Mechanism of Action of Ca2+ Sensitizers—Update 2001

Ca²⁺ sensitizers act on the central mechanism (Ca²⁺ binding affinity of troponin C) and/or downstream mechanisms (thin filament regulation of actin and direct action on crossbridge cycling) of cardiac E–C coupling. Ca²⁺ sensitizers have mechanistic and energetic advantages over the agents that act through the upstream mechanism (intracellular Ca²⁺ mobilization). Ca²⁺ sensitizers and the agents that act through cyclic AMP-mediated signaling process have been postulated to belong to different classes, however, recent experimental findings revealed that certain Ca²⁺ sensitizers, such as levosimendan, OR 1896 and UD-CG 212 Cl, require cyclic AMP-mediated signaling for induction of the Ca²⁺ sensitizing effect. No clinically available agents act primarily via Ca²⁺ sensitization, but the positive inotropic effect of pimobendan and levosimendan is partly due to an increase in myofilament Ca²⁺ sensitivity. These agents are the hybrid of Ca²⁺ sensitizer and PDE III inhibitor. The extent of contribution of Ca²⁺ sensitizing effect of these agents to the clinical effectiveness to improve the hemodynamics in patients with heart failure is uncertain. Nevertheless pieces of evidence have been accumulating that these agents with Ca²⁺ sensitizing effect are clinically more effective than the agents that act purely via the upstream mechanism.     

Elevated level of circulating CD4+Helios+FoxP3+ cells in primary Sjogren’s syndrome patients

Objective: This study was designed to investigate the expression of regulatory T cells in primary Sjögren’s Syndrome (pSS) and to evaluate the clinical role of CD4?⁺?Helios⁺?FoxP3⁺ cells in pSS patients.

Methods: CD4?⁺?FoxP3^+?T cells in the peripheral blood of 39 pSS patients and 30 healthy controls were measured by flow cytometry and CD25 and Helios expression were also analyzed. The repression ability of CD4?⁺?CD25^hi cells was tested in vitro. Clinical information of pSS patients was retrospectively collected and their correlations with circulating Treg cells were analyzed. Cytokine levels in plasma were measured by ELISA and correlations with Helios⁺?FoxP3^+?cells were also detected.

Results: Circulating FoxP3^+?and Helios⁺?FoxP3^+?cells were elevated in pSS patients compared with controls. The suppression function of CD4?⁺?CD25^hi cells is not different between two groups. There are inverse correlations between Helios⁺?FoxP3^+?percentage and ESR, IgG, IgM and ESSDAI. Anti-SSB^? patients possess higher level of Helios⁺?FoxP3^+?cells than anti-SSB^+?patients. IL-6, IFNγ and IFNα levels were increased in pSS plasma and there were positive correlations between the levels of IFNγ/IFNα and percentage of Helios⁺?FoxP3^+?cells.

Conclusion: Circulating Helios ⁺?FoxP3⁺ cells were elevated in pSS patients and may contribute to suppressing autoimmunity in pSS patients.     

Reduced sarcoplasmic reticulum Ca2+ uptake and increased Na+–Ca2+ exchanger expression in left ventricle myocardium of dogs with progression of heart failure

Alteration of intracellular Ca²⁺ homeostasis in failing cardiomyocytes is associated with changes in regulatory proteins located in the sarcoplasmic reticulum (SR) and sarcolemma, which participate in Ca²⁺ fluxes across the membrane during the cardiac cycle. These regulatory proteins include Ca²⁺-ATPase (SERCA 2A), phospholamban (PLB), ryanodine-sensitive Ca²⁺ release channels (RR), and the sarcolemmal Na⁺–Ca²⁺ exchanger (NCX). Although their status is known in failed myocardium, it is poorly understood during the progression of heart failure (HF), particularly in large animals. We studied the left ventricular (LV) myocardium of six dogs with moderate HF and six with severe HF produced by multiple intracoronary microembolizations, compared with six normal dogs (NL). Oxalate-dependent SR Ca²⁺ uptake and expression of SERCA 2A, PLB, phosphorylated PLB at serine 16 (PLB-Ser) and threonine 17 (PLB-Thr), RR, and NCX were determined. Percent LV ejection fraction declined by 47% compared with NL (34.1% ± 1% vs 64% ± 2%) in dogs with moderate HF (HF-2W) 2 weeks after the last embolization and by 42% (20.5% ± 1% vs 34.1% ± 1%) in dogs with severe HF (HF-4M) at 4 months compared with HF-2W. Left ventricular pressure during isovolumic contraction (+dP/dt, mmHg/s) and relaxation (–dP/dt, mmHg/s) was significantly reduced in severe compared with moderate HF. Oxalate-dependent SR Ca²⁺ uptake (nmol ⁴⁵Ca²⁺ accumulated/min per milligram noncollagen protein) declined by 25% (21.3 ± 1 vs 28.5 ± 2) in HF-2W and 49% in HF-4M. Protein expression of SERCA 2A and PLB decreased by 67% and 35%, respectively, in HF-2W compared with NL, whereas SERCA 2A expression increased by 167% and PLB decreased by 40% in HF-4M compared with HF-2W. However, SERCA 2A protein was still significantly lower in HF-4M compared with NL. PLB-Ser and PLB-Thr increased significantly in HF-2W but decreased in HF-4M compared with NL. Similar changes in mRNA encoding PLB and SERCA 2A were observed in dogs with moderate and severe HF. The RR protein level declined in dogs with moderate and severe HF, whereas NCX protein did not change with moderate HF but increased with sever HF. These results suggest that the regulatory proteins responsible for Ca²⁺ uptake, Ca²⁺ release, and Na⁺–Ca²⁺ exchange are critically associated with the deterioration of LV function during the progression of HF.     

Involvement of actin filament in the generation of Ca2+ mobilizing messengers in glucose-induced Ca2+ signaling in pancreatic β-cells

Glucose is a metabolic regulator of insulin secretion from pancreatic β-cells, which is regulated by intracellular Ca²⁺ signaling. We and others previously demonstrated that glucose activates CD38/ADP-ribosyl cyclase (ADPR-cyclase) to produce two Ca²⁺ second messengers, cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP). Although F-actin remodeling is known to be an important step in glucose stimulated insulin secretion, the role of actin cytoskeleton in regulating Ca²⁺ signaling in pancreatic β-cells remain to be solved. Here, we show that actin filaments are involved in the activation of CD38/ADPR-cyclase in pancreatic β-cells. Glucose induces a sequential formation of cADPR and NAADP. Pretreatment with jasplakinolide, an actin polymerizing agent, or a myosin heavy chain IIA (MHCIIA) blocker, blebbistatin, inhibited glucose-induced CD38 internalization, an essential step for cADPR formation. Blocking actin disassembly with jasplakinolide also abrogates glucose-induced cADPR and NAADP formation and sustained Ca²⁺ signals. These results indicate that actin filaments along with MHCIIA play an important role in CD38 internalization for the generation of Ca²⁺ mobilizing messengers for glucose-induced Ca²⁺ signaling in pancreatic β-cells.     

Involvement of actin filament in the generation of Ca2+ mobilizing messengers in glucose-induced Ca2+ signaling in pancreatic β-cells

Glucose is a metabolic regulator of insulin secretion from pancreatic β-cells, which is regulated by intracellular Ca(2+) signaling. We and others previously demonstrated that glucose activates CD38/ADP-ribosyl cyclase (ADPR-cyclase) to produce two Ca(2+) second messengers, cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP). Although F-actin remodeling is known to be an important step in glucose stimulated insulin secretion, the role of actin cytoskeleton in regulating Ca(2+) signaling in pancreatic β-cells remain to be solved. Here, we show that actin filaments are involved in the activation of CD38/ADPR-cyclase in pancreatic β-cells. Glucose induces a sequential formation of cADPR and NAADP. Pretreatment with jasplakinolide, an actin polymerizing agent, or a myosin heavy chain IIA (MHCIIA) blocker, blebbistatin, inhibited glucose-induced CD38 internalization, an essential step for cADPR formation. Blocking actin disassembly with jasplakinolide also abrogates glucose-induced cADPR and NAADP formation and sustained Ca(2+) signals. These results indicate that actin filaments along with MHCIIA play an important role in CD38 internalization for the generation of Ca(2+) mobilizing messengers for glucose-induced Ca(2+) signaling in pancreatic β-cells.     

Decreased number of granzyme B+ activated CD8+ cytotoxic T lymphocytes in the inflammatory background of HIV-associated Hodgkin’s lymphoma

This study aimed to assess the differences in the cellular composition of the inflammatory reactive background around tumoral cells of classical Hodgkins lymphomas (cHL) inside and outside the HIV settings. This retrospective study evaluates the infiltrating T lymphocytes (CD4 and CD8), natural killer cells (CD57⁺ cells), and more especially cytotoxic cells [granzyme B (GrB) and TIA-1⁺ cells] in the background of 99 EBV⁺ cHL. Sections from paraffin-embedded tumor samples from nine HIV-infected cHL patients were immunostained, using standard immunohistochemical protocols and were compared to a control group of 90 HIV-noninfected cHL patients. Our clinical and histological data indicate that HIV-infected cHL patients present a higher frequency of mixed cellularity (MC) histological subtypes, more advanced disease stages, a poor response to treatment, and a poor overall survival compared to control patients. In controls, CD4/CD8 and GrB/TIA-1 ratios were determined as 2:1 and 1:2, respectively. The inflammatory infiltrate of HIV-infected patients had a significant reduction of CD4⁺ T lymphocytes (CD4/CD8 ratio 1:23), a decrease in infiltrating GrB⁺ cells (activated cytotoxic cells) and an increase in infiltrating TIA⁺ T cells (mainly nonactivated cytotoxic cells) in these patients (GrB/TIA-1 ratio 1:12). In conclusion, this study highlights an important intratumoral loss of CD4⁺ T cells (striking inversion in the CD4/CD8 ratio) and a decrease in intratumoral activated cytotoxic T lymphocytes in HIV-associated cHL patients. Further studies are required to confirm these results and to determine the role of these findings on the antitumoral immune response observed in HIV-associated cHL.     

Inhibition of Na+–H+ exchange before resuscitation following hemorrhagic shock is cardioprotective in rats

Background

Stimulation of the Na⁺–H⁺ exchanger during resuscitation following hemorrhagic shock results in myocardial injury and dysfunction. Inhibition of the Na⁺–H⁺ exchanger appears to be a new pharmacological tool for myocardial protection following ischemia–reperfusion. Our lab showed that inhibition of the Na⁺–H⁺ exchanger, using amiloride and dimethyl amiloride, before ex vivo resuscitation of isolated perfused hearts protected the myocardium and improved the post-resuscitation myocardial function. The purpose of the present study was to examine the myocardial protective effects of treating the hemorrhagic shocked rats by intra-arterial injection of 20 μM dimethyl amiloride (DMA), a specific Na⁺–H⁺ exchanger blocker, before in vivo resuscitation.

Methods

Sprague–Dawley rats were assigned to hemorrhagic treated or untreated groups (n = 4 per group). After 60 min of hemorrhagic shock, rats were treated or not by injection of 20 μM 5-(N,N-dimethyl)-amiloride (DMA) intra-arterially. Rats were then resuscitated in vivo and monitored for 30 min. Then hearts were harvested and perfused in the Langendorff system for 60 min for measurements of hemodynamic function.

Results

Administration of DMA before in vivo resuscitation following 60 min of hemorrhagic shock and 30 min of in vivo resuscitation, 20 μM DMA intra-arterially significantly improved post-resuscitation myocardial function.

Conclusion

Our results suggest that DMA protects the heart against post-resuscitation myocardial injury.     

Dofetilide Enhances the Contractility of Rat Ventricular Myocytes via Augmentation of Na+–Ca2+ Exchange

Purpose  Dofetilide (DOF), a novel Class III antiarrhythmic drug, prolongs the action potential duration (APD) and shows a positive inotropic effect in guinea pig papillary muscle. The present experiments were designed to study the positive inotropic effect of DOF on rat ventricle and explore its possible mechanism(s). Methods  Hearts from male Wistar rats (260–320 g) were divided into five groups and perfused in Langendorff mode. Ventricular myocytes were enzymatically isolated from male Wistar rats. Whole-cell voltage-clamping technique was used to test the Na⁺–Ca²⁺ exchange (NCE) current (I _NCX); Calcium transients and cell shortening provoked by field stimulation or using calcium current command waveform were observed synchronously with an ionic imaging system. Results  DOF (0.03–1.0 μM) increased left ventricular function in isolated rat hearts in a concentration-dependent manner. DOF (0.03–1.0 μM) also concentration-dependently increased both inward and outward I _NCX in isolated rat ventricular cells. The EC₅₀ values of DOF were 0.149 μM for the inward I _NCX and 0.249 μM for outward I _NCX, respectively. DOF 0.2 μM significantly enhanced Ca²⁺ transient and cell shortening in single rat ventricular myocytes driven by field electric stimulation. When the patch clamp system was connected to the ionic imaging system, Ca²⁺ current (I _Ca), Ca²⁺ transient and cell shortening amplitude in a same cell were recorded synchronously. Application of DOF 0.2 μM had no effect on I _Ca, but significantly increased Ca²⁺ transient and cell shortening. NCX inhibitor KB-R7943 0.6 μM significantly depressed the effects of DOF on Ca²⁺ transient and cell shortening. Conclusions  We conclude that DOF enhanced contractility of rat ventricular myocytes. The enhancement of NCE may be involved in the positive inotropic action of DOF.     

Quantification of lymph node transit times reveals differences in antigen surveillance strategies of na?ve CD4+ and CD8+ T cells

Naïve T cells continually recirculate between blood and secondary lymphoid organs, scanning dendritic cells (DC) for foreign antigen. Despite its importance for understanding how adaptive immune responses are efficiently initiated from rare precursors, a detailed quantitative analysis of this fundamental process has not been reported. Here we measure lymph node (LN) entry, transit, and exit rates for naïve CD4⁺ and CD8⁺ T cells, then use intravital imaging and mathematical modeling to relate cell–cell interaction dynamics to population behavior. Our studies reveal marked differences between CD4⁺ vs. CD8⁺ T cells. CD4⁺ T cells recirculate more rapidly, homing to LNs more efficiently, traversing LNs twice as quickly, and spending ∼1/3 of their transit time interacting with MHCII on DC. In contrast, adoptively transferred CD8⁺ T cells enter and leave the LN more slowly, with a transit time unaffected by the absence of MHCI molecules on host cells. Together, these data reveal an unexpectedly asymmetric role for MHC interactions in controlling CD4⁺ vs. CD8⁺ T lymphocyte recirculation, as well as distinct contributions of T cell receptor (TCR)-independent factors to the LN transit time, exposing the divergent surveillance strategies used by the two lymphocyte populations in scanning for foreign antigen.     

Flow Cytometric Analysis of Expression of Transforming Growth Factor-β and Glucocorticoid-Induced Tumor Necrosis Factor Receptor on CD4+ CD25+ T Cells of Patients with Inflammatory Bowel Disease

To determine whether human CD4+CD25+ cells express glucocorticoid-induced tumor necrosis factor receptor (GITR) and transforming growth factor-β (TGF-β) and the difference in CD4+CD25+ cells between patients with inflammatory bowel diseases and healthy subjects, peripheral blood lymphocytes were obtained from patients with ulcerative colitis (UC; n = 50), Crohn's disease (CD; n = 49), and healthy volunteers (control; n = 50) and flow cytometric analysis was performed. In control subjects, the expression of GITR on CD4+CD25+ cells (41.8 ± 10.5%) was significantly higher than on CD4+CD25– cells (11.1 ± 7.4%). Similarly, TGF-β expression on CD4+CD25+ cells (5.3 ± 4.6%) was higher than on CD4+CD25– cells (1.2 ± 1.4%). There were no significant differences among UC, CD, and control in CD4+CD25+/CD4+ ratio. However, there was a significant difference in the CD4+CD25+TGF-β+/CD4+CD25+ ratio between active UC and inactive UC (2.7 ± 2.6 and 7.2 ± 3.9%, respectively). The results suggest that TGF-β is involved in the induction or sustained remission of UC.     

Preassociated apocalmodulin mediates Ca2+-dependent sensitization of activation and inactivation of TMEM16A/16B Ca2+-gated Cl? channels

Ca²⁺-activated chloride currents carried via transmembrane proteins TMEM16A and TMEM16B regulate diverse processes including mucus secretion, neuronal excitability, smooth muscle contraction, olfactory signal transduction, and cell proliferation. Understanding how TMEM16A/16B are regulated by Ca²⁺ is critical for defining their (patho)/physiological roles and for rationally targeting them therapeutically. Here, using a bioengineering approach—channel inactivation induced by membrane-tethering of an associated protein (ChIMP)—we discovered that Ca²⁺-free calmodulin (apoCaM) is preassociated with TMEM16A/16B channel complexes. The resident apoCaM mediates two distinct Ca²⁺-dependent effects on TMEM16A, as revealed by expression of dominant-negative CaM₁₂₃₄. These effects are Ca²⁺-dependent sensitization of activation (CDSA) and Ca²⁺-dependent inactivation (CDI). CDI and CDSA are independently mediated by the N and C lobes of CaM, respectively. TMEM16A alternative splicing provides a mechanism for tuning apoCaM effects. Channels lacking splice segment b selectively lost CDI, and segment a is necessary for apoCaM preassociation with TMEM16A. The results reveal multidimensional regulation of TMEM16A/16B by preassociated apoCaM and introduce ChIMP as a versatile tool to probe the macromolecular complex and function of Ca²⁺-activated chloride channels.Calcium (Ca²⁺)-activated chloride (Cl^–) channels (CaCCs) broadly expressed in mammalian cells regulate diverse physiological functions including: epithelial mucus secretion (1, 2), neuronal excitability (3–5), smooth muscle contraction (6), olfactory transduction (7, 8), and cell proliferation (9, 10). Drugs targeting CaCCs are being pursued as therapies for hypertension, cystic fibrosis, asthma, and cancer (1, 9, 11).Three laboratories independently identified the transmembrane protein TMEM16A as the molecular component of a CaCC (12–14). TMEM16A belongs to a protein family with 10 members encoded by distinct genes (15–18). There is universal agreement that TMEM16A, and the closely related TMEM16B, are bona fide CaCCs (2, 12–14, 19). Consistent with this, TMEM16A knockout mice displayed defective CaCC activity in a variety of epithelia (20–22), and the olfactory CaCC current was completely abolished in TMEM16B knockout mice (23). Hydropathy analyses suggest TMEM16 proteins have a similar topology with cytosolic N and C termini and eight predicted transmembrane helices (2, 19). Human TMEM16A has four alternatively spliced segments (a–d), differential inclusion of which modify voltage and Ca²⁺ sensitivity of resultant channel splice variants (24).CaCCs are highly sensitive to intracellular [Ca²⁺], displaying graded increases in Cl⁻ current (I_Cl) amplitude as [Ca²⁺]_i is raised from resting levels (∼100 nM) to the 1- to 2-μM range. In some cases, high [Ca²⁺]_i (>10 μM) leads to decreased I_Cl amplitude (inactivation) (25–27). The Ca²⁺ sensor(s) for Ca²⁺-dependent activation and inactivation (CDA and CDI) of TMEM16A/16B is unknown. There are two possible nonexclusive mechanisms: (i) direct Ca²⁺ binding to the channel or (ii) Ca²⁺ binding through a separate Ca²⁺-sensing protein. The TMEM16A sequence does not reveal any canonical Ca²⁺-binding EF hand motifs (14, 16, 17). A sequence in the first intracellular loop of TMEM16A resembling the “Ca²⁺ bowl” in large conductance Ca²⁺-activated K⁺ (BK) channels was disqualified by mutagenesis as the Ca²⁺ sensor responsible for CDA of TMEM16A (28). A revised TMEM16A topological model suggests the originally predicted extracellular loop 4 is located intracellularly (29), and mutating E702 and E705 within this loop markedly alter Ca²⁺ sensitivity of TMEM16A (29, 30).Some reports have suggested involvement of calmodulin (CaM) in distinct aspects of Ca²⁺-dependent regulation of CaCCs. Tian et al. reported that inhibiting CaM with trifluoperazine or J-8 markedly suppressed CDA of TMEM16A(abc) in HEK293 cells, and mapped the CaM binding site to splice segment b (31). They concluded that CaM is essential for TMEM16A activation. However, this suggestion is contradicted by the robust CDA of TMEM16A(ac), a splice variant lacking the putative CaM binding site on splice segment b (2, 24). Recently, Ca²⁺–CaM was found to bind TMEM16A(ac) in a Ca²⁺-dependent manner and result in an increased permeability of the channel to HCO₃⁻ (32). Deleting the Ca²⁺–CaM binding site did not affect CDA of TMEM16A(ac). Ca²⁺–CaM regulation of TMEM16A HCO₃⁻ permeability conforms to a traditional signaling mode where Ca²⁺ binds to freely diffusing CaM to form a Ca²⁺–CaM complex that then interacts with a target protein.There are several examples of an alternative mode of CaM signaling in which Ca²⁺-free CaM (apoCaM) is preassociated with target proteins under resting [Ca²⁺]_i conditions and acts as a resident Ca²⁺ sensor to regulate function of the host protein in response to increased [Ca²⁺]_i (33). This mode of CaM signaling is used as the activating mechanism for small conductance K⁺ channels (34) and Ca²⁺-dependent regulation of high voltage-gated Ca²⁺ (Ca_V1 and Ca_V2) channels (35, 36). A distinguishing feature of this mode of CaM signaling is that it is impervious to pharmacological inhibitors of Ca²⁺–CaM, but can be eliminated in dominant negative fashion by a CaM mutant, CaM₁₂₃₄, in which all four EF hands have been mutated so they no longer bind Ca²⁺ (34, 36). Whether apocalmodulin preassociates with TMEM16A/TMEM16B channel complexes and participates in Ca²⁺-dependent regulation of these channels is controversial (30, 37, 38).Using a recently developed bioengineering approach—channel inactivation induced by membrane-tethering of an associated protein (ChIMP) (39)—we discovered that apoCaM is functionally preassociated with TMEM16A/16B channel complexes. Whereas the resident apoCaM is not necessary for CDA, it does mediate two distinct Ca²⁺-dependent processes in TMEM16A. First, it causes a leftward shift in the Ca²⁺-dependent activation curve at [Ca²⁺]_i ≤ 1 μM, an effect we term Ca²⁺-dependent “sensitization” of activation (CDSA). Second, it is the Ca²⁺ sensor for CDI observed at [Ca²⁺]_i > 10 μM. The two opposite effects are independently mediated by the two lobes of preassociated apoCaM— Ca²⁺ occupancy of the N lobe leads to CDI, whereas the C lobe mediates CDSA. Alternative splicing of TMEM16A provides a mechanism for regulating apoCaM binding and signaling. TMEM16A splice variants lacking segment a lost apoCaM binding altogether, eliminating both CDSA and CDI, whereas variants specifically lacking segment b were selectively deficient in CDI. Finally, TMEM16A variants defective in apoCaM binding displayed dramatically decreased trafficking to the cell surface.     

Elevation of CD16+CD56+ NK-cells and down-regulation of serum interleukin-21 (IL-21) and IL-1α after splenectomy in relapsed hemophagocytic lymphohistiocytosis of unknown cause

Objectives: Encouraging progress has been made in application of splenectomy in the treatment of relapsed hemophagocytic lymphohistiocytosis (HLH) of unknown cause. The aim was to determine the roles of lymphocyte subpopulations and inflammatory cytokines in splenectomy.

Methods: We retrospectively analyzed changes in lymphocyte subpopulations and levels of inflammatory cytokines at different time-points before and after splenectomy in the patients with relapsed HLH of unknown cause, as well as the correlations between these changes and the disease prognosis.

Results: During the period from June 2006 to June 2016, we enrolled 107 patients with relapsed HLH of unknown cause, of whom 29 were treated with splenectomy. Among the 29 patients, 7 cases were non-Hodgkin lymphomas based on spleen pathology, 1 case withdrew and the remaining 21 non-lymphoma cases were available for analysis. Results showed a significant increase in both percentage of CD16⁺CD56⁺ NK cells (P?=?0.003) and NK cell activity (P?=?0.028) at 24?wk after splenectomy compared to their baseline pre-surgery levels. We also examined seven patients for the changes in cytokine levels before and after splenectomy and found that IL-21 and IL-1α decreased at 4?wk after splenectomy (P?P?=?0.001) compared to the 24 patients with relapsed HLH of unknown cause who were also determined as NR but not treated by splenectomy.

Discussion: Splenectomy can improve clinical symptoms and survival of patients with relapsed HLH of unknown cause. The mechanism is likely related to the changes in percent NK cells and cytokines (IL-21 and IL-1α) after surgery.     

Effect of gold therapy on CD5+ B-cells and TCR γδ + T-cells in patients with rheumatoid arthritis

Summary Circulating CD5⁺ B-cell levels in 15 patients with rheumatoid arthritis (RA) not receiving remittive therapy was significantly increased when compared to 17 normal controls (mean±SE: RA, 19.7±2.85%; controls, 11.6±1.67%; P<0.02). In contrast, 24 patients with RA receiving gold sodium thiomalate therapy (GST) had similar CD5⁺ B-cell levels (11.88±1.65) when compared to controls and significantly reduced levels when compared to the RA group not receiving remittive agents (P<0.01). Furthermore, TCR ⁺ T-cell levels were also assessed in these patients groups. These values were not significantly different between any of the groups (controls, 4.46±1.36%; GST, 6.88±1.73%; RA, 2.73±0.55%), although 42% of the GST treated group had ⁺ T-cell levels higher than the entire untreated RA group. No correlation was observed between the levels of TCR ⁺ T-cells and CD5⁺ B-cells in any of these groups. These results suggested that therapy does influence the level of CD5⁺ B-cells and ⁺ T-cells in these patients.     

Optical detection of rate-determining ion-modulated conformational changes of the ether-à-go-go K+ channel voltage sensor

In voltage-dependent ether-à-go-go (eag) K+ channels, the process of activation is modulated by Mg2+ and other divalent cations, which bind to a site in the voltage sensor and slow channel opening. Previous analysis of eag ionic and gating currents indicated that Mg2+ has a much larger effect on ionic than gating current kinetics. From this, we hypothesized that ion binding modulates voltage sensor conformational changes that are poorly represented in gating current recordings. We have now tested this proposal by using a combined electrophysiological and optical approach. We find that a fluorescent probe attached near S4 in the voltage sensor reports on two phases of the activation process. One component of the optical signal corresponds to the main charge-moving conformational changes of the voltage sensor. This is the phase of activation that is well represented in gating current recordings. Another component of the optical signal reflects voltage sensor conformational changes that occur at more hyperpolarized potentials. These transitions, which are rate-determining for activation and highly modulated by Mg2+, have not been detected in gating current recordings. Our results demonstrate that the eag voltage sensor undergoes conformational changes that have gone undetected in electrical measurements. These transitions account for the time course of eag activation in the presence and absence of extracellular Mg2+.     

Expression of α-Defensins,CD20+ B-lymphocytes,and Intraepithelial CD3+ T-lymphocytes in the Intestinal Mucosa of Patients with Liver Cirrhosis: Emerging Mediators of Intestinal Barrier Function

Aim

The present study investigates the role of innate and adaptive immune system of intestinal mucosal barrier function in cirrhosis.

Methods

Forty patients with decompensated (n?=?40, group A), 27 with compensated cirrhosis (n?=?27, group B), and 27 controls (n?=?27, group C) were subjected to duodenal biopsy. Expression of α-defensins 5 and 6 at the intestinal crypts was evaluated by immunohistochemistry and immunofluorescence. Serum endotoxin, intestinal T-intraepithelial, and lamina propria B-lymphocytes were quantified.

Results

Cirrhotic patients presented higher endotoxin concentrations (p?<?0.0001) and diminished HD5 and HD6 expression compared to healthy controls (p?=?0.000287, p?=?0.000314, respectively). The diminished HD5 and HD6 expressions were also apparent among the decompensated patients compared to compensated group (p?=?0.025, p?=?0.041, respectively). HD5 and HD6 expressions were correlated with endotoxin levels (r?=?-0.790, p?<?0.0001, r?=???0.777, p?<?0.0001, respectively). Although intraepithelial T-lymphocytes were decreased in group A compared to group C (p?=?0.002), no notable alterations between groups B and C were observed. The B-lymphocytic infiltrate did not differ among the investigated groups.

Conclusions

These data demonstrate that decreased expression of antimicrobial peptides may be considered as a potential pathophysiological mechanism of intestinal barrier dysfunction in liver cirrhosis, while remodeling of gut-associated lymphoid tissue as an acquired immune response to bio-pathogens remains an open field to illuminate.

     

In vivo monitoring of intracellular Ca2+ dynamics in the pancreatic β-cells of zebrafish embryos

ABSTRACT

Assessing the response of pancreatic islet cells to glucose stimulation is important for understanding β-cell function. Zebrafish are a promising model for studies of metabolism in general, including stimulus-secretion coupling in the pancreas. We used transgenic zebrafish embryos expressing a genetically-encoded Ca²⁺ sensor in pancreatic β-cells to monitor a key step in glucose induced insulin secretion; the elevations of intracellular [Ca²⁺]_i. In vivo and ex vivo analyses of [Ca²⁺]_i demonstrate that β-cell responsiveness to glucose is well established in late embryogenesis and that embryonic β-cells also respond to free fatty acid and amino acid challenges. In vivo imaging of whole embryos further shows that indirect glucose administration, for example by yolk injection, results in a slow and asynchronous induction of β-cell [Ca²⁺]_i responses, while intravenous glucose injections cause immediate and islet-wide synchronized [Ca²⁺]_i fluctuations. Finally, we demonstrate that embryos with disrupted mutation of the Ca_V1.2 channel gene cacna1c are hyperglycemic and that this phenotype is associated with glucose-independent [Ca²⁺]_i fluctuation in β-cells. The data reveal a novel central role of cacna1c in β-cell specific stimulus-secretion coupling in zebrafish and demonstrate that the novel approach we propose – to monitor the [Ca²⁺]_i dynamics in embryonic β-cells in vivo – will help to expand the understanding of β-cell physiological functions in healthy and diseased states.     

Evaluation of T-cell receptor CD3+γδ in gestational diabetes mellitus

Few studies have shown a significant increase of D3⁺ T-cell receptor (TCR) γδ in the early phases of type 1 diabetes. We wished to determine if CD3⁺ TCR γδ is involved in the pathogenesis of gestational diabetes mellitus (GDM). We studied 29 GDM patients and 21 normal pregnant women. Lymphocyte subpopulations (CD3⁺ TCR αδ, CD3⁺ TCR γδ), islet cell antibodies (ICA), glutamic acid decarboxylase antibodies (GAD) and protein tyrosine phosphatase antibodies (IA₂-Ab) were evaluated in all patients. The percentage of CD3⁺ TCR γδ was significantly higher in GDM women than in the control group (5.1 ± 2.9% vs. 3.7 ± 1.7%, p < 0.05%). No abnormalities of the other lymphocyte subpopulations were found. All subjects were negative for ICA; 2 GDM patients wer positive for GAD, but no relationship was found between GAD positivity and CD3⁺γδ levels in these 2 patients. Further follow-up studies of these patients are required to verify if the CD3⁺ TCR γδ receptor is a useful marker for diabetes development. Received: 3 June 2000 / Accepted in revised form: 3 March 2001