as the region of strongest association. In GRIN2B fine-mapping with seven additional SNPs consolidated C2664T as causal genetic variation. Gender stratification of patients revealed differences in the variability in AO attributable to the CAG repeat number and highly significant differences in the AO association with the C2664T and rs8057394/ rs2650427 variations. Addition of the corresponding genotype variations to the effect of CAG repeat lengths resulted in a significant increase of the R2 values only in females. The sex-specific effect for C2664T is underscored by differences in the genotype and allele frequencies observed for female versus male HD patients (P = 0.01) caused by decreased CC frequency in females. Overall, female HD patients homozygous for the CC genotype tended to have later AO compared to the other two genotypes. Stratification of the results by presumed menopausal status demonstrated that the significant findings were predominantly observed in pre-menopausal patients. We speculate that altered hormone levels herald protective effects of this genotype. Together, GRIN2A and GRIN2B genotype variations explain 7.2% additional variance in AO for HD.
OBJECTIVE: To assess the impact of laquinimod treatment on monocytes and to investigate the underlying immunomodulatory mechanisms in MS. METHODS: In this cross-sectional study, we performed in vivo ...and in vitro analyses of cluster of differentiation (CD14(+)) monocytes isolated from healthy donors (n = 15), untreated (n = 13), and laquinimod-treated patients with MS (n = 14). Their frequency and the expression of surface activation markers were assessed by flow cytometry and the viability by calcein staining. Cytokine concentrations in the supernatants of lipopolysaccharide (LPS)-stimulated monocytes were determined by flow cytometry. The messenger ribonucleic acid (mRNA) expression level of genes involved in cytokine expression was measured by quantitative PCR. The LPS-mediated nuclear factor kappa-light-chain-enhancer of activated B-cell (NF-kappaB) activation was determined by the quantification of the phosphorylation level of the p65 subunit. Laquinimod-treated monocytes were cocultured with CD4(+) T cells, and the resulting cytokine production was analyzed by flow cytometry after intracellular cytokine staining. The interleukin (IL)-17A concentration of the supernatant was assessed by ELISA.RESULTS: Laquinimod did not alter the frequency or viability of circulating monocytes, but led to an upregulation of CD86 expression. LPS-stimulated monocytes of laquinimod-treated patients with MS secreted less IL-1beta following a downregulation of IL-1beta gene expression. Phosphorylation levels of the NF-kappaB p65 subunit were reduced after laquinimod treatment, indicating a laquinimod-associated inhibition of the NF-kappaB pathway. T cells primed with laquinimod-treated monocytes differentiated significantly less into IL-17A-producing T helper (Th)-17 cells.CONCLUSIONS: Our findings suggest that inhibited NF-kappaB signaling and downregulation of IL-1beta expression in monocytes contributes to the immunomodulatory effects of laquinimod and that the impairment of Th17 polarization might mediate its disease-modifying activity in MS.
Abstract We report long-baseline interferometric observations with the CHARA Array that resolve six previously known double-lined spectroscopic binary systems in the Hyades cluster, with orbital ...periods ranging from 3 to 358 days: HD 27483, HD 283882, HD 26874, HD 27149, HD 30676, and HD 28545. We combine those observations with new and existing radial-velocity measurements, to infer the dynamical masses for the components as well as the orbital parallaxes. For most stars, the masses are determined to be better than 1%. Our work significantly increases the number of systems with mass determinations in the cluster. We find that, while current models of stellar evolution for the age and metallicity of the Hyades are able to reproduce the overall shape of the empirical mass–luminosity relation, they overestimate the V -band fluxes by about 0.1 mag between 0.5 and 1.4 M ⊙ . The disagreement is smaller in H , and near zero in K , and depends somewhat on the model. We also make use of the TESS light curves to estimate rotation periods for our targets, and detect numerous flares in one of them (HD 283882), estimating an average flaring rate of 0.44 events per day.
The 30 yr orbit of the Cepheid Polaris has been followed with observations by the Center for High Angular Resolution Astronomy (CHARA) Array from 2016 through 2021. An additional measurement has been ...made with speckle interferometry at the Apache Point Observatory. Detection of the companion is complicated by its comparative faintness—an extreme flux ratio. Angular diameter measurements appear to show some variation with pulsation phase. Astrometric positions of the companion were measured with a custom grid-based model-fitting procedure and confirmed with the CANDID software. These positions were combined with the extensive radial velocities (RVs) discussed by Torres to fit an orbit. Because of the imbalance of the sizes of the astrometry and RV data sets, several methods of weighting are discussed. The resulting mass of the Cepheid is 5.13 ± 0.28 M⊙. Because of the comparatively large eccentricity of the orbit (0.63), the mass derived is sensitive to the value found for the eccentricity. The mass combined with the distance shows that the Cepheid is more luminous than predicted for this mass from evolutionary tracks. The identification of surface spots is discussed. This would give credence to the identification of a radial velocity variation with a period of approximately 120 days as a rotation period. Polaris has some unusual properties (rapid period change, a phase jump, variable amplitude, and unusual polarization). However, a pulsation scenario involving pulsation mode, orbital periastron passage, and low pulsation amplitude can explain these characteristics within the framework of pulsation seen in Cepheids.
We assessed the value of sonography in predicting intraoperative difficulties for patients undergoing laparoscopic cholecystectomy and in identifying indicators for conversion to conventional ...cholecystectomy.
Upper abdominal sonography was performed (according to a checklist) in 75 consecutive patients before laparoscopic cholecystectomy. Sonographic findings were verified by the surgeon in the operating room.
Conversion from laparoscopic surgery to laparotomy was performed in five patients (6.7%). Of 75 patients, 19 had sonograms revealing gallbladder wall thickening (>4 mm); surgical preparation difficulties in 16 of these patients led to laparotomy in four patients. Sensitivity, specificity, positive predictive value, and accuracy of wall thickening as an indicator of technical difficulties were 66.7%, 94.1%, 84.2%, and 85.3%, respectively. Sensitivity, specificity, positive predictive value, and accuracy of wall thickening as an indicator of surgical conversion were 80.0%, 78.6%, 21.1%, and 78.7%, respectively. Technical difficulties at laparoscopy occurred in all five patients with pericholecystic fluid on sonography (sensitivity, 20.8%; specificity, 100%; positive predictive value, 100%; accuracy, 74.7%) and led to laparotomy in three patients (sensitivity 60.0%, specificity 97.1%, positive predictive value 60%, accuracy 94.7%). The accuracy of sonography for cholecystolithiasis was 100%.
On sonography, gallbladder wall thickening is the most sensitive indicator and pericholecystic fluid is the most specific indicator of technical difficulties during laparoscopic cholecystectomy. Such difficulties may require conversion to laparotomy.
Little is known about substitutions that mediate resistance of hepatitis C virus (HCV) to direct-acting antivirals (DAAs), due to the small number of patients with treatment failure in approval ...studies. It is important to identify resistance patterns to select effective salvage treatments.
We performed a comprehensive analysis for resistance-associated substitutions (RASs) in HCV genes (nonstructural protein NS3, NS5A, NS5B) targeted by DAAs. We compared NS3, NS5A, and NS5B sequences from 626 patients in Europe with DAA failure with sequences from 2322 DAA-naïve patients, infected with HCV genotypes 1 to 4. We considered RASs to be relevant if they were associated with DAA failure in patients or conferred a greater than twofold change in susceptibility compared with a reference strain in in vitro replicon assays. Data were collected on pretreatment status, DAA regimen, the treatment initiation date and duration, and virologic response. Patients who received at least 4 weeks of antiviral treatment were included in the analysis.
RASs in NS3 associated with simeprevir or paritaprevir failure include R155K and D168E/V. In addition, several RASs were specifically associated with failure of simeprevir (Q80K/R in patients with genotype 1a or 4) or paritaprevir (Y56H in combination with D168V in patients with genotype 1b). Y93H in NS5A was the RAS most frequently associated with failure of daclatasvir, ledipasvir, or ombitasvir in patients with genotype 1b infection, and L31M was associated with failure of daclatasvir or ledipasvir, but not ombitasvir. RASs in NS5A were heterogeneous among patients with HCV genotype 1a or genotype 4 infections. In patients with HCV genotype 3, Y93H was associated with resistance to daclatasvir, but no RASs were associated with ledipasvir failure, pointing to a limited efficacy of ledipasvir in patients with genotype 3. Among patients failed by sofosbuvir-containing regimens, L159F was enriched in patients with genotype 1b (together with C316N) or genotype 3 infection, whereas the RAS S282T was rarely observed.
We compared RASs in NS3, NS5A, and NS5B among patients failed by DAA therapy. Theses varied with the HCV genotype and subtype, and the different drug classes. These findings might be used to select salvage therapies.
ABSTRACT
We report near-infrared long-baseline interferometric observations of the Hyades multiple system HD 284163, made with the Center for High Angular Resolution Astronomy array, as well as ...almost 43 yr of high-resolution spectroscopic monitoring at the Center for Astrophysics. Both types of observations resolve the 2.39 d inner binary, and also an outer companion in a 43.1 yr orbit. Our observations, combined with others from the literature, allow us to solve for the 3D inner and outer orbits, which are found to be at nearly right angles to each other. We determine the dynamical masses of the three stars (good to better than 1.4 per cent for the inner pair), as well as the orbital parallax. The secondary component (0.5245 ± 0.0047 M⊙) is now the lowest mass star with a dynamical mass measurement in the cluster. A comparison of these measurements with current stellar evolution models for the age and metallicity of the Hyades shows good agreement. All three stars display significant levels of chromospheric activity, consistent with the classification of HD 284163 as an RS CVn object. We present evidence that a more distant fourth star is physically associated, making this a hierarchical quadruple system.
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