PIP2 Mediated Inhibition of TREK Potassium Currents by Bradykinin in Mouse Sympathetic Neurons.

Bradykinin (BK), a hormone inducing pain and inflammation, is known to inhibit potassium M-currents (IM) and to increase the excitability of the superior cervical ganglion (SCG) neurons by activating the Ca2+-calmodulin pathway. M-current is also reduced by muscarinic agonists through the depletion of membrane phosphatidylinositol 4,5-biphosphate (PIP2). Similarly, the activation of muscarinic receptors inhibits the current through two-pore domain potassium channels (K2P) of the "Tandem of pore-domains in a Weakly Inward rectifying K+ channel (TWIK)-related channels" (TREK) subfamily by reducing PIP2 in mouse SCG neurons (mSCG). The aim of this work was to test and characterize the modulation of TREK channels by bradykinin. We used the perforated-patch technique to investigate riluzole (RIL) activated currents in voltage- and current-clamp experiments. RIL is a drug used in the palliative treatment of amyotrophic lateral sclerosis and, in addition to blocking voltage-dependent sodium channels, it also selectively activates the K2P channels of the TREK subfamily. A cell-attached patch-clamp was also used to investigate TREK-2 single channel currents. We report here that BK reduces spike frequency adaptation (SFA), inhibits the riluzole-activated current (IRIL), which flows mainly through TREK-2 channels, by about 45%, and reduces the open probability of identified single TREK-2 channels in cultured mSCG cells. The effect of BK on IRIL was precluded by the bradykinin receptor (B2R) antagonist HOE-140 (d-Arg-[Hyp3, Thi5, d-Tic7, Oic8]BK) but also by diC8PIP2 which prevents PIP2 depletion when phospholipase C (PLC) is activated. On the contrary, antagonizing inositol triphosphate receptors (IP3R) using 2-aminoethoxydiphenylborane (2-APB) or inhibiting protein kinase C (PKC) with bisindolylmaleimide did not affect the inhibition of IRIL by BK. In conclusion, bradykinin inhibits TREK-2 channels through the activation of B2Rs resulting in PIP2 depletion, much like we have demonstrated for muscarinic agonists. This mechanism implies that TREK channels must be relevant for the capture of information about pain and visceral inflammation.

Ion Channels and Thermosensitivity: TRP, TREK, or Both?

Controlling body temperature is a matter of life or death for most animals, and in mammals the complex thermoregulatory system is comprised of thermoreceptors, thermosensors, and effectors. The activity of thermoreceptors and thermoeffectors has been studied for many years, yet only recently have we begun to obtain a clear picture of the thermosensors and the molecular mechanisms involved in thermosensory reception. An important step in this direction was the discovery of the thermosensitive transient receptor potential (TRP) cationic channels, some of which are activated by increases in temperature and others by a drop in temperature, potentially converting the cells in which they are expressed into heat and cold receptors. More recently, the TWIK-related potassium (TREK) channels were seen to be strongly activated by increases in temperature. Hence, in this review we want to assess the hypothesis that both these groups of channels can collaborate, possibly along with other channels, to generate the wide range of thermal sensations that the nervous system is capable of handling.

Turbot (Scophthalmus maximus) Nk-lysin induces protection against the pathogenic parasite Philasterides dicentrarchi via membrane disruption.

P. dicentrarchi is one of the most threatening pathogens for turbot aquaculture. This protozoan ciliate is a causative agent of scuticociliatosis, which is a disease with important economic consequences for the sector. Neither vaccines nor therapeutic treatments are commercially available to combat this infection. Numerous antimicrobial peptides (AMPs) have demonstrated broad-spectrum activity against bacteria, viruses, fungi, parasites and even tumor cells; an example is Nk-lysin (Nkl), which is an AMP belonging to the saposin-like protein (SAPLIP) family with an ability to interact with biological membranes. Following the recent characterization of turbot Nkl, an expression plasmid encoding Nkl was constructed and an anti-Nkl polyclonal antibody was successfully tested. Using these tools, we demonstrated that although infection did not clearly affect nkl mRNA expression, it induced changes at the protein level. Turbot Nkl had the ability to inhibit proliferation of the P. dicentrarchi parasite both in vivo and in vitro. Moreover, a shortened peptide containing the active core of turbot Nkl (Nkl71-100) was synthesized and showed high antiparasitic activity with a direct effect on parasite viability that probably occurred via membrane disruption. Therefore, the nkl gene may be a good candidate for genetic breeding selection of fish, and either the encoded peptide or its shortened analog is a promising antiparasitic treatment in aquaculture.

Protocol for cryopreservation of the turbot parasite Philasterides dicentrarchi (Ciliophora, Scuticociliatia).

Philasterides dicentrarchi is a free-living marine ciliate that can become an endoparasite that causes a severe disease called scuticociliatosis in cultured fish. Long-term maintenance of this scuticociliate in the laboratory is currently only possible by subculture, with periodic passage in fish to maintain the virulence of the isolates. In this study, we developed and optimized a cryopreservation protocol similar to that used for the long-term storage of scuticociliates of the genus Miamiensis. The cryogenic medium comprised ATCC medium 1651 and a combination of 11% dimethylsulfoxide and 5% glycerol. We have verified that the most important factor ensuring the efficiency of the cryopreservation procedure is the growth phase of the culture, and that ciliates should be cryopreserved at the stationary phase (around the sixth day of culture). The cryopreservation protocol described here can be used for all strains of P. dicentrarchi as well as commercial strains of Miamiensis and enables the virulence of the strains to be maintained. Finally, this cryopreservation protocol has been shown to be more effective than others routinely applied to scuticociliates, yielding a higher survival rate with a lower initial concentration of ciliates. The results obtained indicate that the cropreservation protocol enables the long-term storage of scuticociliate parasites while maintaining the virulence of the isolates. The protocol is therefore suitable for use in vaccine production and related studies.

Combined antiparasitic and anti-inflammatory effects of the natural polyphenol curcumin on turbot scuticociliatosis.

The histiophagous scuticociliate Philasterides dicentrarchi is the aetiological agent of scuticociliatosis, a parasitic disease of farmed turbot. Curcumin, a polyphenol from Curcuma longa (turmeric), is known to have antioxidant and anti-inflammatory properties. We investigated the in vitro effects of curcumin on the growth of P. dicentrarchi and on the production of pro-inflammatory cytokines in turbot leucocytes activated by parasite cysteine proteases. At 100 µm, curcumin had a cytotoxic effect and completely inhibited the growth of the parasite. At 50 µm, curcumin inhibited the protease activity of the parasite and expression of genes encoding two virulence-associated proteases: leishmanolysin-like peptidase and cathepsin L-like. At concentrations between 25 and 50 µm, curcumin inhibited the expression of S-adenosyl-L-homocysteine hydrolase, an enzyme involved in the biosynthesis of the amino acids methionine and cysteine. At 100 µm, curcumin inhibited the expression of the cytokines tumour necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1ß) produced in turbot leucocytes activated by parasite proteases. Results show that curcumin has a dual effect on scuticociliatosis: an antiparasitic effect on the catabolism and anabolism of ciliate proteins, and an anti-inflammatory effect that inhibits the production of proinflammatory cytokines in the host. The present findings suggest the potential usefulness of this polyphenol in treating scuticociliatosis.

Long-term drug survival of biological agents in patients with rheumatoid arthritis in clinical practice.

OBJECTIVES: To assess and compare the long-term drug survival (time to drug discontinuation) of biological agents (BA) in patients with rheumatoid arthritis (RA) in clinical practice. Factors associated with discontinuation of BAs were also investigated. METHOD: We conducted an observational longitudinal study of RA patients taking BAs from 1999 to 2013. The primary endpoint was BA discontinuation due to: adverse drug reactions (ADRs), inefficacy, and other causes. Incidence rates of discontinuation (IRs) per 100 patient-years were estimated using survival techniques. Comparisons between BA discontinuation rates and other associated factors were made using Cox regression models. RESULTS: We included 851 courses of BA therapy (1869 patient-years). Adalimumab (33%) was the BA most frequently used, followed by etanercept (24.4%), infliximab, and rituximab. Treatment was suspended in 558 cases [IR 29.8, 95% confidence interval (CI) 27-32]. In the first year of therapy 68% continued on BAs, and after 10 years the retention rate did not exceed 10%. The IR due to inefficacy was 12.1 (95% CI 10.6-13.8) and the IR of ADRs was 13.6 (95% CI 12-15). The unadjusted IR was higher for rituximab than for tumour necrosis factor (TNF) antagonists. In multivariate analysis, infliximab was the BA with the highest risk of discontinuation, compared to adalimumab. Calendar period, taking subsequent courses of BAs, concomitant therapy, and specific comorbidities were also independent factors associated with discontinuation. CONCLUSIONS: After several years of BA treatment in clinical practice, the survival rate was low, mainly as a result of ADRs and inefficacy. We also found differences between the discontinuation rates of BAs and other clinical factors that modify their survival.

Muscarinic modulation of TREK currents in mouse sympathetic superior cervical ganglion neurons.

Muscarinic receptors play a key role in the control of neurotransmission in the autonomic ganglia, which has mainly been ascribed to the regulation of potassium M-currents and voltage-dependent calcium currents. Muscarinic agonists provoke depolarization of the membrane potential and a reduction in spike frequency adaptation in postganglionic neurons, effects that may be explained by M-current inhibition. Here, we report the presence of a riluzole-activated current (IRIL ) that flows through the TREK-2 channels, and that is also inhibited by muscarinic agonists in neurons of the mouse superior cervical ganglion (mSCG). The muscarinic agonist oxotremorine-M (Oxo-M) inhibited the IRIL by 50%, an effect that was abolished by pretreatment with atropine or pirenzepine, but was unaffected in the presence of himbacine. Moreover, these antagonists had similar effects on single-channel TREK-2 currents. IRIL inhibition was unaffected by pretreatment with pertussis toxin. The protein kinase C blocker bisindolylmaleimide did not have an effect, and neither did the inositol triphosphate antagonist 2-aminoethoxydiphenylborane. Nevertheless, the IRIL was markedly attenuated by the phospholipase C (PLC) inhibitor ET-18-OCH3. Finally, the phosphatidylinositol-3-kinase/phosphatidylinositol-4-kinase inhibitor wortmannin strongly attenuated the IRIL , whereas blocking phosphatidylinositol 4,5-bisphosphate (PIP2 ) depletion consistently prevented IRIL inhibition by Oxo-M. These results demonstrate that TREK-2 currents in mSCG neurons are inhibited by muscarinic agonists that activate M1 muscarinic receptors, reducing PIP2 levels via a PLC-dependent pathway. The similarities between the signaling pathways regulating the IRIL and the M-current in the same neurons reflect an important role of this new pathway in the control of autonomic ganglia excitability.

Particle size and traffic of phagocytes between the turbot peritoneal cavity and lymphoid organs.

New adjuvants based on microparticles are being developed for use in fish vaccines. The size of the microparticles may affect the immune response generated, as the adjuvant can either be retained at the site of injection or transported to lymphoid organs. The objectives of this study were to evaluate the maximum size of particles that can be exported out of the cavity, to determine the phagocytosis kinetics and to establish the routes whereby particle-containing cells move from the peritoneal cavity after injection. Fish were injected intraperitoneally with fluorescent cyclodextrins or with fluorescent particles of different size (0.1-10 µm). Phagocytes containing beads of size 4 µm or larger did not reach lymphoid organs, although some were able to cross the peritoneal mesothelium. The number of free peritoneal neutrophils and macrophage-like cells containing beads peaked at 6 and 24 h respectively, and the numbers then decreased quickly, indicating migration of cells to the peritoneum or other body areas. Migration of cells containing beads mainly occurs through the visceral peritoneum. These cells were found on the latero-ventral surfaces of the peritoneal folds that connect the visceral organs. Except for some vascularised areas, the surfaces of liver, stomach and intestine were devoid of particle-containing cells. Some cells containing beads were also found attached to the parietal peritoneum, although in lower numbers than in the visceral peritoneum. Such cells were also found in high numbers in the spleen and kidney 6 h post injection. Because cells containing phagocytosed material quickly become attached to the peritoneum or migrate to lymphoid organs, the immune response generated by a vaccine or by an inflammatory stimulus should probably be evaluated in attached cells as well as in free peritoneal cells.

The neuronal serum- and glucocorticoid-regulated kinase 1.1 reduces neuronal excitability and protects against seizures through upregulation of the M-current.

The M-current formed by tetramerization of Kv7.2 and Kv7.3 subunits is a neuronal voltage-gated K(+) conductance that controls resting membrane potential and cell excitability. In Xenopus laevis oocytes, an increase in Kv7.2/3 function by the serum- and glucocorticoid-regulated kinase 1 (SGK1) has been reported previously (Schuetz et al., 2008). We now show that the neuronal isoform of this kinase (SGK1.1), with distinct subcellular localization and modulation, upregulates the Kv7.2/3 current in Xenopus oocytes and mammalian human embryonic kidney HEK293 cells. In contrast to the ubiquitously expressed SGK1, the neuronal isoform SGK1.1 interacts with phosphoinositide-phosphatidylinositol 4,5-bisphosphate (PIP(2)) and is distinctly localized to the plasma membrane (Arteaga et al., 2008). An SGK1.1 mutant with disrupted PIP(2) binding sites produced no effect on Kv7.2/3 current amplitude. SGK1.1 failed to modify the voltage dependence of activation and did not change activation or deactivation kinetics of Kv7.2/3 channels. These results suggest that the kinase increases channel membrane abundance, which was confirmed with flow cytometry assays. To evaluate the effect of the kinase in neuronal excitability, we generated a transgenic mouse (Tg.sgk) expressing a constitutively active form of SGK1.1 (S515D). Superior cervical ganglion (SCG) neurons isolated from Tg.sgk mice showed a significant increase in M-current levels, paralleled by reduced excitability and more negative resting potentials. SGK1.1 effect on M-current in Tg.sgk-SCG neurons was counteracted by muscarinic receptor activation. Transgenic mice with increased SGK1.1 activity also showed diminished sensitivity to kainic acid-induced seizures. Altogether, our results unveil a novel role of SGK1.1 as a physiological regulator of the M-current and neuronal excitability.

Safe switching from a pdFIX (Immunine®) to a rFIX (Bax326).

The ability to switch between coagulation factors safely is of common interest to haemophilia patients and treating physicians. This is the first formal prospective comparative evaluation of safety, efficacy and incremental recovery of a plasma-derived FIX (pdFIX) and a recombinant FIX (rFIX) in the same haemophilia B patients following a switch from pdFIX Immunine® to a recently developed rFIX Bax326 product. Patients (aged <65 years) who completed a pretreatment study which prospectively documented the exposure to Immunine® and monitored FIX inhibitors while receiving prophylactic treatment were transitioned into pivotal (patients aged 12-65 years) and paediatric (patients aged <12 years) clinical studies investigating prophylaxis and treatment of bleeding episodes with Bax326. None of the 44 patients developed inhibitory or specific binding anti-FIX antibodies during the course of the studies. A total of 38 unrelated adverse events (AEs) were occurred in 20/44 (45.5%) subjects during the Immunine® study. Following a switch to Bax326, 51 AEs were reported in 25/44 (56.8%) subjects. The incidence of AEs related to Bax326 treatment (two episodes of dysgeusia in one patient) was low (2.3%); there were no serious adverse reactions. The comparison between Immunine® and Bax326 demonstrated analogous haemostatic characteristics and annualized bleeding rates. Overall, there is direct evidence indicating a safe and clinically effective transition from a pdFIX (Immunine®) to a newly developed rFIX (Bax326(1) ) for prophylaxis and treatment of bleeding in previously treated patients of all age cohorts with severe or moderately severe haemophilia B.

Pharmacokinetics, efficacy and safety of BAX326, a novel recombinant factor IX: a prospective, controlled, multicentre phase I/III study in previously treated patients with severe (FIX level <1%) or moderately severe (FIX level ≤2%) haemophilia B.

BAX326 is a recombinant factor IX (rFIX; nonacog gamma) manufactured without the addition of any materials of human or animal origin, and with two viral inactivation steps (solvent/detergent treatment and 15 nm nanofiltration). The aim of this prospective trial was to investigate the pharmacokinetics, haemostatic efficacy and safety of BAX326 in previously treated patients aged 12-65 years with severe or moderately severe haemophilia B. BAX326 was safe and well tolerated in all 73 treated subjects; adverse events considered related to treatment (2.7% incidence, all non-serious) were transient and mild, and no hypersensitivity reactions, inhibitor formation or thrombotic events were observed. Pharmacokinetic (PK) equivalence (n = 28) between BAX326 and a licensed rFIX was confirmed in terms of the ratio of geometric mean AUC(0-72) h per dose. Twice-weekly prophylaxis [mean duration 6.2 (±0.7) months; 1.8 (±0.1) infusions per week, 49.5 (±4.8) IU kg(-1) per infusion] was effective in preventing bleeding episodes, with a significantly lower (79%, P < 0.001) annualized bleed rate (4.2) compared to an on-demand treatment in a historical control group (20.0); 24 of 56 subjects on prophylaxis (43%) did not bleed throughout the study observation period. Of 249 total acute bleeds, 211 (84.7%) were controlled with one to two infusions of BAX326. Haemostatic efficacy at resolution of bleed was rated excellent or good in 96.0% of all treated bleeding episodes. The results of this study indicate that BAX326 is safe and efficacious in treating bleeds and routine prophylaxis in patients aged 12 years and older with haemophilia B.

Inflammatory responses and side effects generated by several adjuvant-containing vaccines in turbot.

Several of the adjuvants used in fish vaccines cause adhesions in internal organs when they are injected intraperitoneally. We describe the damage caused by vaccines containing different adjuvants in the turbot Scophthalmus maximus and show that internal adhesions can be greatly reduced by injecting the fish in a specific way. Injection of fish with the needle directed towards the anterior part of the peritoneal cavity induced formation of a single cell-vaccine mass (CVM) that became attached to the parietal peritoneum. However, injection of the fish with the needle pointing in the opposite direction generated many small CVM that became attached to the visceral and parietal peritoneum and in some cases caused internal adhesions. We describe the structural and cellular changes in the adjuvant-induced CVMs. The CVMs mainly comprised neutrophils and macrophages, although most of the former underwent apoptosis, which was particularly evident from day 3 post-injection. The apoptotic cells were phagocytosed by macrophages, which were the dominant cell type from the first days onwards. All of the vaccines induced angiogenesis in the area of contact between the CVM and the mesothelium. Vaccines containing oil-based adjuvants or microspheres induced the formation of granulomas in the CVM; however, no granulomas were observed in the CVM induced by vaccines containing aluminium hydroxide or Matrix-Q(®) as adjuvants. All of the vaccines induced strong migration of cells to the peritoneal cavity. Although some of these cells remained unattached in the peritoneal cavity, most of them formed part of the CVM. We also observed migration of the cells from the peritoneal cavity to lymphoid organs, indicating bidirectional traffic of cells between the inflamed areas and these organs.

Expression of human endogenous retrovirus HERV-K18 is associated with clinical severity in osteoarthritis patients.

OBJECTIVES: The aim of this study was to evaluate the involvement of human endogenous retrovirus K18 (HERV-K18) in osteoarthritis (OA), by genotyping the HERV-K18 env locus in OA patients and controls, and analysing HERV-K18 RNA expression and its association with OA risk and clinical variables. METHOD: We recruited 558 patients with symptomatic OA and 600 controls. We performed the genotyping by TaqMan assays and the analysis of expression by quantitative real-time polymerase chain reaction (qRT-PCR). Scores on the Western Ontario and McMasters Universities Osteoarthritis Index (WOMAC), the Lequesne index, and the Stanford Health Assessment Questionnaire (HAQ) were analysed with regard to the expression levels of HERV-K18. RESULTS: The 18.3 haplotype tended towards an association with OA risk and concordantly this haplotype was associated with a higher HERV-K18 expression (p = 0.05). We found statistically significant differences when we compared the scores on the WOMAC, the Lequesne index for knee and hip, and the HAQ between OA patients with higher expression [normalization ratio (NR) > 10] and OA patients without HERV-K18 expression (p = 0.0003, 0.0005, 0.002, and 0.05, respectively), and also when the comparison was made between OA patients with higher expression (NR > 10) and OA patients with low expression of HERV-K18 (NR = 1) for the WOMAC and the Lequesne index for knee and hip (p = 0.002, 0.013, and 0.006, respectively). CONCLUSIONS: We found an association between health status measurement systems and severity index for OA and the levels of expression of HERV-K18. These results suggest the possible involvement of HERV-K18 in the aetiopathogenesis of the disease.

Biodegradable microparticles covalently linked to surface antigens of the scuticociliate parasite P. dicentrarchi promote innate immune responses in vitro.

The histiophagous scuticociliate endoparasite Philasterides dicentrarchi is an emerging pathogen that infects the turbot Scophthalmus maximus and thus causes important economic losses in turbot aquaculture. This in vitro study investigated the adjuvant capacity of biodegradable microspheres (MS) composed of two polymers (chitosan and Gantrez(®)) covalently coupled to surface antigens (Ag) of P. dicentrarchi. The coupled MS-Ag significantly stimulated the phagocytic response of both murine macrophages (RAW 264.7 cells) and leukocytes from the anterior kidney of turbot (HLK), at a level similar to that induced by zymosan A. The MS-Ag also significantly increased production of reactive oxygen and nitrogen species, as shown by the increased O(2) consumption and stimulation of the respiratory burst and nitric oxide production by murine and in particular by turbot HLK. The MS-Ag stimulated the production of the proinflammatory cytokine tumour necrosis factor alpha (TNFα) by murine and turbot HLK. The results confirm the high adjuvant capacity of biodegradable MS covalently bound to Ag as regards stimulating the innate immune response, and they justify the use of MS in the production of safe and effective vaccines against fish pathogens.

Interlaboratory Evaluation of Multiple LC-MS/MS Methods and a Commercial ELISA Method for Determination of Tetrodotoxin in Oysters and Mussels.

BACKGROUND: Given the recent detection of tetrodotoxin (TTX) in bivalve molluscs but the absence of a full collaborative validation study for TTX determination in a large number of shellfish samples, interlaboratory assessment of method performance was required to better understand current capabilities for accurate and reproducible TTX quantitation using chemical and immunoassay methods. OBJECTIVE: The aim was to conduct an interlaboratory study with multiple laboratories, using results to assess method performance and acceptability of different TTX testing methods. METHODS: Homogenous and stable mussel and oyster materials were assessed by participants using a range of published and in-house detection methods to determine mean TTX concentrations. Data were used to calculate recoveries, repeatability, and reproducibility, together with participant acceptability z-scores. RESULTS: Method performance characteristics were good, showing excellent sensitivity, recovery, and repeatability. Acceptable reproducibility was evidenced by HorRat values for all LC-MS/MS and ELISA methods being less than the 2.0 limit of acceptability. Method differences between the LC-MS/MS participants did not result in statistically different results. Method performance characteristics compared well with previously published single-laboratory validated methods and no statistical difference was found in results returned by ELISA in comparison with LC-MS/MS. CONCLUSION: The results from this study demonstrate that current LC-MS/MS methods and ELISA are on the whole capable of sensitive, accurate, and reproducible TTX quantitation in shellfish. Further work is recommended to expand the number of laboratories testing ELISA and to standardize an LC-MS/MS protocol to further improve interlaboratory precision. HIGHLIGHTS: Multiple mass spectrometric methods and a commercial ELISA have been successfully assessed through an interlaboratory study, demonstrating excellent performance.

Activation of TREK currents by the neuroprotective agent riluzole in mouse sympathetic neurons.

Background K2P channels play a key role in stabilizing the resting membrane potential, thereby modulating cell excitability in the central and peripheral somatic nervous system. Whole-cell experiments revealed a riluzole-activated current (I(RIL)), transported by potassium, in mouse superior cervical ganglion (mSCG) neurons. The activation of this current by riluzole, linoleic acid, membrane stretch, and internal acidification, its open rectification and insensitivity to most classic potassium channel blockers, indicated that I(RIL) flows through channels of the TREK [two-pore domain weak inwardly rectifying K channel (TWIK)-related K channel] subfamily. Whole-ganglia and single-cell reverse transcription-PCR demonstrated the presence of TREK-1, TREK-2, and TRAAK (TWIK-related arachidonic acid-activated K(+) channel) mRNA, and the expression of these three proteins was confirmed by immunocytochemistry in mSCG neurons. I(RIL) was enhanced by zinc, inhibited by barium and fluoxetine, but unaffected by quinine and ruthenium red, strongly suggesting that it was carried through TREK-1/2 channels. Consistently, a channel with properties identical with the heterologously expressed TREK-2 was recorded in most (75%) cell-attached patches. These results provide the first evidence for the expression of K2P channels in the mammalian autonomic nervous system, and they extend the impact of these channels to the entire nervous system.

A vaccine based on biodegradable microspheres induces protective immunity against scuticociliatosis without producing side effects in turbot.

The histiophagous scuticociliate parasite Philasterides dicentrarchi is an emergent pathogen in aquaculture and causes significant economic losses on turbot (Scophthalmus maximus) farms. In this study, the surface antigens (Ag) of the parasite were encapsulated and covalently linked to a polymeric microparticle formulation composed of two biodegradable polymers (chitosan and Gantrez). The antigenicity of the formulation and the protection provided were compared in mice and turbot. This formulation induced a higher antibody (Ab) response in mice at doses of 5mg of microspheres (MS) conjugated with approximately 230 µg of Ag (MS-Ag(c)). However, Ab levels were significantly lower than in mice vaccinated with the same concentration of Ag in complete Freund's adjuvant (FCA). In turbot, the MS-Ag(c) formulation induced a higher level of Abs than that induced by the same vaccine emulsified in FCA. The challenge experiments performed with P. dicentrarchi and vaccinated turbot also showed a clear correlation between Ab levels and survival levels. Growth was significantly affected in fish vaccinated with FCA, but not in fish vaccinated with MS. The high adjuvant capacity of MS, together with its biodegradability and low toxicity to fish, makes this new vaccine an economical, effective and safe alternative to oil-based adjuvants for the immunoprophylaxis of scuticociliatosis in turbot.

Differences in the in vitro susceptibility to resveratrol and other chemical compounds among several Philasterides dicentrarchi isolates from turbot.

Philasterides dicentrarchi is a histophagous scuticociliate that causes important losses in aquaculture. Several strains that differ in morphological, genetic and serological characteristics and virulence have been isolated from outbreaks of turbot scuticociliatosis. In the present study, seven isolates of the ciliate were exposed in vitro to formalin, hydrogen peroxide and resveratrol (a phytoalexin produced by plants) in order to evaluate the susceptibility of the isolates to the different compounds. The LD50 values for the three compounds tested varied widely among the isolates. The LD100 values were similar among isolates for formalin (25-30 ppm) and resveratrol (60-70 ppm) but were very different for hydrogen peroxide (25->80 ppm). The results indicate that there are many physiological differences among isolates and even among specimens of the same isolates, which must be taken into account in designing control programmes. The naturally occurring resveratrol may be a good alternative to other compounds for reducing the amounts of viable ciliates in water.

Antiarrhythmic calcium channel blocker verapamil inhibits trek currents in sympathetic neurons.

Background and Purpose: Verapamil, a drug widely used in certain cardiac pathologies, exert its therapeutic effect mainly through the blockade of cardiac L-type calcium channels. However, we also know that both voltage-dependent and certain potassium channels are blocked by verapamil. Because sympathetic neurons of the superior cervical ganglion (SCG) are known to express a good variety of potassium currents, and to finely tune cardiac activity, we speculated that the effect of verapamil on these SCG potassium channels could explain part of the therapeutic action of this drug. To address this question, we decided to study, the effects of verapamil on three different potassium currents observed in SCG neurons: delayed rectifier, A-type and TREK (a subfamily of K2P channels) currents. We also investigated the effect of verapamil on the electrical behavior of sympathetic SCG neurons. Experimental Approach: We employed the Patch-Clamp technique to mouse SCG neurons in culture. Key Results: We found that verapamil depolarizes of the resting membrane potential of SCG neurons. Moreover, we demonstrated that this drug also inhibits A-type potassium currents. Finally, and most importantly, we revealed that the current driven through TREK channels is also inhibited in the presence of verapamil. Conclusion and Implications: We have shown that verapamil causes a clear alteration of excitability in sympathetic nerve cells. This fact undoubtedly leads to an alteration of the sympathetic-parasympathetic balance which may affect cardiac function. Therefore, we propose that these possible peripheral alterations in the autonomic system should be taken into consideration in the prescription of this drug.

Role of scuticociliate proteinases in infection success in turbot, Psetta maxima (L.).

Scuticociliatosis caused by Philasterides dicentrarchi is one of the most severe diseases of farmed turbot, Psetta maxima (L.). Immunized fish showed elevated levels of specific antibodies (Ab), which caused the destruction of parasites through the activation of complement by the alternative and classical pathways. By using affinity chromatography on bacitracin-sepharose columns, we demonstrated the existence of high levels of parasite proteinases in the serum and, to a lesser extent, in the ascitic fluid of experimentally infected fish, and the absence of such proteinases in the serum of uninfected fish. Serum from uninfected fish displayed haemolytic activity against sheep red blood cells. However, incubation of this serum with parasite proteinases led to a decrease in serum haemolytic activity, suggesting that proteinases are able to destroy fish complement. Proteinases isolated from serum or ascitic fluid of infected fish were also able to degrade turbot Ab. Preincubation of turbot serum containing specific Ab for P. dicentrarchi with the proteinases led to a significant decrease in the killing activity of the serum. The results confirm that P. dicentrarchi proteinases in serum from infected fish may provide a mechanism for circumventing normal host immunity by inactivating the Ab and complement factors required for complement activation.