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In recent years, the field of additive manufacturing (AM), often referred to as 3D printing, has seen tremendous growth and radically changed the means by which we describe valid 3D models for production. In particular, it is now conceivable to produce composite structures consisting of smoothly varying oriented anisotropic constitutive materials. In the present work, we propose a sensitivity driven method for the generation of transverse isotropic fiber reinforced structures having smooth spatially varying orientations. Our approach builds upon finite element analysis (FEA) and density-based topology optimization (TO). The local material orientations are formulated as design variables in a stiffness maximization problem, and solved with a non-convex gradient-based optimization scheme. Length-scale control is achieved through the use of filters for regularization. We demonstrate the ability of the proposed approach to handle large-scale 3D problems with synchronous optimization of material densities and orientations yielding millions of design variables on multiple load case scenarios. The method is shown to be compatible with compliant mechanism optimization as well as local volume constraints. Finally, the approach is extended with an additional design variable dictating the ratio of anisotropy for each element, thereby delegating the choice of material type to the optimization scheme.

Immunocompromised patients may be at increased risk for complications of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) infection. However, comprehensive data of SARS‐CoV‐2 infection in solid organ transplant (SOT) recipients are still lacking. We performed a multicenter nationwide observational study within the Swiss Transplant Cohort Study (STCS) to describe the epidemiology, clinical presentation, treatment and outcomes of the first microbiologically documented SARS‐CoV‐2 infection among SOT recipients. Overall, 21 patients were included with a median age of 56 years (10 kidney, 5 liver, 1 pancreas, 1 lung, 1 heart and 3 combined transplantations). The most common presenting symptoms were fever (76%), dry cough (57%), nausea (33%) and diarrhea (33%). Ninety‐five percent and 24% of patients required hospital and ICU admission, respectively, and 19% were intubated. After a median of 33 days of follow‐up, 16 patients were discharged, 3 were still hospitalized and 2 patients died. These data suggest that clinical manifestations of SARS‐CoV‐2 infection in middle‐aged SOT recipients appear to be similar to the general population without an apparent higher rate of complications. These results need to be confirmed in larger cohorts.

Allergy transfer upon solid organ transplantation has been reported in the literature, although only few data are available as to the frequency, significance, and management of these cases. Based on a review of 577 consecutive deceased donors from the Swisstransplant Donor‐Registry, 3 cases (0.5%) of fatal anaphylaxis were identified, 2 because of peanut and 1 of wasp allergy. The sera of all 3 donors and their 10 paired recipients, prospectively collected before and after transplantation for the Swiss Transplant Cohort Study, were retrospectively processed using a commercial protein microarray fluorescent test. As early as 5 days posttransplantation, newly acquired peanut‐specific IgE were transiently detected from 1 donor to 3 recipients, of whom 1 liver and lung recipients developed grade III anaphylaxis. Yet, to define how allergy testing should be performed in transplant recipients and to better understand the impact of immunosuppressive therapy on IgE sensitization, we prospectively studied 5 atopic living‐donor kidney recipients. All pollen‐specific IgE and >90% of skin prick tests remained positive 7 days and 3 months after transplantation, indicating that early diagnosis of donor‐derived IgE sensitization is possible. Importantly, we propose recommendations with respect to safety for recipients undergoing solid‐organ transplantation from donors with a history of fatal anaphylaxis., Based on the Swisstransplant donor registry, the Swiss‐Transplant‐Cohort‐Study, and a prospective analysis on allergy maintenance in atopic recipients, this study makes new recommendations for the management of allergy transfer upon solid organ transplantation, emphasizing the poor effect of immunosuppression on IgE sensitization and the need of early allergological investigation in the donor and respective recipients.

Background Asymptomatic bacteriuria is frequent in kidney transplant recipients (KTRs). However, there is no consensus on diagnosis or management. We conducted a European survey to explore current practice related to the diagnosis and management of asymptomatic bacteriuria in adult KTRs. Methods A panel of experts from the European Renal Association-European Dialysis Transplant Association/Developing Education Science and Care for Renal Transplantation in European States working group and the European Study Group for Infections in Compromised Hosts of the European Society of Clinical Microbiology and Infectious Diseases designed this cross-sectional, questionnaire-based, self-administered survey. Invitations to participate were e-mailed to European physicians involved in the care of KTRs. Results Two hundred and forty-four participants from 138 institutions in 25 countries answered the survey (response rate 30%). Most participants [72% (176/244)] said they always screen for asymptomatic bacteriuria in KTRs. Six per cent (15/240) reported never treating asymptomatic bacteriuria with antibiotics. When antimicrobial treatment was used, 24% of the participants (53/224) said they would start with empirical antibiotics. For an episode of asymptomatic bacteriuria caused by a fully susceptible microorganism and despite no contraindications, a majority of participants (121/223) said they would use a fluoroquinolone (n = 56), amoxicillin/clavulanic acid (n = 38) or oral cephalosporins (n = 27). Conclusions Screening for and treating asymptomatic bacteriuria are common in KTRs despite uncertainties around the benefits and harms. In an era of antimicrobial resistance, further studies are needed to address the diagnosis and management of asymptomatic bacteriuria in these patients.

BACKGROUND: Early and long-term use of cyclosporine A (CsA) leads to increased risks of renal toxicity. We hypothesized that administration of daclizumab in combination with mycophenolate mofetil (MMF) allows a relevant reduction in the dose of CsA. METHODS: We carried out a 3-year, prospective, randomized, controlled clinical multi-centre trial in 156 patients. The patients were randomized to standard treatment (CsA, MMF, steroids) or to high-dose daclizumab (first dose: 2 mg/kg), in combination with low-dose CsA, MMF and steroids. We maintained the mean CsA levels of daclizumab patients at 57% of standard patients (132 versus 216 ng/ml) on Day 7 post-transplant, and 84% by 6 months. RESULTS: Primary outcome, creatinine clearance (with imputation of informative dropouts) at 12 months, was significantly better in daclizumab-treated (34 +/- 17) than standard patients (29 +/- 17; P = 0.028, two sided). Only 5 cases of BPAR were recorded in the daclizumab compared to 22 in the standard group (P = 0.0016). Daclizumab patients had 91% event-free survival after 1 year compared to 66% in standard patients (P = 0.00017). CONCLUSION: We demonstrate here that high-dose daclizumab in combination with lower CsA levels in adult renal transplant recipients is as or more effective than standard regimen (CsA, MMF, steroids) and may result in better outcomes at 12 months post-transplant with no increase in adverse reactions.

Copyright © 2015 by the American Society of Nephrology. Proteinuria and hyperphosphatemia are cardiovascular risk factors independent of GFR. We hypothesized that proteinuria induces relative phosphate retention via increased proximal tubule phosphate reabsorption. To test the clinical relevance of this hypothesiswe studied phosphate handling in nephrotic children and patients with CKD. Plasma fibroblast growth factor 23 (FGF 23) concentration plasma phosphate concentration and tubular reabsorption of phosphate increased during the proteinuric phase compared with the remission phase in nephrotic children. Cross sectional analysis of a cohort of 1738 patients with CKD showed that albuminuria 300 mg/24 hours is predictive of higher phosphate levels independent of GFR and other confounding factors. Albuminuric patients also displayed higher plasma FGF 23 and parathyroid hormone levels. To understand the molecular mechanisms underlying these observations we induced glomerular proteinuria in two animal models. Rats with puromycin aminonucleoside induced nephrotic proteinuria displayed higher renal protein expression of the sodium phosphate co transporter NaPi IIa lower renal Klotho protein expression and decreased phosphorylation of FGF receptor substrate 2a a major FGF 23 receptor substrate. These findings were confirmed in transgenicmice that develop nephrotic range proteinuria resulting from podocyte depletion. In vitro albumin did not directly alter phosphate uptake in cultured proximal tubule OK cells. In conclusion we show that proteinuria increases plasma phosphate concentration independent of GFR. This effect relies on increased proximal tubuleNaPi IIa expression secondary to decreased FGF 23 biologic activity. Proteinuria induces elevation of both plasma phosphate and FGF 23 concentrations potentially contributing to cardiovascular disease.

International audience; Evidence before this study We searched PubMed on June 30, 2016, for English-language cohort studies published since Jan 1, 1980, of patients with invasive listeriosis worldwide with the keywords " listeria " , " listeriosis " , " maternal " , and " neurolisteriosis ". Studies had to include epidemiological or clinical data on listeriosis. All clinical forms of infection were included (bacteraemia, neurolisteriosis, and maternal–neonatal infection). Host risk factors for listeriosis have been well identified, but the clinical features and prognostic factors of the disease are based on retrospective studies compiling heterogeneous data or random collected cases. Furthermore, no clinical trial has ever been done and medical management is not evidence based. Added value of the study Our study is the first prospective clinical study focusing on all forms of invasive listeriosis. The study is based on a national mandatory system that allowed the nearly complete capture of microbiologically proven cases. The study shows a higher burden of listeriosis than reported before: more than 80% of infected mothers experienced major fetal or neonatal complications (fetal loss, very high prematurity, early or late onset disease); only 39% of patients with neurolisteriosis survived and fully recovered. The study provides important new data to improve management and predict outcome in listeriosis, such as determination of the time window for fetal losses (

Contaminated surface water poses a risk to human populations and is a challenge for public health authorities. Climatic change, intensification of agriculture, urban development of coastal areas, a...

From 2001 to 2005, numerous cases of seafood poisonings were reported in a tribe from Lifou (Loyalty Islands Province, New Caledonia) of which 35 were thoroughly examined. Observations outlined by the epidemiological and clinical data (including severity and rapid onset of certain symptoms following consumption of either giant clams ( Tridacna spp.) or grazing and molluscivorous fish together with the apparent inefficacy of traditional remedies, were not in favour of a classical Ciguatera Fish Poisoning (CFP) outbreak. From 2005 onwards, an environmental offshore survey of the affected area was conducted. Screening of the damaged coral area revealed the presence of large populations of cyanobacteria identified as Hydrocoleum Kutzing, but the absence of Gambierdiscus spp., the well-known dinoflagellate causative agent of CFP. In vivo and in vitro toxicological studies of extracts obtained from cyanobacteria and giant clams, strongly suggested the co-occurrence of ciguatoxin-like, anatoxin-like and paralytic shellfish toxins in these samples. These new findings shed new light on the complexity of the CFP symptomatology and treatment and also on the diversity and origin of the CFP toxins. Furthermore they provide new evidence of the overall variability of seafood poisonings following the ingestion of different sea products living in a marine environment where significant harmful populations of microalgae and cyanobacteria coexist. This is the first report on the involvement of cyanobacteria in CFP-like outbreaks following the consumption of giant clams or fish specimens. Consequently, it is recommended that CFP risk assessment programs now include monitoring of cyanobacteria besides the obvious screening of CFP-promoting dinoflagellates.

The effects and the mode of action of hypericin (1) were studied, in the dark, on the action potential (AP) and the L-type Ca2+ channel of frog atrial heart muscle, using intracellular microelectrode and patch-clamp techniques, respectively. In the presence of Ca2+ in Ringer solution, hypericin (1 to 4 microM) did not markedly modify the AP. Total replacement of Ca2+ by Sr2+ in the solution (Ringer Sr2+) revealed that hypericin (4 microM) prolonged the AP duration (APD). Hypericin dose-dependently increased the magnitude of the Sr2+current, which develops through L-type Ca2+ channels in the Ringer solution containing tetrodotoxin (0.7 microM) and tetraethylammonium (10 mM), but did not modify the kinetics of activation and inactivation. This revealed that hypericin increased L-type Ca2+ channel conductance, which accounted for the APD lengthening. The hypericin-induced APD lengthening recorded in the Ringer Sr2+ was not prevented by (i) a blockade of alpha- and beta-adrenoceptors by yohimbine (1 microM), urapidil (1 microM), and propanolol (50 microM), respectively, and (ii) PKC blockade by staurosporine (1 microM). The hypericin-induced APD lengthening recorded in the Ringer Sr2+ was prevented by blocking soluble guanylate cyclase (sGC) activity by 1H-[1,2,4]-oxadiazolo[4,3-a]quinoxalin-1-one (13 microM), which mimicked the effects of hypericin. Hypericin decreased the cellular cGMP level by 69% in atrial myocytes. The compound also decreased the cellular cGMP level by inhibiting sGC, thus cancelling the nucleotide inhibitory effect on the cardiac L-type Ca2+ channel.

Using intracellular microelectrodes, we studied transmembrane resting and action potentials (AP) of left ventricle papillary muscles isolated from the heart of adult lindane-treated (TMG) and untreated (UMG) male genitor rat offspring, obtained by mating untreated female with males chronically treated and untreated with lindane (2 ppb) trace concentrations through beverage. The AP magnitude and duration (APD) were similar in both groups and their response to low temperature (22 °C) unchanged. Lowering the external Ca 2+ concentration from 2.5 to 0.625 mM prolonged APD in the TMG group but not in the UMG group. In the TMG group, (i) cumulative addition of Sr 2+ (1 mM) to the physiological solution prolonged APD; (ii) apamin (4 μM) and charybdotoxin (4 μM) prolonged the APD. In conclusion, our data revealed that an altered sensitivity of the Ca 2+ -induced Ca 2+ inactivation of L-type Ca 2+ channels and of Ca 2+ -activated K + channels to Ca 2+ has been transferred to TMG offspring.

We demonstrate the direct and noninvasive imaging of functional neurons by ionic contrast terahertz near-field microscopy. This technique provides quantitative measurements of ionic concentrations in both the intracellular and extracellular compartments and opens the way to direct noninvasive imaging of neurons during electrical, toxin, or thermal stresses. Furthermore, neuronal activity results from both a precise control of transient variations in ionic conductances and a much less studied water exchange between the extracellular matrix and the intraaxonal compartment. The developed ionic contrast terahertz microscopy technique associated with a full three-dimensional simulation of the axon-aperture near-field system allows a precise measurement of the axon geometry and therefore the direct visualization of neuron swelling induced by temperature change or neurotoxin poisoning. Water influx as small as 20 fl per μm of axonal length can be measured. This technique should then provide grounds for the development of advanced functional neuroimaging methods based on diffusion anisotropy of water molecules.

The mechanisms underlying the swelling of frog red blood cells (RBC), induced by Pacific (P-CTX-1) and Caribbean (C-CTX-1) ciguatoxins (CTXs), were investigated by measuring the length, width and surface of their elliptic shape. P-CTX-1 (0.5 to 5 nM) and C-CTX-1 (1 nM) induced RBC swelling within 60 min. The CTXs-induced RBC swelling was blocked by apamin (1 microM) and by Sr(2+) (1 mM). P-CTX-1-induced RBC swelling was prevented and inhibited by H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (27 microM), an inhibitor of soluble guanylate cyclase (sGC), and NOS blockade by NG methyl-l-arginine (l-NMA; 10 microM). Cytochalasin D (cytD, 10 microM) increased RBC surface and mimicked CTX effect but did not prevent the P-CTX-1-induced l-NMA-sensitive extra increase. Calculations revealed that P-CTX-1 and cytD increase RBC total surface envelop and volume. These data strongly suggest that the molecular mechanisms underlying CTXs-induced RBC swelling involve the NO pathway by an activation of the inducible NOS, leading to sGC activation which modulates intracellular cGMP and regulates L-type Ca(2+) channels. The resulting increase in intracellular Ca(2+) content, in turn, disrupts the actin cytoskeleton, which causes a water influx and triggers a Ca(2+)-activated K(+) current through SK2 isoform channels.

The heart of adult rat offspring, born to mothers treated with trace concentrations of lindane (0.5 to 2 ppb) through a beverage and to mothers chronically treated with lindane (CL-T) with the same trace concentration, also through a beverage, during lactation and growth has a round shape and accumulates lindane. The left ventricle (LV) presents a hypertrophied area, atrophied papillary muscles, and unorganized collagen bundles and layers. These observations led us to study the electrical activity of their left ventricle papillary muscles (LVPM) by recording action potential using intracellular microelectrodes. CL-T shortened LVPM action potential duration (APD): 1 ppb shortened the plateau; 2 ppb shortened the plateau and the slow repolarizing phase. In CL-T (2 ppb) and untreated groups, low temperature (22 °C) decreased the resting potential and prolonged APD. TEA (tetraethylammonium; 1-2 mmol/L) partially lengthened CL-T (2 ppb lindane) APD. Quinidine (0.2 mmol/L) and E-4031 (10 nmol/L) prolonged CL-T APD, suggesting that the rapid delayed outward K+ current (IKr) was increased. Our results indicate the silent effects of chronic exposure to trace concentrations of lindane on the morphological and electrical activity of heart muscle. They demonstrate that chronic lindane treatment of female rats alters the tissue integrity and electrical activity in the LV of their offspring.Key words: heart muscle, membrane potential, lindane, K+ channel.

International audience; The effects of lepadiformine, a natural marine alkaloid isolated from the ascidians Clavelina lepadifonllis (Muller) and C. moluccensis (Sluiter), were studied in vivo by arterial blood pressure (aBP) recordings and electrocardiograms (ECG) in anaesthetised rats and in situ by peripheral vascular pressure recordings on perfused rabbit ear. Transmembrane resting (RP) and action (AP) potentials were also recorded by intracellular microelectrodes on electrically stimulated left ventricular papillary muscle and spontaneously beating atrium isolated from rat and frog hearts, respectively. Intravenous injection of lepadiformine (6 mg/kg) produced marked bradycardia and a lengthening of ECG intervals as well as a transient decrease of aBP, which rapidly returned to normal. The decrease of aBP may have been related to a vasoconstrictor effect observed in the perfused ear experiment. Lepadiformine did not alter RP, bur significantly lengthened the repolarising phase of AP in rat papillary muscle and frog atrium. Lepadiformine also mimicked the effect of Ba2+ (0.2 mM) on the rat AP repolarising phase. Moreover, the lengthening of the AP in frog atrium induced by lepadiformine still developed after the delayed outward K+ current (I-K) was blocked by tetraethylammonium (10 mM). These observations suggest that lepadiformine-induced lengthening of AP duration was not due to a decrease of I-K, but may reasonably be attributed to a reduction of the inward rectifying K+ current (I-K1). This blockade of I-K1 could account for the cardiovascular effects of lepadiformine in vivo and in vitro and suggests that lepadiformine: has antiarrhythmic properties. (C) 2001 Elsevier Science Ltd. AH rights reserved.

The effects of trachynilysin (TLY), a protein toxin isolated from stonefish (Synanceia trachynis) venom, were studied on the electrical and mechanical activities of frog atrial fibres. TLY (1 microg/ml) hyperpolarized the membrane, shortened the action potential (AP) duration (APD), exerted a negative inotropic effect and elicited contracture. These effects did not develop in the presence of atropine. TLY shortened the APD of fibres isolated from a frog completely paralyzed with botulinum type A toxin, in the presence of Ca2+ but not when Ca2+ was replaced by Sr2+. TLY increased the basal and the peak of the fluorescence ratio of stimulated fibres loaded with fura-2. Confocal laser scanning microscopy revealed the existence of a diffuse innervation in atrial tissue. Our results suggest that TLY enhances the release of acetylcholine from atrial cholinergic nerve terminals and activates indirectly muscarinic receptors leading to a shortening of APD. They also show that the mechanical effects induced by TLY are due to an increase of the Ca2+ influx and to a rise in intracellular Ca2+ levels which leads to (i) a slowing of the Na+/Ca2+ exchange activity, which accounts for the contracture and (ii) the activation of a Ca2+-dependent K+ current involved in the APD shortening.

Halothane has many effects on the resting membrane potential (Vm) of excitable cells and exerts numerous effects on skeletal muscle one of which is the enhancement of Ca2+ release by the sarcoplasmic reticulum (SR) resulting in a sustained contracture. The aim of this study was to analyse the effects of clinical doses of halothane on Vm, recorded using intracellular microelectrodes on cleaned and non stimulated sartorius muscle which was freshly isolated from the leg of the frog Rana esculenta. We assessed the mechanism of effects of superfused halothane on Vm by the administration of selective antagonists of membrane bound Na+, K+ and Cl− channels and by inhibition of SR Ca2+ release. Halothane (3%) induced an early and transient depolarization (4.5 mV within 7 min) and a delayed and sustained hyperpolarization (about 11 mV within 15 min) of Vm. The halothane-induced transient depolarization was sensitive to ryanodine (10 μM) and to 4-acetamido-4′-isothiocyanatostilbene 2,2′ disulphonic acid (SITS, 1 mM). The hyperpolarization of Vm induced by halothane (0.1–3%) was dose-dependent and reversible. It was insensitive to SITS (1 mM), tetrodotoxin (0.6 μM), and tetraethylammonium (10 mM) but was blocked and/or prevented by ryanodine (10 μM), charybdotoxin (CTX, 1 μM), and glibenclamide (10 nM). Our observations revealed that the effects of halothane on Vm may be related to the increase in intracellular Ca2+ concentration produced by the ryanodine-sensitive Ca2+ release from the SR induced by the anaesthetic. The depolarization may be attributed to the activation of Ca2+-dependent Cl− (blocked by SITS) channels and the hyperpolarization to the activation of large conductance Ca2+-dependent K+ channels, blocked by CTX, and to the opening of ATP-sensitive K+ channels, inhibited by glibenclamide. Keywords: Anaesthetics, halothane, membrane resting potential, skeletal muscle, electrophysiology Introduction Volatile anaesthetic agents have been reported to exert numerous effects on the central nervous system. Synaptic transmission is more sensitive to general anaesthetics than axonal conduction (Pocock & Richards, 1993). Voltage-gated Na+ and K+ channels are generally thought to be very insensitive to general anaesthetics; voltage-gated fast transient K+ currents are quite insensitive to volatile agents while voltage gated Ca2+ channels are thought to be probably the most anaesthetic-sensitive of all voltage-gated channels (Franks & Lieb, 1998). The general anaesthetic halothane is a well known inhalational anaesthetic. Electrophysiological reports have shown that halothane blocks the Na+ current without inducing a shift in the voltage-dependence of its steady-state inactivation (Haydon et al., 1984) and reduces voltage-activated K+ currents at least as much, if not more, than Na+ current in the squid giant axon (Urban & Haydon, 1987). Halothane depresses the delayed outward K+ current and slightly reduces the inward rectifier K+ current in cardiac myocytes (Pancrazio et al., 1993). Halothane also exerts varied effects on the resting membrane potential (Vm) of excitable cells. It does not affect Vm in mouse diaphragm (Gage & Hamill, 1976), crayfish axon (Bean et al., 1981), hyperpolarizes the membrane in frog motoneurons and in central neurons (Nicoll & Madison, 1982), and in hippocampal CA1 neurons (Maclver & Kendig, 1991), produces various effects (depolarization or hyperpolarization) on cat respiratory neurons (Takeda & Haji, 1992), tends to depolarize the membrane in squid axon (Haydon et al., 1984; 1988; Urban & Haydon, 1987; Haydon & Simon, 1988). The sarcoplasmic reticulum (SR) is one of the intracellular sites of action of halothane which has been shown to increase Ca2+ release from the SR in skeletal muscle (Palade et al., 1989). Halothane prolonged the opening of isolated ryanodine receptor Ca2+ release channels from the SR (Bull & Marengo, 1994; Connelly & Coronado, 1994). The ryanodine receptor Ca2+ release channel or the α1-subunit of the dihydropyridine receptor are thought to be involved in malignant hyperthermia, a pharmacogenetic disorder. The effects of halothane on muscle membrane protein regulatory Ca2+-homeostasis have shown that halothane influenced Ca2+-ATPases (Karon et al., 1995; Lopez & Kosk-Kosicka, 1995); induced oligomerization of the terminal cisternae Ca2+-binding protein calsequestrin, the junctional ryanodine receptor Ca2+-release channel and the transverse-tubular α1-dihydropyridine receptor of rabbit skeletal muscle (Froemming et al., 1999). Anaesthetic effects on ion channels are modulated by the electrolyte environment and may depend on membrane potential (Urban & Friederich, 1998). The aim of the present work was to study the effects of clinically effective concentrations of halothane on the resting membrane potential of frog skeletal muscle using conventional microelectrodes and to determine the mechanism by which halothane altered the membrane potential. We report that, in frog skeletal muscle, halothane induced a ryanodine-sensitive hyperpolarization which was also sensitive to charybdotoxin and to glibenclamide, and suggests that Ca2+-dependent and ATP-sensitive K+ channels are involved in the hyperpolarizing effect induced by the anaesthetic.

Mediterranean strains of Prorocentrum minimum do not appear to have the same toxic component as Japanese strains since they showed no cytotoxicity for hepatocytes in culture. However, their toxic components, which appear to block calcium channels, were detectable by the immobilisation test on Diptera larvae. A bio-accumulation experiment in the laboratory showed that the toxins could accumulate in nearly equivalent amounts in the hepatopancreas and meat of cultured mussels. The same toxicity was found in natural samples collected in a period of bloom of P. minimum. These results suggest that P. minimum could be responsible for shellfish toxicity in the natural environment and thus present a risk for human health.

The goal of the present review is to report information concerning cardiac innervation or more precisely to approach the modulation of cardiac electrical and mechanical activity by parasympathetic innervation. Acetylcholine (ACh) release by nerve endings from the vagus nerve hyperpolarizes the membrane, shortens action potential (AP) duration and has a negative inotropic effect on cardiac muscle. Toxins are usefull tools in the study of membrane signals. The Caribbean ciguatoxin (C-CTX-1) has a muscarinic effect on frog atrial fibres. The toxin evokes the release of ACh from motoneuron nerve terminals innervating this tissue which allows us to propose a model, similar to the one of the neuromuscular junction (nmj), to describe the events occurring during the triggering and release of ACh. Trachynilysin (TLY) is a proteic toxin which causes an influx of Ca2+ into the cells and releases ACh from nmj synaptic vesicles. TLY has a muscarinic effect on atrial fibres which is explicated in the release of neurotransmitter from the nerve endings generated by the TLY-induced Ca2+ influx. It is known that ACh release from nmj is known to be due to exocytosis of synaptic vesicles via the activation of a proteic complex blocked by botulinum toxins. One of these proteins SNAP-25 is the target of type A botulinum toxin (BoNT/A). The study of hearts isolated from BoNT/A poisoned frogs show that atrial AP is lengthened and reveals the presence of SNAP-25 in nerve endings of this tissue. Moreover, the electrical activity of ventricular muscle is markedly altered; in BoNT/A treated frog, an important outward current activated by internal Ca2+ develops. ACh released from nerve terminals binds to a G protein coupled membrane receptor and activates a K+ channel and other effectors. Five subtypes of muscarinic receptors have been cloned from different tissue (M1, M2, M3, M4) subtypes have been identified in cardiac tissues throughout many species. These receptors coupled with different G-proteins activate different effectors. M1 receptors modulate the cardiac plateau and therefore the magnitude of the peak contraction. M2 receptors are mainly involved in the repolarization phase of the AP and modulate the duration of the peak contraction. The roles of M3 and M4 are not yet clearly defined; however, they may activate K+ currents. In conclusion, ACh releases from parasympathetic nerve endings which innervate cardiac cells follows to similar events (Ca2+ influx; presence of a SNAP-25 protein) to those which produce ACh release from nmj, stimulates different G proteins coupled muscarinic receptors, and activates different effectors involved in the modulation of cardiac electrical and mechanical activity.

M.-P. Sauviat, P. Garnier, F. Goudey-Perriere and C. Perriere. Does crude venom of the stonefish (Synanceia verrucosa) activate β-adrenoceptors in the frog heart muscle ? Toxicon 33, 1207-1213, 1995.-Venom isolated from the stonefish Synanceia verrucosa was assayed in concentrations of 0.07 and 5.7 μg/ml on frog atrial fibres and myocytes. Venom, less than 2.9 μg/ml, dose-dependently increased the amplitude and the duration of the stimulated peak tension, lengthened the time constant of the relaxation phase and shortened the duration of the action potential (AP). The concentration of venom 5.7 μg/ml decreased the amplitude of the peak tension, induced a contracture, reduced the amplitude of the plateau and shortened its duration as well as the repolarizing phase of the AP. The positive inotropic effect induced by the venom (2.9 μg/ml) on the contraction was inhibited dose-dependently by propranolol but was unchanged by the α-adrenergic antagonists urapidil and yohimbine, the adenyl cyclase activity remaining sensitive to forskolin. Venom, adrenalin and propranolol competed for a common site. Venom (2.9 μg/ml) increased both the Ca and the delayed outward K currents of enzymatically isolated atrial myocytes. The data suggest that the venom activates adrenoceptors, essentially β-adrenoceptors.

1. The effects of palytoxin (PTX) on transmembrane potentials and currents of frog skeletal muscle were analyzed by intracellular microelectrode techniques and the double sucrose-gap voltage clamp method. 2. PTX irreversibly depolarized the membrane. The depolarization was Na-sensitive. 3. Under voltage clamp, PTX induced an inward resting current which did not inactivate, was inhibited by external Na+ removal and was a function of external Na concentration. 4. This resting current could be carried either by Na+, Li+, K+ or by guanidinium according to the permeability sequence K+ less than Li+ less than Na+ less than Gua+. 5. The PTX-induced current was only weakly sensitive to tetrodotoxin. It was reversibly and dose-dependently inhibited by amiloride with a one to one stoichiometry and a KD of 0.3 mM. 6. Acidic pH partially inhibited the current induced by PTX which was also highly sensitive to external Cd2+ and La3+. The inhibitory sequence for divalent cations was: Mg2+ less than Ca2+ = Ba2+ = Mn2+ less than Cd2+; with La3+ greater than Cd2+. 7. The amplitude of the PTX-induced I(rest) was markedly reduced in the absence of external Ca2+. 8. PTX induced a Na+ resting conductance in frog skeletal muscle. The size of the channel induced by PTX is larger than the guanidinium ion. External membrane Ca2+ might be a cofactor involved in the mode of action of PTX.

Fumonisin, a toxin isolated from the fungus Fusarium moniliforme sheld, blocks both the calcium current and the mechanical activity in frog atrial muscle. Toxicon 29, 1025–1031, 1991.—The effects of fumonisin, a toxin isolated from the fungus Fusarium moniliforme sheld were studied on transmembrane potentials and currents of the frog heart muscle using the double sucrose gap technique. Fumonisin (280 μM) shortened the plateau duration of the action potential. Under voltage clamp conditions, fumonisin inhibited the Ca current. The block occurred without alteration of either the kinetic parameters or the apparent reversal potential of the current suggesting that the toxin blocked the maximal Ca conductance. Fumonisin reduced mainly the phasic component of the peak tension. The time to peak tension was unchanged whereas its relaxation phase was accelerated at positive membrane potentials suggesting that Na-Ca exchange was accelerated by fumonisin. Dose-response curves for the Ca current and the peak tension indicated half inhibition at about 100 μM fumonisin and a stoichiometric parameter of 1.7, suggesting that more than one toxin molecule interacted with the Ca channel. The data suggested that the effect of fumonisin on the Ca current and the tension might account for cardiac failure reported in animal intoxication by Fusarium moniliforme .

M.-P. Sauviat . Effect of phoratoxin B, a toxin isolated from mistletoe, on frog skeletal muscle fibres. Toxicon 28, 83–89, 1990.—The effect of phoratoxin B on transmembrane potentials and currents of frog skeletal muscle was studied using intracellular microelectrode recording and double sucrose gap voltage clamp techniques. Phoratoxin B irreversibly depolarized the membrane. The depolarization was insensitive to tetrodotoxin, tetraethylammonium, 4-aminopyridine, cesium, cadmium and D-600. In voltage clamp experiments, phoratoxin B induced an inward resting current ( I rest ) which did not inactivate. I rest was blocked by increasing the external calcium concentration in the Ringer solution; it was not blocked when Sr 2+ replaced Ca 2+ . Analysis of the peak sodium current indicated that while both Ca 2+ and Sr 2+ screen membrane surface charges, Ca 2+ bound and reversed the phoratoxin B induced I rest whereas Sr 2+ did not. The inward I rest induced by phoratoxin B developed in the presence of external chloride and remained unchanged in the presence of bicarbonate. The data suggest that the depolarizing action of the toxin might be attributed to an increase in the non-selective leak current and that it might act as a detergent.

We demonstrated the direct and noninvasive imaging of functional neurons, 1 as well as auricular heart muscle electrical activity 2 by Ionic Contrast Terahertz (ICT) near-field microscopy. This technique provides quantitative measurements of ionic concentrations in both the intracellular and extracellular compartments and opens the way to direct noninvasive imaging of neurons during electrical, toxin, or thermal stresses. Furthermore, neuronal activity results from both a precise control of transient variations in ionic conductances and a much less studied water exchange between the extracellular matrix and the intraaxonal compartment. The developed ICT technique associated with a full three-dimensional simulation of the axon-aperture near-field system allows a precise measurement of the axon geometry and therefore the direct visualization of neuron swelling induced by temperature change or neurotoxin poisoning. This technique should then provide grounds for the development of advanced functional neuroimaging methods based on diffusion anisotropy of water molecules.

Three marine alkaloids, purified from Clavelina moluccensis, were structurally identified as lepadiformines A, B, and C and studied on frog atrial myocytes I(K1), using the patch-clamp technique. Lepadiformine A (0.4 to 3.3 microM) blocked I(K1) dose-dependently with an apparent dissociation constant (K(D)) equal to 1.42 microM and a stoichiometry of 0.77. The block is voltage-dependent, suggesting that lepadiformine A occupies a receptor site located at about two-thirds of the membrane depth. The shortening of the aliphatic chain at position C13 of lepadiformine B decreased the potency of the molecule to block I(K1) but not the affinity (K(D) = 1.56 microM) and stoichiometry (0.72). Additional deletion of the oxygenated side chain at C2 in lepadiformine C markedly decreased the inhibitory effect of the molecule. In conclusion, lepadiformine modulates I(K1) response in cardiac muscle. The oxygenated side chain in C2 is implicated in the affinity of lepadiformine, which behaved as an amine, for a receptor located near or inside the I(K1) pore, and the aliphatic chain length at position C13 is involved in the degree of I(K1) blockage.

Background: We characterize the application of multiphoton microscopy to the observation of the extracellular matrix of fresh unstained vessels. Method: Combined two-photon-excited fluorescence (2PEF) and second harmonic generation (SHG) imaging of large arteries reveals the architecture of elastin and collagen fibers in the vessel wall with remarkable specificity. Results: We present elastin/collagen imaging in unstained rat vessels at both micrometer and whole vessel scales, and we characterize the optical properties of rat carotid artery and aorta walls. We apply this method to evidence deleterious effects of residual doses of a pesticide on the vessel wall. Conclusion: This study illustrates the potential of 2PEF/ SHG microscopy for pharmacological studies in unlabeled arteries. q 2005 International Society for Analytical Cytology

The effects of 31 plant extracts, which most are traditionally used to treat ciguatera fish poisoning in the Pacific area, were studied on the cytotoxicity of mouse neuroblastoma cells produced by ouabain, veratridine and/or brevetoxin-3 or Pacific ciguatoxin-1. The cell viability was determined using a quantitative colorimetric method. A marked cytotoxicity of seven of the 31 plant extracts studied, was observed. Despite this, these plant extracts were suspected to contain active compound(s) against the cytotoxicity produced by brevetoxin (2 extracts), brevetoxin, ouabain and/or veratridine (3 extracts), or only against that of ouabain and/or veratridine (2 extracts). Among the 24 plant extracts that exhibited by themselves no cytotoxicity, 22 were active against the effect of brevetoxin or against that of both veratridine and brevetoxin. Similar results were obtained when the seven most active plant extracts were reassayed using ciguatoxin instead of brevetoxin. In conclusion, the present work reports the first activity assessment of some plant extracts, achieved in vitro on a quite large scale. The fact that 27 plant extracts were found to exert, in vitro, a protective effect against the action of ciguatoxin and/or brevetoxin, paves the way for finding new active compounds to treat ciguatera fish poisoning, provided these compounds also reverse the effects of sodium channel activators.

International audience; We extend second-harmonic generation (SHG) microscopy to the measurement of sarcomere length in unstained living cardiac myocytes with 20-nm accuracy. We quantify individual sarcomere shortening in the presence of saxitoxin and find that it is in agreement with mechanical measurements of atrial tissue contracture. This functional application of SHG microscopy is generally applicable to quantify the physiological effects of drugs on contractile tissue. Our data also suggest that packed myosin heads in sarcomere thick filaments are responsible for the large second-harmonic endogenous signal in muscle tissue.

International audience; The effects of Caribbean ciguatoxin (C-CTX-1) isolated from horse-eye jack (Caranx latus) on the electrical and mechanical activities of frog auricle were studied. C-CTX-1 (1pM-50nM) dose-dependently shortened the duration of the plateau and the repolarizing phase of the action potential (AP). The AP shortening induced by C-CTX-1 (50nM) was suppressed or prevented either by tetrodotoxin (TTX; 0.6nM) or by atropine (0.1mM). C-CTX-1 (50nM) prolonged the TTX (0.6nM)-sensitive electrical response of the vagus nerve branches, which innervate the auricle. The C-CTX-1 (50nM)-induced shortening of the plateau and of the repolarization phase were prevented or reversed by gallamine (20µM) and pirenzepine (0.5µM), respectively. C-CTX-1 (50nM) decreased the amplitude of the peak contraction and shortened its duration. In the presence of gallamine (20µM), C-CTX-1 decreased the amplitude of the peak contraction and shortened its duration in the presence of pirenzepine (0.5µM). C-CTX-1 (50nM) decreased the time constant of the relaxation phase of the peak contraction suggesting that it increased the Na+/Ca2+ exchange activity. Acetylcholine (ACh; 1pM) shortened APD, decreased the peak contraction and mimics the effects of C-CTX-1. In conclusion, the presented data show that C-CTX-1 released ACh from atrial cholinergic nerve terminals which activated M1 and M2 muscarinic receptors subtype (mAChR). Our findings suggest that M1 and M2 mAChR are present in frog atrial tissue and play a previously unrecognized role in the modulation of the AP duration and of the mechanical activity of cardiac tissue. Cop. 2002 Elsevier Science Ltd. All rights reserved.

Charged submicron emulsions are a priori interesting candidates for the delivery of drugs in and/or through the skin. In the present study, it was possible by using stearylamine or deoxycholic acid (DCA) to incorporate either econazole or miconazole nitrate, respectively, in positively and negatively charged submicron emulsions. The investigation of the relationship between the physicochemical properties of the vehicles, especially the charge of the emulsion and skin permeation, was conducted ex vivo during percutaneous absorption experiments using hairless female rat skin. In addition, drug quantification was carried out using two different analytical techniques (HPLC and radioactivity measurements) in order to examine if the drug analysis approach might affect the results. The results clearly indicate that the surface-modified droplets have a significant influence on the diffusion through the skin. Furthermore, the method of preparation of the formulation and subsequently the analytical method of drug concentration measurement are able to influence the results of percutaneous experiments.

Freshly purified but unstable verrucotoxin (VTX) and a more stable proteic complex of the toxin (p-VTX) were isolated from the venom of the stonefish Synanceia verrucosa and applied to frog atrial fibres. VTX and p-VTX decreased the amplitude and the duration of the stimulated peak tension and accelerated the relaxation phase of the contraction. The negative inotropic effect of p-VTX decreased with increasing the external Ca concentration ([Ca]o) in the Ringer solution. The negative chronotropic effect induced by p-VTX was insensitive to change in [Ca]o. It is reversed by glibenclamide. p-VTX shortened the duration of the plateau and the repolarizing phase of the action potential. Glibenclamide but not tetraethylammonium reversed the p-VTX-induced shortening of the AP repolarizing phase. The data suggest that the toxin isolated from the venom of S. verrucosa inhibits Ca channels and might activate ATP-sensitive potassium channels in frog atrial heart muscle.

The effects of an unknown toxin, isolated along with okadaic acid from the hepatopancreas of French mussels contaminated by Dinophysis sp., producing ataxia, neurologic symptoms, bradycardia, arrhythmias, electrocardiographic changes, and cardiac arrest, have been studied in terms of the electrical and mechanical activity of frog atrial heart muscle. The toxin, in a dose-dependent manner, decreased the amplitude of the stimulated peak tension of isolated fibers. The toxin (1-36 micrograms/ml) did not modify the membrane resting potential but decreased the amplitude of the plateau and shortened the duration of the action potential. The toxin inhibited the Cd-sensitive L-type Ca current and increased a 4-aminopyridine-sensitive outward current in voltage-clamped cardiac myocytes. The data show that the cardiac effect of the toxin is markedly different from that of okadaic acid.

The effects of a proteic extract (PachyTX), isolated from the sponge Pachymatisma johnstonii, were studied respectively with intracellular microelectrode and the "whole cell" patch clamp techniques on fibers and myocytes isolated from the sinoatrial region of frog heart. PachyTX significantly and reversibly shortened the action potential duration and decreased the stimulated peak contraction. Under voltage clamp, PachyTX increased, in a dose-dependent manner, a tetraethylammonium-insensitive but glibenclamide-inhibited outward current. This current failed to develop when adenosine 5'-triphosphate (ATP) was intracellularly applied to the cell by diffusion through the patch electrode. PachyTX mimics the effect of the ATP-sensitive K channel opener SR 44866 on frog atrial action potential and peak contraction. From this evidence, it can be concluded that PachyTX is a new natural potent opener of ATP-sensitive K channels in frog atrial heart muscle and that the opening of these channels is associated with the inhibition of the contraction of cardiac muscle.

Publisher Summary This chapter discusses palytoxin (PTX) and its electrophysiology. It also presents the characteristics of the PTX-induced channel, its electrical properties, and mode of action in excitable cells. PTX is a powerful water-soluble toxin produced by coelenterate species belonging to the genus Palythoa. The toxin has a unique structure and produces multiple effects on both non-excitable and excitable cells. The chapter summarizes the exceptional effects of the toxin. PTX induces release of potassium and is hemolytic in erythrocytes. It depolarizes excitable cells and increases the intracellular calcium content in both heart and smooth muscles. PTX also belongs to the non-TPA type of tumor promoters and stimulates arachidonic acid metabolism. A common effect of PTX in excitable tissues is depolarization of membranes. The chapter also discusses the effect of PTX on calcium current and mechanical activity. PTX is a unique type of cytolysin with complex action. The first step of the toxin action is the binding of the toxin to a membrane receptor, which leads to the formation of cationic pores principally permeable to Na + . The conductance induced by the toxin plays a key role in a cascade of events that affect other membrane conductance and induce metabolic alterations.

The effects of lindane, a gamma-isomer of hexachlorocyclohexane, were studied on transmembrane potentials and currents of frog atrial heart muscle using intracellular microelectrodes and the whole cell voltage-clamp technique. Lindane (0.34 microM to 6.8 microM) dose-dependently shortened the action potential duration (APD). Under voltage-clamp conditions, lindane (1.7 microM) increased the amplitude of the outward current (Iout) which developed in Ringer solution containing TTX (0.6 microM), Cd2+ (1 mM) and TEA (10 mM). The lindane-increased Iout was not sensitive to Sr2+ (5 mM). It was blocked by subsequent addition of quinidine (0.5 mM) or E-4031 (1 microM). E-4031 lengthened the APD; it prevented or blocked the lindane-induced APD shortening. In conclusion, our data revealed that lindane increased the quinidine and E-4031-sensitive rapid delayed outward K+ current which contributed to the AP repolarization in frog atrial muscle.

M. -P. Sauviat , C. Pater and J. Berton . Does palytoxin open a sodium-sensitive channel in cardiac muscle? Toxicon25, 695 – 704, 1987.—The effect of palytoxin (PTX) on transmembrane potentials and currents of frog atrial fibres was studied using the double sucrose gap technique. PTX irreversibly depolarized the membrane. This depolarization was reversed only when Na ions were removed from the Ringer solution and replaced by a non-permeant cation such as choline. The depolarization was tetrodotoxin (TTX) insensitive and a function of the external Na concentration. In voltage clamp experiments PTX induced the development of a large inward resting current which did not inactivate and was insensitive to ouabain and to a lowering of the temperature. PTX and ouabain did not share the same receptor. Dose - response curves indicated a stoichiometry of 2, which suggested the aggregation of 2 molecules of PTX to form a channel. The channel formed by PTX remained insensitive to TTX, 4 aminopyridine, tetramethyl-ammonium, Cs and Cd, the classical blockers of Na, K and Ca conductances. PTX reduced the Na current, but not the apparent reversal potential for Na ions. It was concluded that PTX might act on frog atrial fibres as a Na ionophore.

1. The effect of palytoxin (PTX) on the Ca current (ICa) and the mechanical activity of frog atrial fibres was studied by use of the double sucrose gap voltage clamp technique. 2. In normal Ringer solution, PTX transiently increased the electrically-evoked peak tension which then decreased while a major contracture developed. PTX slowed the time course of the relaxation phase of the evoked tension. 3. Evidence is presented which suggests that the toxin also increased the entry of Ca and Sr via the Na-Ca exchange mechanism. It also induced the development of a Ca-dependent outward current which was inhibited by Sr. 4. In Na-free solution, PTX increased ICa and shifted the reversal potential for Ca towards more negative membrane potentials, thus suggesting that the internal Ca concentration had increased. Current-voltage, tension-voltage, time to peak-voltage and inactivation time constant-membrane potential curves were all shifted towards more negative membrane potentials in the presence of PTX. 5. These effects of PTX are similar to those caused by the increase in internal Ca concentration induced by Na ionophores by way of voltage-dependent Ca influx of the Na-Ca exchange mechanism.

The effects of diclofurime on the electrical activity of the rabbit sinus node, rabbit atria and frog atrial fibres were studied using microelectrode and the double sucrose gap voltage-clamp techniques respectively. In rabbit sinus node, diclofurime (10(-7) M to 10(-6) M) decreased the action potential (AP) amplitude and maximum rate of depolarization (Vmax), increased the AP duration and slowed the sinus rate. In rabbit atria, the drug reduced the amplitude of the depolarizing phase and Vmax, lengthened the AP duration and decreased the resting membrane potential. In frog atrial fibres, the drug (10(-5) M) depolarized the resting membrane potential, decreased Vmax as well as the plateau amplitude. It inhibited the sodium current (INa) with a dissociation constant of 3.7 X 10(-6) M and a one to one relationship between the drug molecule and the Na channel. Diclofurime did not alter the apparent reversal potential for the fast Na current (ENa) but it inhibited the sodium conductance (GNa) in a frequency-dependent manner. Diclofurime also blocked the slow inward current (Islow) without alteration of Eslow. The block of Islow occurred with a dissociation constant of 2 X 10(-5) M and unity stoichiometry. The data suggest that diclofurime might be effective in the control of cardiac arrythmias since it exhibited both local anaesthetic-like and calcium antagonistic properties.

Two cases of human intoxication caused by the lyophilized powder of Lissoclinum bistratum Sluiter, a New Caledonian ascidian, are reported. The symptoms observed were caused by a substance designated bistramide A (C40H68N2O8) of hitherto unknown chemical structure. Preliminary toxicological investigations indicate that bistramide A may effect the central nervous system, leading to paresthesia and loss of muscle tone. A progressive decrease in cardiac rhythm was also observed in animals. Bistramide A (1.4 x 10(-6) M) did not alter the resting potential of frog heart and skeletal muscle but reduced the amplitude and duration of cardiac action potential and prolonged the interval between action potentials. Bistramide A also has a marked cytotoxic effect on cancer cells KB (IC50 = 4.5 x 10(-8) M) and P 388 (IC50 = 2.0 x 10(-8) M) and on normal endothelial cells (IC50 = 2.2 x 10(-8) M). However, it has not been possible to relate the cytotoxic property to the symptoms of intoxication. Bistramide A may originate from the urochordate itself or from symbiotic algae.

Ladies and Gentlemen, Dear Colleagues: I speak to you today about my great-grandfather, Dr. Pierre Marie, whom I always admired. I am delighted to honor with you tonight the memory of this humble man. But first I would like to thank Dr. Richard Robbins and Prof. Roger Guillemin for their kind invitation. Since I met Prof. Guillemin last year, we have established an exchange of information and documents concerning my great-grandfather.

The effects of gymnochrome A were tested on the electrical activity of the frog atrial heart muscle. Gymnochrome A (1-5 microM) did not alter the resting potential. Gymnochrome A (5 microM) slowed the initial depolarizing phase of the spontaneously beating action potential. Under voltage-clamp conditions gymnochrome A (5 microM) did not affect the electrical constant of the membrane and the kinetic parameters of the peak Na+ current (INa) recorded in the Ringer solution containing tetraethylammonium (2 mM) and Cd2+ (1 mM) but shifted the membrane potential at which the current both activated and reached its maximal value toward more negative membrane potentials. It did not alter the reversal potential for INa, indicating that the selectivity of the Na+ channels had not changed. These observations suggest that gymnochrome A binds to the membrane and shifts the activation of INa on the voltage axis by modifying the free negative fixed charges present at the membrane surface rather than by occupying a specific site on the Na+ channel. Photoexcited gymnochrome A transiently triggered an early outward current which lengthened the time-to-peak of INa and decreased its amplitude. In addition, photoexcited gymnochrome A blocked the background K+ current. This is, to our knowledge, the first time that such effects are reported on the cardiac muscle. These observations suggest that the photoexcitation of gymnochrome produces physico-chemical effects which lead to intracellular changes. Further experiments are required to determine their nature.

There is a paucity of data on posaconazole (PCZ) dosing and therapeutic-drug-monitoring (TDM) in allogeneic hematopoietic cell transplant recipients (allogeneic-HCTr). This was a 3-year retrospective multicenter study (January 1, 2016 to December 31, 2018) in adult allogeneic-HCTr who received PCZ (intravenously, IV and/or as delayed-release tablet, DRT) as prophylaxis or treatment for ≥7 consecutive days (D) with at least 1-PCZ-level available using data of the Swiss Transplant Cohort Study. The primary objective was to describe the distribution of PCZ-level and identify predictors of therapeutic PCZ-level and associations between PCZ-dosing and PCZ-level. A total of 288 patients were included: 194 (67.4%) and 94 (32.6%) received PCZ as prophylaxis and treatment, respectively, for a median of 90 days (interquartile range, IQR: 42–188.5). There were 1944 PCZ-level measurements performed, with a median PCZ level of 1.3 mg/L (IQR: 0.8-1.96). PCZ-level was

Background The burden and timeline of posttransplant infections are not comprehensively documented in the current era of immunosuppression and prophylaxis. Methods In this prospective study nested within the Swiss Transplant Cohort Study (STCS), all clinically relevant infections were identified by transplant–infectious diseases physicians in persons receiving solid organ transplant (SOT) between May 2008 and December 2014 with ≥12 months of follow-up. Results Among 3541 SOT recipients, 2761 (1612 kidney, 577 liver, 286 lung, 213 heart, and 73 kidney-pancreas) had ≥12 months of follow-up; 1520 patients (55%) suffered 3520 infections during the first year posttransplantation. Burden and timelines of clinically relevant infections differed between transplantations. Bacteria were responsible for 2202 infections (63%) prevailing throughout the year, with a predominance of Enterobacteriaceae (54%) as urinary pathogens in heart, lung, and kidney transplant recipients, and as digestive tract pathogens in liver transplant recipients. Enterococcus spp (20%) occurred as urinary tract pathogens in kidney transplant recipients and as digestive tract pathogens in liver transplant recipients, and Pseudomonas aeruginosa (9%) in lung transplant recipients. Among 1039 viral infections, herpesviruses predominated (51%) in kidney, liver, and heart transplant recipients. Among 263 fungal infections, Candida spp (60%) prevailed as digestive tract pathogens in liver transplant recipients. Opportunistic pathogens, including Aspergillus fumigatus (1.4%) and cytomegalovirus (6%), were rare, scattering over 12 months across all SOT recipients. Conclusions In the current era of immunosuppression and prophylaxis, SOT recipients experience a high burden of infections throughout the first year posttransplantation, with rare opportunistic pathogens and a predominance of bacteria., Data on burden and timeline of infections following solid organ transplantation are currently lacking. This Swiss nationwide cohort study found a high burden of infections throughout the first year posttransplantation, with rare opportunistic pathogens and a predominance of bacteria.

Background The burden of antimicrobial resistance is high in solid organ transplant (SOT) recipients. Among Swiss SOT recipients, we assessed temporal trends of ESBL-producing Enterobacterales (ESBL-E), identified risk factors for ESBL-E, and assessed the impact of resistance on patient outcome. Methods Data from the Swiss Transplant Cohort Study (STCS), a nationwide prospective cohort of SOT-recipients, were analysed. Temporal trends were described for ESBL-detection among Escherichia coli and non-Escherichia coli. In a nested case–control study, cases with ESBL-E infection were 1:1 matched (by time since transplantation, organ transplant, pathogen) to controls infected with non-ESBL-E. Factors associated with resistance and with unfavourable 30-day outcome (death, infection relapse, graft loss) were assessed. Results From 2012 to 2018, we identified 1′212 infection episodes caused by Enterobacterales in 1′074 patients, thereof 11.4% (138/1′212) caused by ESBL-E. The proportion of ESBL-production among Escherichia coli remained stable over time (p = 0.93) but increased for non-E. coli (p = 0.02) Enterobacterales. In the case–control study (n = 102), antibiotic pre-treatment was independently associated with ESBL-production (aOR = 2.6, 95%-CI: 1.0–6.8, p = 0.046). Unfavourable outcome occurred in 24/51 (47%) cases and 9/51 (18%) controls (p = 0.003). Appropriate empiric antibiotic therapy was the only modifiable factor associated with unfavourable outcome. Conclusions In Swiss SOT-recipients, proportion of infections with ESBL-producing non-E. coli Enterobacterales increased in recent years. Antibiotic pre-treatment represents a risk factor for ESBL-E. Improving appropriateness of empiric antibiotic treatment might be an important measure to reduce unfavourable outcome, which was observed in almost half of SOT-recipients with ESBL-E infections.

Acute antibody-mediated rejection (AMR) remains a challenge after kidney transplantation (KT). As there is no clear-cut treatment recommendation, accurate information on current therapeutic strategies in real-life practice is needed. KT recipients from the multicenter Swiss Transplant Cohort Study treated for acute AMR during the first post-transplant year were included retrospectively. We aimed at describing the anti-rejection protocols used routinely, as well as patient and graft outcomes, with focus on infectious complications. Overall, 65/1669 (3.9%) KT recipients were treated for 75 episodes of acute AMR. In addition to corticosteroid boluses, most common therapies included plasmapheresis (56.0%), intravenous immunoglobulins (IVIg) (38.7%), rituximab (25.3%), and antithymocyte globulin (22.7%). At least one infectious complication occurred within 6 months from AMR treatment in 63.6% of patients. Plasmapheresis increased the risk of overall (hazard ratio [HR]: 2.89; P-value = 0.002) and opportunistic infection (HR: 5.32; P-value = 0.033). IVIg exerted a protective effect for bacterial infection (HR: 0.29; P-value = 0.053). The recovery of renal function was complete at 3 months after AMR treatment in 67% of episodes. One-year death-censored graft survival was 90.9%. Four patients (6.2%) died during the first year (two due to severe infection). In this nationwide cohort we found significant heterogeneity in therapeutic approaches for acute AMR. Infectious complications were common, particularly among KT recipients receiving plasmapheresis.

MAIN PROBLEM Increasing evidence indicates a role of vitamin D in the immune system affecting response to infections. We aimed to characterize the role of vitamin D status, i.e. deficiency (25-OH vitamin D [25-OHD] < 50nmol/l) and no deficiency (25-OHD ≥ 50nmol/l) in incident infections after liver transplantation. METHODS In 135 liver transplant recipients blood samples drawn at time of liver transplantation and 6 months afterwards were used to determine 25-OHD levels. Incident infections episodes were prospectively collected within the STCS database. Poisson regression was applied to address associations between vitamin D status and incident infections. RESULTS Vitamin D deficiency was common at time of transplantation and 6 months afterwards without a significant change in median 25-OHD levels. In univariable analyses vitamin D deficiency was a risk factor for incident infections in the first 6 months post-transplant (IRR 1.52, 95% CI 1.08-2.15, P=0.018) and for bacterial infections occurring after 6 up to 30 months post-transplant (IRR 2.29, 95% CI 1.06-4.94, P=0.034). These associations were not detectable in multivariable analysis with adjustment for multiple confounders. CONCLUSIONS Efforts to optimize vitamin D supplementation in liver transplant recipients are needed. Our data question the role of vitamin D deficiency in incident infections. This article is protected by copyright. All rights reserved.

Objective To identify the determinants of antibody responses to adjuvanted split influenza A (H1N1) vaccines in patients with inflammatory rheumatic diseases. Methods One hundred seventy-three patients (82 with rheumatoid arthritis, 45 with spondylarthritis, and 46 with other inflammatory rheumatic diseases) and 138 control subjects were enrolled in this prospective single-center study. Controls received 1 dose of adjuvanted influenza A/09/H1N1 vaccine, and patients received 2 doses of the vaccine. Antibody responses were measured by hemagglutination inhibition assay before and 3–4 weeks after each dose. Geometric mean titers (GMTs) and rates of seroprotection (GMT ≥40) were calculated. A comprehensive medical questionnaire was used to identify the determinants of vaccine responses and adverse events. Results Baseline influenza A/09/H1N1 antibody levels were low in patients and controls (seroprotection rates 14.8% and 14.2%, respectively). A significant response to dose 1 was observed in both groups. However, the GMT and the seroprotection rate remained significantly lower in patients (GMT 146 versus 340, seroprotection rate 74.6% versus 87%; both P < 0.001). The second dose markedly increased antibody titers in patients, with achievement of a similar GMT and seroprotection rate as elicited with a single dose in healthy controls. By multivariate regression analysis, increasing age, use of disease-modifying antirheumatic drugs (DMARDs) (except hydroxychloroquine and sulfasalazine), and recent (within 3 months) B cell depletion treatment were identified as the main determinants of vaccine responses; tumor necrosis factor α antagonist treatment was not identified as a major determinant. Immunization was well tolerated, without any adverse effect on disease activity. Conclusion DMARDs exert distinct influences on influenza vaccine responses in patients with inflammatory rheumatic diseases. Two doses of adjuvanted vaccine were necessary and sufficient to elicit responses in patients similar to those achieved with 1 dose in healthy controls.

BACKGROUND Responses to influenza vaccines are poorly characterized in immunocompromised patients. The goal of this study was to assess the efficacy of the AS03 adjuvanted influenza H1N1/A/09 vaccine in allogeneic hematopoietic stem cell transplant recipients. DESIGN AND METHODS We enrolled 65 patients and 138 controls in an open prospective study. Controls received one dose and patients 2 doses of the AS03 adjuvanted influenza H1N1/A/09 vaccine at a 3 week interval. Geometric mean titers and seroprotection/seroconversion rates were determined by hemagglutination inhibition before and four weeks after the last immunization. Clinical and biological markers including immunoglobulins CD3+ CD4+ CD8+ and naïve CD4+ T cell counts were assessed in all patients. RESULTS Baseline seroprotection rates were low in patients (6.6) and controls (14.8). After 2 doses patients (n=57 92.3) achieved similar seroprotection rates (84 vs. 87 P=0.65) and antibody titers (305 vs. 340 P=0.88) as controls (n=131 93.9) after one dose. In univariate analysis transplant to vaccination interval less than 12 months active graft versus host disease immunosuppressive drugs hemoglobin less than 12 g/L lymphopenia less than 1 G/L IgG less than 4 g/L IgA less than 0.5 g/L IgM less than 0.5 g/L and naive CD4+ T cells less than 150/µL were significantly associated with weaker responses. Multivariate analysis identified transplant to vaccination interval and active graft versus host disease as the most powerful negative predictors of antibody responses (P=0.04 and P=0.002 respectively). Vaccination was well tolerated in both cohorts. CONCLUSIONS In allogeneic hematopoietic stem cell transplant recipients 2 doses of an adjuvanted influenza vaccine elicited comparable responses to a single dose in healthy individuals. However vaccine responses remained poor in patients with ongoing graft versus host disease supporting the need for additional strategies in this high risk patient population. (ClinicalTrials.gov Identifier: NCT01022905).