Cristina Arias

Origin and differentiation of dendritic cells

Despite extensive, recent research on the development of dendritic cells (DCs), their origin is a controversial issue in immunology, with important implications regarding their use in cancer immunotherapy. Although, under defined experimental conditions, DCs can be generated from myeloid or lymphoid precursors, the differentiation pathways that generate DCs in vivo remain unknown largely. Indeed, experimental results suggest that the in vivo differentiation of a particular DC subpopulation could be unrelated to its possible experimental generation. Nevertheless, the analysis of DC differentiation by in vivo and in vitro experimental systems could provide important insights into the control of the physiological development of DCs and constitutes the basis of a model of common DC differentiation that we propose.

Characterization of a common precursor population for dendritic cells

Dendritic cells (DCs) are essential for the establishment of immune responses against pathogens and tumour cells, and thus have great potential as tools for vaccination and cancer immunotherapy trials. Experimental evidence has led to a dual DC differentiation model, which involves the existence of both myeloid- and lymphoid-derived DCs. But this concept has been challenged by recent reports demonstrating that both CD8- and CD8+ DCs, considered in mice as archetypes of myeloid and lymphoid DCs respectively, can be generated from either lymphoid or myeloid progenitors. The issue of DC physiological derivation therefore remains an open question. Here we report the characterization of a DC-committed precursor population, which has the capacity to generate all the DC subpopulations present in mouse lymphoid organs—including CD8- and CD8+ DCs, as well as the B220+ DC subset—but which is devoid of myeloid or lymphoid differentiation potential. These data support an alternative model of DC development, in which there is an independent, common DC differentiation pathway.

Spironolactone and Its Main Metabolite, Canrenoic Acid, Block Human Ether-a-Go-Go-Related Gene Channels

Background—It has been demonstrated that spironolactone (SP) decreases the QT dispersion in chronic heart failure. In this study, the effects of SP and its metabolite, canrenoic acid (CA), on human ether-a-go-go–related gene (HERG) currents were analyzed. Methods and Results—HERG currents elicited in stably transfected Chinese hamster ovary cells were measured with the whole-cell patch-clamp technique. SP decreased HERG currents in a concentration-dependent manner (IC50=23.0±1.5 &mgr;mol/L) and shifted the midpoint of the activation curve to more negative potentials (Vh=−13.1±3.4 versus −18.9±3.6 mV, P <0.05) without modifying the activation and deactivation kinetics. SP-induced block (1 &mgr;mol/L) appeared at the range of membrane potentials coinciding with that of channel activation, and thereafter, it remained constant, reaching 24.7±3.8% at +60 mV (n=6, P <0.05). CA (0.01 nmol/L to 500 &mgr;mol/L) blocked HERG channels in a voltage- and frequency-independent manner. CA at 1 nmol/L shifted the midpoint of the activation curve to −19.9±1.8 mV and accelerated the time course of channel activation (&tgr;=1064±125 versus 820±93 ms, n=11, P <0.01). The envelope of the tail test demonstrated that at the very beginning of the pulses to +40 mV (25 ms), a certain amount of block was apparent (31.3±9.9%). CA did not modify the voltage-dependence of HERG channel inactivation (Vh=−60.8±5.6 versus −62.9±3.1 mV, n=6, P >0.05) or the kinetics of the reactivation process at any potential tested. CA and aldosterone also blocked the native IKr in guinea-pig ventricular myocytes. Conclusions—At concentrations reached after administration of therapeutic doses of SP, CA blocked the HERG channels by binding to both the closed and open states of the channel.

Effects of levobupivacaine, ropivacaine and bupivacaine on HERG channels: stereoselective bupivacaine block

Levobupivacaine and ropivacaine are the pure S(−) enantiomers of N‐butyl‐ and N‐propyl‐2′,6′‐pipecoloxylidide, developed as less cardiotoxic alternatives to bupivacaine. In the present study, we have analysed the effects of levobupivacaine, ropivacaine and bupivacaine on HERG channels stably expressed in CHO cells. The three drugs blocked HERG channels in a concentration‐, time‐ and state‐dependent manner. Block measured at the end of 5 s pulses to −10 mV induced by 20 μM bupivacaine (52.7±2.0%, n=15) and ropivacaine (55.5±2.7%, n=13) was similar (P>0.05) and both lower than that induced by levobupivacaine (67.5±4.2%, n=11) (P<0.05). Dextrobupivacaine (20 μM) was less potent (47.2±5.2%, n=10) than levobupivacaine (P<0.05), indicating stereoselective HERG channel block. Block induced by the three local anaesthetics exhibited a steep voltage dependence in the range of channel activation. In all cases, block measured at the maximum peak current at a test potential of 0 mV after promoting recovery from inactivation (I→O) was lower than that observed at the end of 5‐s pulses (I+O). Levobupivacaine, ropivacaine and bupivacaine accelerated HERG inactivation kinetics, slowed the recovery from inactivation and shifted the inactivation curve towards more negative membrane potentials. The three local anaesthetics induced a rapid time‐dependent decline after using a protocol that quickly activates HERG channels. All these results suggest that: (1) these drugs bind to the open and the inactivated states of HERG channels, (2) they stabilize HERG channels in the inactivated state, and (3) block induced by bupivacaine enantiomers is stereoselective.

Effects of propafenone and its main metabolite, 5-hydroxypropafenone, on HERG channels

OBJECTIVES Propafenone is a class Ic antiarrhythmic drug used to maintain sinus rhythm in patients with atrial fibrillation. During chronic therapy, it undergoes extensive first-pass hepatic metabolism to 5-hydroxypropafenone. In the present study we have analysed the effects of propafenone and 5-hydroxypropafenone on HERG current. METHODS The whole-cell configuration of the patch-clamp technique was used in CHO cells stably transfected with the gene encoding HERG channels. RESULTS Propafenone and 5-hydroxypropafenone (2 microM) inhibited HERG current by 78.7+/-2.3% (n=7) and 71.1+/-4.1% (n=7, P>0.05) when measured at the end of 5-s depolarizing pulses to -10 mV. Block measured at the maximum peak of tail currents recorded at -60 mV was similar for propafenone (78.3+/-2.0%, n=7, P>0.05) and higher for 5-hydroxypropafenone (79.3+/-1.5%, n=7, P<0.05). Propafenone and 5-hydroxypropafenone shifted the midpoint of the activation curve by -10.2+/-0.9 mV (n=7, P<0.01) and -7.4+/-1.1 mV (n=10, P<0.01), respectively. Both drugs accelerated the deactivation and the inactivation process of HERG current. Propafenone, but not 5-hydroxypropafenone, inhibited to a higher extent HERG current at the end of 5-s depolarizing pulses to 0 mV than after promoting the transition of HERG channels from the inactivated to the opened state. CONCLUSIONS These results indicate that propafenone and its main active metabolite, 5-hydroxypropafenone, block HERG channels to a similar extent by binding predominantly to the open state of the channel.

Kvβ1.3 Reduces the Degree of Stereoselective Bupivacaine Block of Kv1.5 Channels

Background:Kvβ1.3 subunit modifies the gating and the pharmacology of Kv1.5 channels, decreasing their sensitivity to block induced by drugs, suggesting that Kvβ1.3 competes with them for a binding site at Kv1.5 channels. Methods:Currents generated by the activation of Kv1.5 and Kv1.5 + Kvβ1.3 channels expressed in HEK293 cells and Xenopus oocytes were recorded by using whole cell patch clamp and voltage clamp techniques. Results:Block of Kv1.5, but not that produced on Kv1.5 + Kvβ1.3 channels, was voltage dependent. In both channels, bupivacaine block was time dependent. R(+)- and S(−)-bupivacaine blocked Kv1.5 with IC50 4.4 ± 0.5 &mgr;m (n = 15) and 39.8 ± 8.2 &mgr;m (n = 16; P < 0.05), respectively. These values increased fourfold for R(+)-bupivacaine (17.2 ± 2.2 &mgr;m) and twofold for S(−)-bupivacaine (71.9 ± 11.5 &mgr;m) in Kv1.5 + Kvβ1.3 channels. Therefore, the degree of stereoselectivity (&thgr;) decreased from 9 to 4 in the presence of Kvβ1.3. The decrease in potency to block Kv1.5 + Kvβ1.3 channels was the result of a less stable interaction between bupivacaine enantiomers and channels. Differences in stereoselectivity in each situation were due to a more favorable interaction between the channel and R(+)-bupivacaine. In the presence of Kvβ1.3, stereoselectivity was abolished for V514A mutant channels (involved in bupivacaine binding but not in Kvβ1.3 binding) but not for L510A (part of Kvβ1.3 binding site). Conclusions:The degree of stereoselective block of Kv1.5 decreases from 9 to 4 when Kvβ1.3 is present. L510 is determinant for the modulation of bupivacaine block, because it is the only residue of the S6 segment that binds to both bupivacaine and Kvβ1.3. These findings support an overlapping binding site for drugs and Kvβ1.3.

corrigendum: Characterization of a common precursor population for dendritic cells

This corrects the article DOI: 4151043a