Dual video microscopic imaging of membrane potential and cytosolic calcium of immunoidentified embryonic rat cortical cells

Membrane potential (MP) and cytosolic Ca2+ (Ca2+(c)) constitute important components involved in the physiological regulation of a myriad of cell functions in eukaryotic organisms. In particular, during development of the central nervous system, both properties are thought to be important in the regulation of cell cycle, cell migration, cell differentiation, cell-cell communication, and naturally occurring cell death. However, obtaining insight into the precise relationship between these two parameters of cell function is relatively limited either by technical difficulties inherent in using electrical recordings of membrane properties in conjunction with optical imaging of single cells or by employing optical imaging of either one or another property alone. Here, we describe in detail a novel strategy to record changes in both MP and Ca2+(c) from many intact single cells in a noninvasive manner using digital video microscopy. This method involves double-loading the cells with voltage- and calcium-sensitive fluorescent indicator dyes, green oxonol, and fura-2, which can be sequentially excited with a mercury arc lamp filtered at appropriate wavelengths and their resulting emissions can be captured with an intensified charged-coupled device camera at 1-s intervals. As an example of the utility of dual-recording strategy, we present data on a distinct functional expression of excitable membrane and cytoplasmic calcium properties in proliferating and differentiating embryonic rat cerebral cortical cells.     

Antibodies to citrullinated proteins and differences in clinical progression of rheumatoid arthritis

Antibodies to citrullinated proteins (anti-cyclic-citrullinated peptide [anti-CCP] antibodies) are highly specific for rheumatoid arthritis (RA) and precede the onset of disease symptoms, indicating a pathogenetic role for these antibodies in RA. We recently showed that distinct genetic risk factors are associated with either anti-CCP-positive disease or anti-CCP-negative disease. These data are important as they indicate that distinct pathogenic mechanisms are underlying anti-CCP-positive disease or anti-CCP-negative disease. Likewise, these observations raise the question of whether anti-CCP-positive RA and anti-CCP-negative RA are clinically different disease entities. We therefore investigated whether RA patients with anti-CCP antibodies have a different clinical presentation and disease course compared with patients without these autoantibodies. In a cohort of 454 incident patients with RA, 228 patients were anti-CCP-positive and 226 patients were anti-CCP-negative. The early symptoms, tender and swollen joint count, and C-reactive protein level at inclusion, as well as the swollen joint count and radiological destruction during 4 years of follow-up, were compared for the two groups. There were no differences in morning stiffness, type, location and distribution of early symptoms, patients' rated disease activity and C-reactive protein at inclusion between RA patients with and without anti-CCP antibodies. The mean tender and swollen joint count for the different joints at inclusion was similar. At follow-up, patients with anti-CCP antibodies had more swollen joints and more severe radiological destruction. Nevertheless, the distribution of affected joints, for swelling, bone erosions and joint space narrowing, was similar. In conclusion, the phenotype of RA patients with or without anti-CCP antibodies is similar with respect to clinical presentation but differs with respect to disease course.     

Cell volume kinetics of adherent epithelial cells measured by laser scanning reflection microscopy: determination of water permeability changes of renal principal cells

The water channel aquaporin-2 (AQP2), a key component of the antidiuretic machinery in the kidney, is rapidly regulated by the antidiuretic hormone vasopressin. The hormone exerts its action by inducing a translocation of AQP2 from intracellular vesicles to the cell membrane. This step requires the elevation of intracellular cyclic AMP. We describe here a new method, laser scanning reflection microscopy (LSRM), suitable for determining cellular osmotic water permeability coefficient changes in primary cultured inner medullary collecting duct (IMCD) cells. The recording of vertical-reflection-mode x-z-scan section areas of unstained, living IMCD cells proved useful and valid for the investigation of osmotic water permeability changes. The time-dependent increases of reflection-mode x-z-scan section areas of swelling cells were fitted to a single-exponential equation. The analysis of the time constants of these processes indicates a twofold increase in osmotic water permeability of IMCD cells after treatment of the cells both with forskolin, a cyclic AMP-elevating agent, and with Clostridium difficile toxin B, an inhibitor of Rho proteins that leads to depolymerization of F-actin-containing stress fibers. This indicates that both agents lead to the functional insertion of AQP2 into the cell membrane. Thus, we have established a new functional assay for the study of the regulation of the water permeability at the cellular level.     

Cost-effectiveness of abatacept,rituximab, and TNFi treatment after previous failure with TNFi treatment in rheumatoid arthritis: a pragmatic multi-centre randomised trial

IntroductionFor patients with rheumatoid arthritis (RA) whose treatment with a tumour necrosis factor inhibitor (TNFi) is failing, several biological treatment options are available. Often, another TNFi or a biological with another mode of action is prescribed. The objective of this study was to compare the effectiveness and cost-effectiveness of three biologic treatments with different modes of action in patients with RA whose TNFi therapy is failing.MethodsWe conducted a pragmatic, 1-year randomised trial in a multicentre setting. Patients with active RA despite previous TNFi treatment were randomised to receive abatacept, rituximab or a different TNFi. The primary outcome (Disease Activity Score in 28 joints) and the secondary outcomes (Health Assessment Questionnaire Disability Index and 36-item Short Form Health Survey scores) were analysed using linear mixed models. Cost-effectiveness was analysed on the basis of incremental net monetary benefit, which was based on quality-adjusted life-years (calculated using EQ-5D scores), and all medication expenditures consumed in 1 year. All analyses were also corrected for possible confounders.ResultsOf 144 randomised patients, 5 were excluded and 139 started taking abatacept (43 patients), rituximab (46 patients) or a different TNFi (50 patients). There were no significant differences between the three groups with respect to multiple measures of RA outcomes. However, our analysis revealed that rituximab therapy is significantly more cost-effective than both abatacept and TNFi over a willingness-to-pay range of 0 to 80,000 euros.ConclusionsAll three treatment options were similarly effective; however, when costs were factored into the treatment decision, rituximab was the best option available to patients whose first TNFi treatment failed. However, generalization of these costs to other countries should be undertaken carefully.

Trial registration

Netherlands Trial Register number NTR1605. Registered 24 December 2008.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-015-0630-5) contains supplementary material, which is available to authorized users.     

Elevated global SUMOylation in Ubc9 transgenic mice protects their brains against focal cerebral ischemic damage

We have previously shown that a massive increase in global SUMOylation occurs during torpor in ground squirrels, and that overexpression of Ubc9 and/or SUMO-1 in cell lines and cortical neurons protects against oxygen and glucose deprivation. To examine whether increased global SUMOylation protects against ischemic brain damage, we have generated transgenic mice in which Ubc9 is expressed strongly in all tissues under the chicken β-actin promoter. Ubc9 expression levels in 10 founder lines ranged from 2 to 30 times the endogenous level, and lines that expressed Ubc9 at modestly increased levels showed robust resistance to brain ischemia compared to wild type mice. The infarction size was inversely correlated with the Ubc9 expression levels for up to five times the endogenous level. Although further increases showed no additional benefit, the Ubc9 expression level was highly correlated with global SUMO-1 conjugation levels (and SUMO-2,3 levels to a lesser extent) up to a five-fold Ubc9 increase. Most importantly, there were striking reciprocal relationships between SUMO-1 (and SUMO-2,3) conjugation levels and cerebral infarction volumes among all tested animals, suggesting that the limit in cytoprotection by global SUMOylation remains undefined. These results support efforts to further augment global protein SUMOylation in brain ischemia.     

Lysosomal thiol reductase negatively regulates autophagy by altering glutathione synthesis and oxidation

Redox regulation is critical for a number of cellular functions and has been implicated in the etiology and progression of several diseases, such as cardiovascular diseases, neurodegenerative diseases, and cancer. It has been shown that, in the absence of gamma-interferon inducible lysosomal thiol reductase (GILT), cells are under increased oxidative stress with higher superoxide levels and decreased stability, expression, and function of mitochondrial manganese superoxide dismutase (SOD2). Here, we further elucidate the role of GILT in the homeostatic regulation of oxidative stress. We show that GILT-deficient fibroblasts exhibit reduced glutathione levels, shift in GSSG/GSH ratio toward the oxidized form, and accumulate dysfunctional mitochondria. Redox-sensitive pathways involving Erk1/2 activation and nuclear high mobility group box 1 (HMGB1) protein cytosolic translocation are both activated and associated with increased autophagy in GILT−/− fibroblasts. We hypothesize that these events are responsible for degrading the damaged mitochondria and mitochondrial SOD2 in the absence of GILT. This is the first time to our knowledge that a lysosomal enzyme has been implicated in global effects within the cell.     

Unexpected role for granzyme K in CD56bright NK cell-mediated immunoregulation of multiple sclerosis

Functional NK cell deficiencies are associated with autoimmune diseases, including multiple sclerosis. NK cells can promote or inhibit adaptive immunity via either cytokine production or cytotoxicity toward immature dendritic cells and activated T cells. In humans, this immunoregulatory role resides in the CD56(bright) NK cell subset, which is selectively expanded by daclizumab, a CD25-blocking Ab that suppresses multiple sclerosis-associated inflammation. The objective of this study was to investigate the molecular mechanisms underlying the cytotoxicity of NK cells toward activated T cells. We demonstrated that NK cells induce caspase-independent apoptosis that requires NK cell degranulation and causes mitochondrial dysfunction in activated T cells. Although both granzyme A and granzyme K (GrK) can mediate this form of apoptosis, quantitatively we observed preferential transfer of GrK to target cells. Consequently, gene silencing of GrK in the NK-92 cell line, which retains functional characteristics of CD56(bright) NK cells, profoundly inhibited the ability of NK-92 cells to kill activated syngeneic T cells. Finally, we demonstrated that daclizumab treatment significantly enhanced this newly defined mechanism of cytotoxicity by CD56(bright) NK cells. Our study describes the important physiological role that GrK plays in immunoregulation of adaptive immunity in humans and indicates that therapeutic exploitation of this pathway is beneficial in controlling autoimmunity.     

17beta-Estradiol supplementation reduces tubulointerstitial fibrosis by increasing MMP activity in the diabetic kidney

We previously reported that supplementation with 17beta-estradiol (E2) attenuates albuminuria, glomerulosclerosis, and tubulointerstitial fibrosis in diabetic nephropathy. The present study examined the mechanisms by which E2 regulates extracellular matrix (ECM) metabolism, a process that contributes to the development of glomerulosclerosis and tubulointerstitial fibrosis. The study was performed in female nondiabetic (ND), streptozotocin-induced diabetic (D), and diabetic with E2 supplementation (D+E2) Sprague-Dawley rats for 12 wk. Diabetes was associated with an increase in the renal expression of collagen alpha type IV [ND, 0.22 +/- 0.02; D, 0.99 +/- 0.09 relative optical density (ROD); P < 0.05] and fibronectin protein (ND, 0.36 +/- 0.08; D, 1.47 +/- 0.08 ROD; P < 0.05), as measured by Western blot analysis. E2 supplementation partially attenuated this increase in collagen alpha type IV (D+E2, 0.47 +/- 0.10 ROD) and fibronectin (D+E2, 0.71 +/- 0.16 ROD) protein expression associated with D. Diabetes was also associated with a decrease in the expression of matrix metalloproteinase (MMP) isoform MMP-2 (ND, 0.79 +/- 0.01; D, 0.62 +/- 0.06 ROD; P < 0.05) and MMP-9 protein (ND, 0.49 +/- 0.02; D, 0.33 +/- 0.03 ROD; P < 0.05). E2 supplementation restored MMP-2 and MMP-9 protein to levels similar or even greater than in the ND kidneys (MMP-2, 0.75 +/- 0.06; MMP-9, 0.73 +/- 0.01 ROD). The activities of MMP-2 (ND, 7.88 +/- 0.44; D, 5.60 +/- 0.54 ROD; P < 0.05) and MMP-9 (ND, 29.9 +/- 1.8; D, 12.9 +/- 2.3 ROD; P < 0.05), as measured by zymography, were also decreased with D. E2 supplementation restored MMP-2 and MMP-9 activity to levels similar to that in ND kidneys (MMP-2, 7.66 +/- 0.35; MMP-9, 21.4 +/- 2.9 ROD). We conclude that E2 supplementation is renoprotective by attenuating glomerulosclerosis and tubulointerstitial fibrosis by reducing ECM synthesis and increasing ECM degradation.