Kinetic Mechanism of the Ca2+-Dependent Switch-On and Switch-Off of Cardiac Troponin in Myofibrils

The kinetics of Ca2+-dependent conformational changes of human cardiac troponin (cTn) were studied on isolated cTn and within the sarcomeric environment of myofibrils. Human cTnC was selectively labeled on cysteine 84 with N-((2-(iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-1,3-diazole and reconstituted with cTnI and cTnT to the cTn complex, which was incorporated into guinea pig cardiac myofibrils. These exchanged myofibrils, or the isolated cTn, were rapidly mixed in a stopped-flow apparatus with different [Ca2+] or the Ca2+-buffer 1,2-Bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid to determine the kinetics of the switch-on or switch-off, respectively, of cTn. Activation of myofibrils with high [Ca2+] (pCa 4.6) induced a biphasic fluorescence increase with rate constants of >2000 s−1 and ∼330 s−1, respectively. At low [Ca2+] (pCa 6.6), the slower rate was reduced to ∼25 s−1, but was still ∼50-fold higher than the rate constant of Ca2+-induced myofibrillar force development measured in a mechanical setup. Decreasing [Ca2+] from pCa 5.0–7.9 induced a fluorescence decay with a rate constant of 39 s−1, which was approximately fivefold faster than force relaxation. Modeling the data indicates two sequentially coupled conformational changes of cTnC in myofibrils: 1), rapid Ca2+-binding (kB ≈ 120 μM−1 s−1) and dissociation (kD ≈ 550 s−1); and 2), slower switch-on (kon = 390s−1) and switch-off (koff = 36s−1) kinetics. At high [Ca2+], ∼90% of cTnC is switched on. Both switch-on and switch-off kinetics of incorporated cTn were around fourfold faster than those of isolated cTn. In conclusion, the switch kinetics of cTn are sensitively changed by its structural integration in the sarcomere and directly rate-limit neither cardiac myofibrillar contraction nor relaxation

Streptococcus pneumoniae Serotype 1 Capsular Polysaccharide Induces CD8+CD28− Regulatory T Lymphocytes by TCR Crosslinking

Zwitterionic capsular polysaccharides (ZPS) of commensal bacteria are characterized by having both positive and negative charged substituents on each repeating unit of a highly repetitive structure that has an α-helix configuration. In this paper we look at the immune response of CD8+ T cells to ZPSs. Intraperitoneal application of the ZPS Sp1 from Streptococcus pneumoniae serotype 1 induces CD8+CD28− T cells in the spleen and peritoneal cavity of WT mice. However, chemically modified Sp1 (mSp1) without the positive charge and resembling common negatively charged polysaccharides fails to induce CD8+CD28− T lymphocytes. The Sp1-induced CD8+CD28− T lymphocytes are CD122lowCTLA-4+CD39+. They synthesize IL-10 and TGF-β. The Sp1-induced CD8+CD28− T cells exhibit immunosuppressive properties on CD4+ T cells in vivo and in vitro. Experimental approaches to elucidate the mechanism of CD8+ T cell activation by Sp1 demonstrate in a dimeric MHC class I-Ig model that Sp1 induces CD8+ T cell activation by enhancing crosslinking of TCR. The expansion of CD8+CD28− T cells is independent, of direct antigen-presenting cell/T cell contact and, to the specificity of the T cell receptor (TCR). In CD8+CD28− T cells, Sp1 enhances Zap-70 phosphorylation and increasingly involves NF-κB which ultimately results in protection versus apoptosis and cell death and promotes survival and accumulation of the CD8+CD28− population. This is the first description of a naturally occurring bacterial antigen that is able to induce suppressive CD8+CD28− T lymphocytes in vivo and in vitro. The underlying mechanism of CD8+ T cell activation appears to rely on enhanced TCR crosslinking. The data provides evidence that ZPS of commensal bacteria play an important role in peripheral tolerance mechanisms and the maintenance of the homeostasis of the immune system

Latest news on RA-ILD

Rheumatoid arthritis-associated interstitial lung disease (RA-ILD) is of high clinical relevance. It not only affects the quality of life but also makes a significant contribution to the mortality rate of patients with rheumatoid arthritis. RA-ILD can present with all known radiological and histopathological patterns seen in other interstitial pneumonias. Among these pneumonias, diffuse alveolar damage (DAD), followed by usual interstitial pneumonia (UIP) has the worst prognosis. In addition, acute exacerbation of RA-ILD, which can occur at any time during the disease, is highly lethal. An algorithm for the diagnosis and treatment of RA-ILD is pending and will be addressed in the following article. In addition to immunosuppressants and disease-modifying antirheumatic drugs (DMARD), antifibrotics have recently gained importance in the therapy of RA-ILD

Thiazide-Associated Hyponatremia, Report of the Hyponatremia Registry: An Observational Multicenter International Study

Background: Hyponatremia is a frequent and potentially life-threatening adverse side effect of thiazide diuretics. This sub-analysis of the Hyponatremia Registry database focuses on current management practices of thiazide-associated hyponatremia (TAH) and compares differences between TAH and syndrome of inappropriate antidiuretic hormone secretion (SIADH). Methods: We analyzed 477 patients from 225 US and EU sites with euvolemic hyponatremia ([Na+] <= 130 mEq/L) who were receiving a thiazide diuretic. Of these, 118 met criteria for true thiazide-induced hyponatremia (TIH). Results: Thiazide was withdrawn immediately after hyponatremia was diagnosed only in 57% of TAH; in these patients, the median rate of [Na+] change (. daily [Na+]) was significantly higher than those with continued thiazide treatment (3.8 [interquartile range: 4.0] vs. 1.7 [3.8] mEq/L/day). The most frequently employed therapies were isotonic saline (29.6%), fluid restriction (19.9%), the combination of these two (8.2%), and hypertonic saline (5.2%). Hypertonic saline produced the greatest. daily [Na+] (8.0[6.4] mEq/L/day) followed by a combination of fluid restriction and normal saline (4.5 [3.8] mEq/L/day) and normal saline alone (3.6 [3.5] mEq/L/day). Fluid restriction was markedly less effective (2.7 [2.7] mEq/L/day). Overly rapid correction of hyponatremia occurred in 3.1% overall, but in up to 21.4% given hypertonic saline. Although there are highly significant differences in the biochemical profiles between TIH and SIADH, no predictive diagnostic test could be derived. Conclusions: Despite its high incidence and potential risks, the management of TAH is often poor. Immediate withdrawal of the thiazide is crucial for treatment success. Hypertonic saline is most effective in correcting hyponatremia but associated with a high rate of overly rapid correction. We could not establish a diagnostic laboratory-based test to differentiate TIH from SIADH. (C) 2017 The Author(s) Published by S. Karger AG, Base

Euvolemic hyponatremia in cancer patients. Report of the Hyponatremia Registry: an observational multicenter international study

Hyponatremia secondary to SIADH is frequent in cancer patients and potentially deleterious. The aim of this sub-analysis of the Hyponatremia Registry database is to analyze current diagnostic and therapeutic management practices in cancer patients with SIADH. We analyzed 358 cancer patients who had serum sodium concentration ([Na+]) = 130 mEq/L. Overly rapid correction of hyponatremia occurred in 11.7%. Although essential for successful hyponatremia management, appropriate diagnostic testing is not routinely performed in current practice. The most frequently employed monotherapies were often ineffective and sometimes even aggravated hyponatremia. Tolvaptan was used less often but showed significantly greater effectiveness. Despite clear evidence that hyponatremia is associated with poor outcome in oncology patients, most patients were discharged still hyponatremic. Further studies are needed to assess the beneficial impact of hyponatremia correction with effective therapies

Preoperative Short-Term Calorie Restriction for Prevention of Acute Kidney Injury After Cardiac Surgery: A Randomized, Controlled, Open-Label, Pilot Trial

Background-Acute kidney injury is a frequent complication after cardiac surgery and is associated with adverse outcomes. Although short-term calorie restriction (CR) has proven protective in rodent models of acute kidney injury, similar effects have not yet been demonstrated in humans. Methods and Results-CR_KCH (Effect of a Preoperative Calorie Restriction on Renal Function After Cardiac Surgery) is a randomized controlled trial in patients scheduled for cardiac surgery. Patients were randomly assigned to receive either a formula diet containing 60% of the daily energy requirement (CR group) or adlibitum food (control group) for 7days before surgery. In total, 82 patients were enrolled between April 16, 2012, and February 5, 2015. There was no between-group difference in the primary end point of median serum creatinine increment after 24 hours (control group: 0.0 mg/dL [-0.1 - (+0.2) mg/dL]; CR group: 0.0 mg/dL [-0.2 - (+0.2) mg/dL]; P=0.39). CR prevented a rise in median creatinine at 48 hours (control group: +0.1 mg/dL [0.0 - 0.3 mg/dL]; CR group: -0.1 mg/dL [-0.2 - (+0.1) mg/dL]; P=0.03), with most pronounced effects observed in male patients and patients with a body mass index >25. This benefit persisted until discharge: Median creatinine decreased by 0.1 mg/dL (-0.2 - 0.0 mg/dL) in the CR group, whereas it increased by 0.1 mg/dL (0.0 - 0.3 mg/dL; P=0.0006) in the control group. Incidence of acute kidney injury was reduced by 5.8% (41.7% in the CR group compared with 47.5% in the control group). Safety-related events did not differ between groups. Conclusions-Despite disappointing results with respect to creatinine rise within the first 24 hours, the benefits observed at later time points and the subgroup analyses suggest the protective potential of short-term CR in patients at risk for acute kidney injury, warranting further investigation

Modulation of Endocannabinoids by Caloric Restriction Is Conserved in Mice but Is Not Required for Protection from Acute Kidney Injury

Acute kidney injury (AKI) is a frequent and critical complication in the clinical setting. In rodents, AKI can be effectively prevented through caloric restriction (CR), which has also been shown to increase lifespan in many species. In Caenorhabditis elegans (C. elegans), longevity studies revealed that a marked CR-induced reduction of endocannabinoids may be a key mechanism. Thus, we hypothesized that regulation of endocannabinoids, particularly arachidonoyl ethanolamide (AEA), might also play a role in CR-mediated protection from renal ischemia-reperfusion injury (IRI) in mammals including humans. In male C57Bl6J mice, CR significantly reduced renal IRI and led to a significant decrease of AEA. Supplementation of AEA to near-normal serum concentrations by repetitive intraperitoneal administration in CR mice, however, did not abrogate the protective effect of CR. We also analyzed serum samples taken before and after CR from patients of three different pilot trials of dietary interventions. In contrast to mice and C. elegans, we detected an increase of AEA. We conclude that endocannabinoid levels in mice are modulated by CR, but CR-mediated renal protection does not depend on this effect. Moreover, our results indicate that modulation of endocannabinoids by CR in humans may differ fundamentally from the effects in animal models

TUNEL stainings of mouse kidneys.

<p>A) Control kidney without IR damage. B) and C) Mouse kidneys 24 hours after IR. B) Control mouse on standard ad libitum chow and drinking water. C) Mouse treated with twice oral gavage of 250 μl of 10% GlcNAc solution.</p