Semaphorin 3C exacerbates liver fibrosis.

BACKGROUND AND AIMS: Chronic liver disease is a growing epidemic, leading to fibrosis and cirrhosis. TGF-ß is the pivotal profibrogenic cytokine that activates HSC, yet other molecules can modulate TGF-ß signaling during liver fibrosis. Expression of the axon guidance molecules semaphorins (SEMAs), which signal through plexins and neuropilins (NRPs), have been associated with liver fibrosis in HBV-induced chronic hepatitis. This study aims at determining their function in the regulation of HSCs. APPROACH AND RESULTS: We analyzed publicly available patient databases and liver biopsies. We used transgenic mice, in which genes are deleted only in activated HSCs to perform ex vivo analysis and animal models. SEMA3C is the most enriched member of the semaphorin family in liver samples from patients with cirrhosis. Higher expression of SEMA3C in patients with NASH, alcoholic hepatitis, or HBV-induced hepatitis discriminates those with a more profibrotic transcriptomic profile. SEMA3C expression is also elevated in different mouse models of liver fibrosis and in isolated HSCs on activation. In keeping with this, deletion of SEMA3C in activated HSCs reduces myofibroblast marker expression. Conversely, SEMA3C overexpression exacerbates TGF-ß-mediated myofibroblast activation, as shown by increased SMAD2 phosphorylation and target gene expression. Among SEMA3C receptors, only NRP2 expression is maintained on activation of isolated HSCs. Interestingly, lack of NRP2 in those cells reduces myofibroblast marker expression. Finally, deletion of either SEMA3C or NRP2, specifically in activated HSCs, reduces liver fibrosis in mice. CONCLUSION: SEMA3C is a novel marker for activated HSCs that plays a fundamental role in the acquisition of the myofibroblastic phenotype and liver fibrosis.

Quail eggs in artificial nests change their coloration when exposed to ambient conditions: implication for studies on nest predation.

Quail eggs have been widely used in field experiments, mainly to study factors associated with the risk of nest predation. Some shortcomings of using quail eggs in this type of study have been previously addressed (e.g., these eggs might be too big for some predators of eggs of small birds). Here, we show experimental evidence of another shortcoming of the use of these eggs in field experiments. Quail eggs exposed to sunlight rapidly faded in colour after three days, both in the visible and UV spectra, and this change was related to the amount of solar radiation received. This caused changes in the camouflage of the eggs, which may be perceived by predators with different visual systems (dichromatic, trichromatic, and tetrachromatic (for both violet- and UV-sensitive species)). Therefore, the results of field studies of nest predation using quail eggs might be questioned in those cases in which the camouflage has been altered due to the rapid changes in coloration, as this can affect the resulting predation rates. We recommend that researchers planning to use quail eggs should perform a prospective assessment of changes in coloration of eggs exposed to environmental conditions in the nest sites used by the target species.

Comparison of first-line and second-line use of fingolimod in relapsing MS: The open-label EARLIMS study.

BACKGROUND: Treatment of MS often begins with low-efficacy injectable disease-modifying therapy (iDMT). OBJECTIVES: To compare the effect of fingolimod 0.5 mg/day on clinical, MRI, patient-reported, and safety outcomes, in treatment-naïve and previously treated (≥1 iDMT) patients with early MS. METHODS: EARLIMS was a multicentre, open-label, non-randomized, parallel-group phase 3 b/4 study in Australia and Spain. Patients with relapsing-remitting MS, Expanded Disability Status Scale (EDSS) score <4.0, and ≥1-5 years since diagnosis, received daily fingolimod for 48 weeks. The primary endpoint was annualized relapse rate (ARR). RESULTS: Of 347 patients enrolled at 51 sites (treatment-naïve, 200 [57.6%]; previously treated, 147 [42.4%]), 320 completed the study (treatment-naïve, 184 [92.0%]; previously treated, 136 [92.5%]), but the study remained underpowered (planned enrolment, n = 432). Fingolimod reduced ARR to similar levels in both treatment-naïve (mean ARR [95% confidence interval], 0.21 [0.14, 0.29]) and previously treated groups (0.30 [0.20, 0.41]; p = 0.1668). There were no new safety signals. CONCLUSIONS: Fingolimod appeared equally effective as first- or second-line therapy in relapsing MS. There was a trend for better outcomes with fingolimod in treatment-naïve patients than in those previously treated with >1 iDMT.

New Algorithms Improving PML Risk Stratification in MS Patients Treated With Natalizumab.

Overview: We assessed the role of age and disease activity as new factors contributing to establish the risk of progressive multifocal leucoencephalopathy in multiple sclerosis patients treated with natalizumab in 36 University Hospitals in Europe. We performed the study in 1,307 multiple sclerosis patients (70.8% anti-John Cunninghan virus positive antibodies) treated with natalizumab for a median time of 3.28 years. Epidemiological, clinical, and laboratory variables were collected. Lipid-specific IgM oligoclonal band status was available in 277 patients. Factors associated with progressive multifocal leucoencephalopathy onset were explored by uni- and multivariate logistic regression. Results: Thirty-five patients developed progressive multifocal leucoencephalopathy. The multivariate analysis identified anti-John Cunninghan virus antibody indices and relapse rate as the best predictors for the onset of this serious opportunistic infection in the whole cohort. They allowed to stratify progressive multifocal leucoencephalopathy risk before natalizumab initiation in individual patients [area under the curve (AUC) = 0.85]. The risk ranged from <1/3,300 in patients with anti-John Cunninghan virus antibody indices <0.9 and relapse rate >0.5, to 1/50 in the opposite case. In patients with lipid-specific IgM oligoclonal bands assessment, age at natalizumab onset, anti-John Cunninghan virus antibody indices, and lipid-specific IgM oligoclonal band status predicted progressive multifocal leucoencephalopathy risk (AUC = 0.92). The absence of lipid-specific IgM oligoclonal bands was the best individual predictor (OR = 40.94). The individual risk ranged from <1/10,000 in patients younger than 45 years at natalizumab initiation, who showed anti John Cunningham virus antibody indices <0.9 and lipid-specific IgM oligoclonal bands to 1/33 in the opposite case. Conclusions: In a perspective of personalized medicine, disease activity, anti-lipid specific IgM oligoclonal bands, anti Jonh Cunninghan virus antibody levels, and age can help tailor natalizumab therapy in multiple sclerosis patients, as predictors of progressive multifocal leucoencephalopathy.

Does the use of nest materials in a ground-nesting bird result from a compromise between the risk of egg overheating and camouflage?

Many studies addressing the use of nest materials by animals have focused on only one factor to explain its function. However, the consideration of more than one factor could explain the apparently maladaptive choice of nest materials that make nests conspicuous to predators. We experimentally tested whether there is a trade-off in the use of nest materials between the risks of egg predation versus protection from overheating. We studied the ground-nesting Kentish plover, Charadrius alexandrinus, in southern Spain. We added materials differing in thermal properties and coloration to the nests, thus affecting rates of egg heating, nest temperature and camouflage. Before these manipulations, adults selected materials that were lighter than the microhabitat, probably to buffer the risk of egg overheating. However, the adults did not keep the lightest experimental materials, probably because they reduced camouflage, and this could make the nests even more easily detectable to predators. In all nests, adults removed most of the experimental materials independently of their properties, so that egg camouflage returned to the original situation within a week of the experimental treatments. Although the thermal environment may affect the choice of nest materials by plovers, ambient temperatures were not so high at our study site as to determine the acceptance of the lightest experimental materials.

Exposure of black-necked grebes (Podiceps nigricollis) to metal pollution during the moulting period in the Odiel Marshes, Southwest Spain.

European populations of black-necked grebes (Podiceps nigricollis) congregate every year to moult at the salt ponds of the Odiel Marshes (SW Spain). However, the Odiel Marshes are part of one of the most metal-polluted coastal estuaries in the world, which may pose risks to wildlife. We assessed the exposure of grebes to metal pollution during the critical moulting period in the Odiel Marshes and its potential to cause adverse health effects. Levels of metals in red blood pellet (as a biomarker of exposure), plasma carotenoids, eye redness, and body condition (as biomarkers of effects) were studied. Metal content was also analyzed in the brine shrimp Artemia parthenogenetica, the most important food for grebes in this hypersaline ecosystem during the moulting period. Results showed that, in comparison to toxicity thresholds, grebes had relatively high blood levels of arsenic (As), mercury (Hg) and zinc (Zn). The high loads found in Artemia and the way blood levels vary during the moulting period indicate that shrimp consumption may be the main route of metal exposure for grebes. Plasma carotenoids and body condition showed a positive association with exposure to As, while the relationship of lutein-like carotenoids with Hg accumulation was negative at the beginning of the moulting period to become positive afterwards. Moreover, eye redness was negatively affected by As accumulation. Factors including food resource availability, seasonal fluctuations in physiological status, and interannual variations in the degree of environmental contamination should be considered in monitoring efforts when using moult migrant waterbirds as sentinel species.

Latitudinal variation in biophysical characteristics of avian eggshells to cope with differential effects of solar radiation.

Solar radiation is an important driver of animal coloration, not only because of the effects of coloration on body temperature but also because coloration may protect from the deleterious effects of UV radiation. Indeed, dark coloration may protect from UV, but may increase the risk of overheating. In addition, the effect of coloration on thermoregulation should change with egg size, as smaller eggs have higher surface-volume ratios and greater convective coefficients than larger eggs, so that small eggs can dissipate heat quickly. We tested whether the reflectance of eggshells, egg spottiness, and egg size of the ground-nesting Kentish plover Charadrius alexandrinus is affected by maximum ambient temperature and solar radiation at breeding sites. We measured reflectance, both in the UV and human visible spectrum, spottiness, and egg size in photographs from a museum collection of plover eggshells. Eggshells of lower reflectance (darker) were found at higher latitudes. However, in southern localities where solar radiation is very high, eggshells are also of dark coloration. Eggshell coloration had no significant relationship with ambient temperature. Spotiness was site-specific. Small eggs tended to be light-colored. Thermal constraints may drive the observed spatial variation in eggshell coloration, which may be lighter in lower latitudes to diminish the risk of overheating as a result of higher levels of solar radiation. However, in southern localities with very high levels of UV radiation, eggshells are of dark coloration likely to protect embryos from more intense UV radiation. Egg size exhibited variation in relation to coloration, likely through the effect of surface area-to-volume ratios on overheating and cooling rates of eggs. Therefore, differential effects of solar radiation on functions of coloration and size of eggshells may shape latitudinal variations in egg appearance in the Kentish plover.

Long-term disability trajectories in primary progressive MS patients: A latent class growth analysis.

BACKGROUND: Several natural history studies on primary progressive multiple sclerosis (PPMS) patients detected a consistent heterogeneity in the rate of disability accumulation. OBJECTIVES: To identify subgroups of PPMS patients with similar longitudinal trajectories of Expanded Disability Status Scale (EDSS) over time. METHODS: All PPMS patients collected within the MSBase registry, who had their first EDSS assessment within 5 years from onset, were included in the analysis. Longitudinal EDSS scores were modeled by a latent class mixed model (LCMM), using a nonlinear function of time from onset. LCMM is an advanced statistical approach that models heterogeneity between patients by classifying them into unobserved groups showing similar characteristics. RESULTS: A total of 853 PPMS (51.7% females) from 24 countries with a mean age at onset of 42.4 years (standard deviation (SD): 10.8 years), a median baseline EDSS of 4 (interquartile range (IQR): 2.5-5.5), and 2.4 years of disease duration (SD: 1.5 years) were included. LCMM detected three different subgroups of patients with a mild ( n = 143; 16.8%), moderate ( n = 378; 44.3%), or severe ( n = 332; 38.9%) disability trajectory. The probability of reaching EDSS 6 at 10 years was 0%, 46.4%, and 81.9% respectively. CONCLUSION: Applying an LCMM modeling approach to long-term EDSS data, it is possible to identify groups of PPMS patients with different prognosis.

Treatment effectiveness of alemtuzumab compared with natalizumab, fingolimod, and interferon beta in relapsing-remitting multiple sclerosis: a cohort study.

BACKGROUND: Alemtuzumab, an anti-CD52 antibody, is proven to be more efficacious than interferon beta-1a in the treatment of relapsing-remitting multiple sclerosis, but its efficacy relative to more potent immunotherapies is unknown. We compared the effectiveness of alemtuzumab with natalizumab, fingolimod, and interferon beta in patients with relapsing-remitting multiple sclerosis treated for up to 5 years. METHODS: In this international cohort study, we used data from propensity-matched patients with relapsing-remitting multiple sclerosis from the MSBase and six other cohorts. Longitudinal clinical data were obtained from 71 MSBase centres in 21 countries and from six non-MSBase centres in the UK and Germany between Nov 1, 2015, and June 30, 2016. Key inclusion criteria were a diagnosis of definite relapsing-remitting multiple sclerosis, exposure to one of the study therapies (alemtuzumab, interferon beta, fingolimod, or natalizumab), age 65 years or younger, Expanded Disability Status Scale (EDSS) score 6·5 or lower, and no more than 10 years since the first multiple sclerosis symptom. The primary endpoint was annualised relapse rate. The secondary endpoints were cumulative hazards of relapses, disability accumulation, and disability improvement events. We compared relapse rates with negative binomial models, and estimated cumulative hazards with conditional proportional hazards models. FINDINGS: Patients were treated between Aug 1, 1994, and June 30, 2016. The cohorts consisted of 189 patients given alemtuzumab, 2155 patients given interferon beta, 828 patients given fingolimod, and 1160 patients given natalizumab. Alemtuzumab was associated with a lower annualised relapse rate than interferon beta (0·19 [95% CI 0·14-0·23] vs 0·53 [0·46-0·61], p<0·0001) and fingolimod (0·15 [0·10-0·20] vs 0·34 [0·26-0·41], p<0·0001), and was associated with a similar annualised relapse rate as natalizumab (0·20 [0·14-0·26] vs 0·19 [0·15-0·23], p=0·78). For the disability outcomes, alemtuzumab was associated with similar probabilities of disability accumulation as interferon beta (hazard ratio [HR] 0·66 [95% CI 0·36-1·22], p=0·37), fingolimod (1·27 [0·60-2·70], p=0·67), and natalizumab (0·81 [0·47-1·39], p=0·60). Alemtuzumab was associated with similar probabilities of disability improvement as interferon beta (0·98 [0·65-1·49], p=0·93) and fingolimod (0·50 [0·25-1·01], p=0·18), and a lower probability of disability improvement than natalizumab (0·35 [0·20-0·59], p=0·0006). INTERPRETATION: Alemtuzumab and natalizumab seem to have similar effects on annualised relapse rates in relapsing-remitting multiple sclerosis. Alemtuzumab seems superior to fingolimod and interferon beta in mitigating relapse activity. Natalizumab seems superior to alemtuzumab in enabling recovery from disability. Both natalizumab and alemtuzumab seem highly effective and viable immunotherapies for multiple sclerosis. Treatment decisions between alemtuzumab and natalizumab should be primarily governed by their safety profiles. FUNDING: National Health and Medical Research Council, and the University of Melbourne.

Contribution of different relapse phenotypes to disability in multiple sclerosis.

OBJECTIVE: This study evaluated the effect of relapse phenotype on disability accumulation in multiple sclerosis. METHODS: Analysis of prospectively collected data was conducted in 19,504 patients with relapse-onset multiple sclerosis and minimum 1-year prospective follow-up from the MSBase cohort study. Multivariable linear regression models assessed associations between relapse incidence, phenotype and changes in disability (quantified with Expanded Disability Status Scale and its Functional System scores). Sensitivity analyses were conducted. RESULTS: In 34,858 relapses recorded during 136,462 patient-years (median follow-up 5.9 years), higher relapse incidence was associated with greater disability accumulation (ß = 0.16, p < 0.001). Relapses of all phenotypes promoted disability accumulation, with the most pronounced increase associated with pyramidal (ß = 0.27 (0.25-0.29)), cerebellar (ß = 0.35 (0.30-0.39)) and bowel/bladder (ß = 0.42 (0.35-0.49)) phenotypes (mean (95% confidence interval)). Higher incidence of each relapse phenotype was associated with an increase in disability in the corresponding neurological domain, as well as anatomically related domains. CONCLUSION: Relapses are associated with accumulation of neurological disability. Relapses in pyramidal, cerebellar and bowel/bladder systems have the greatest association with disability change. Therefore, prevention of these relapses is an important objective of disease-modifying therapy. The differential impact of relapse phenotypes on disability outcomes could influence management of treatment failure in multiple sclerosis.

Highly active immunomodulatory therapy ameliorates accumulation of disability in moderately advanced and advanced multiple sclerosis.

OBJECTIVE: To evaluate variability and predictability of disability trajectories in moderately advanced and advanced multiple sclerosis (MS), and their modifiability with immunomodulatory therapy. METHODS: The epochs between Expanded Disability Status Scale (EDSS) steps 3-6, 4-6 and 6-6.5 were analysed. Patients with relapse-onset MS and having reached 6-month confirmed baseline EDSS step (3/4/6) were identified in MSBase, a global observational MS cohort study. We used multivariable survival models to examine the impact of disease-modifying therapy, clinical and demographic factors on progression to the outcome EDSS step (6/6.5). Sensitivity analyses with varying outcome definitions and inclusion criteria were conducted. RESULTS: For the EDSS 3-6, 4-6 and 6-6.5 epochs, 1560, 1504 and 1231 patients were identified, respectively. Disability trajectories showed large coefficients of variance prebaseline (0.92-1.11) and postbaseline (2.15-2.50), with no significant correlations. The probability of reaching the outcome step was not associated with prebaseline variables, but was increased by higher relapse rates during each epoch (HRs 1.58-3.07; p<0.001). A greater proportion of each epoch treated with higher efficacy therapies was associated with lower risk of reaching the outcome disability step (HRs 0.72-0.91 per 25%; p≤0.02). 3 sensitivity analyses confirmed these results. CONCLUSIONS: Disease progression during moderately advanced and advanced MS is highly variable and amnesic to prior disease activity. Lower relapse rates and greater time on higher efficacy immunomodulatory therapy after reaching EDSS steps 3, 4 and 6 are associated with a decreased risk of accumulating further disability. Highly effective immunomodulatory therapy ameliorates accumulation of disability in moderately advanced and advanced relapse-onset MS.

Higher latitude is significantly associated with an earlier age of disease onset in multiple sclerosis.

BACKGROUND: Age at onset (AAO) in multiple sclerosis (MS) is an important marker of disease severity and may have prognostic significance. Understanding what factors can influence AAO may shed light on the aetiology of this complex disease, and have applications in the diagnostic process. METHODS: The study cohort of 22 162 eligible patients from 21 countries was extracted from the MSBase registry. Only patients with MS aged ≥16 years were included. To reduce heterogeneity, only centres of largely European descent were included for analysis. AAO was defined as the year of the first symptom suggestive of inflammatory central nervous system demyelination. Predictors of AAO were evaluated by linear regression. RESULTS: Compared with those living in lower latitudes (19.0-39.9°), onset of symptoms was 1.9 years earlier for those at higher latitudes (50.0-56.0°) (p=3.83×10-23). A reciprocal relationship was seen for ambient ultraviolet radiation (UVR), with a significantly increasing AAO for patients with MS per each quartile increment of ambient UVR (p=1.56×10-17). We found that the AAO of female patients was ∼5 months earlier than male patients (p=0.002). AAO of progressive-onset patients with MS were ∼9 years later than relapsing-onset patients (p=1.40×10-265). CONCLUSIONS: An earlier AAO in higher latitude regions was found in this worldwide European-descent cohort and correlated inversely with variation in latitudinal UVR. These results suggest that environmental factors which act at the population level may significantly influence disease severity characteristics in genetically susceptible populations.

Latitudinal-Related Variation in Wintering Population Trends of Greylag Geese (Anser Anser) along the Atlantic Flyway: A Response to Climate Change?

The unusually high quality of census data for large waterbirds in Europe facilitates the study of how population change varies across a broad geographical range and relates to global change. The wintering population of the greylag goose Anser anser in the Atlantic flyway spanning between Sweden and Spain has increased from 120 000 to 610 000 individuals over the past three decades, and expanded its wintering range northwards. Although population sizes recorded in January have increased in all seven countries in the wintering range, we found a pronounced northwards latitudinal effect in which the rate of increase is higher at greater latitudes, causing a constant shift in the centre of gravity for the spatial distribution of wintering geese. Local winter temperatures have a strong influence on goose numbers but in a manner that is also dependent on latitude, with the partial effect of temperature (while controlling for the increasing population trend between years) being negative at the south end and positive at the north end of the flyway. Contrary to assumptions in the literature, the expansion of crops exploited by greylag geese has made little contribution to the increases in population size. Only in one case (expansion of winter cereals in Denmark) did we find evidence of an effect of changing land use. The expanding and shifting greylag population is likely to have increasing impacts on habitats in northern Europe during the course of this century.

Effects of diazoxide in multiple sclerosis: A randomized, double-blind phase 2 clinical trial.

OBJECTIVE: The aim of this study was to test the safety of diazoxide and to search for signs of efficacy in patients with relapsing-remitting multiple sclerosis (RRMS). METHODS: In this multicenter, randomized, placebo-controlled, double-blind trial (treatment allocation was concealed), 102 patients with RRMS were randomized to receive a daily oral dose of diazoxide (0.3 and 4 mg/d) or placebo for 24 weeks (NCT01428726). The primary endpoint was the cumulative number of new T1 gadolinium-enhancing lesions per patient, recorded every 4 weeks from week 4 to week 24. Secondary endpoints included brain MRI variables such as the number of new/enlarging T2 lesions and the percentage brain volume change (PBVC); clinical variables such as the percentage of relapse-free patients, relapse rate, and change in the Expanded Disability Status Scale score; and safety and tolerability. RESULTS: Diazoxide was well-tolerated and it produced no serious adverse events other than 1 case of Hashimoto disease. At the 2 doses tested, diazoxide did not improve the primary endpoint or the MRI and clinical variables related to the presence of new lesions or relapses. Patients treated with diazoxide showed reduced PBVC compared with the placebo group, although such changes could be confounded by the higher disease activity of the treated group and the vascular effects of diazoxide. CONCLUSION: At the doses tested, oral diazoxide did not decrease the appearance of new lesions evident by MRI. The effects in slowing the progression of brain atrophy require further validation. CLASSIFICATION OF EVIDENCE: This study provides Class I evidence that for patients with RRMS, diazoxide (0.3 and 4 mg/d) does not significantly change the number of new MRI T1 gadolinium-enhancing lesions.

Defining reliable disability outcomes in multiple sclerosis.

Prevention of irreversible disability is currently the most important goal of disease modifying therapy for multiple sclerosis. The disability outcomes used in most clinical trials rely on progression of Expanded Disability Status Scale score confirmed over 3 or 6 months. However, sensitivity and stability of this metric has not been extensively evaluated. Using the global MSBase cohort study, we evaluated 48 criteria of disability progression, testing three definitions of baseline disability, two definitions of progression magnitude, two definitions of long-term irreversibility and four definitions of event confirmation period. The study outcomes comprised the rates of detected progression events per 10 years and the proportions of the recorded events persistent at later time points. To evaluate the ratio of progression frequency and stability for each criterion, we calculated the proportion of events persistent over the five subsequent years once progression was achieved. Finally, we evaluated the clinical and demographic determinants characterising progression events and, for those that regressed back to baseline, determinants of their subsequent regression. The study population consisted of 16 636 patients with the minimum of three recorded disability scores, totalling 112 584 patient-years. The progression rates varied between 0.41 and 1.14 events per 10 years, with the length of required confirmation interval as the most important determinant of the observed variance. The concordance among all tested progression criteria was only 17.3%. Regression of disability occurred in 11-34% of the progression events over the five subsequent years. The most important determinant of progression stability was the length of the confirmation period. For the most accurate set of the progression criteria, the proportions of 3-, 6-, 12- or 24-month confirmed events persistent over 5 years reached 70%, 74%, 80% and 89%, respectively. Regression post progression was more common in younger patients, relapsing-remitting disease course, and after a smaller change in disability, and was inflated by higher visit frequency. These results suggest that the disability outcomes based on 3-6-month confirmed disability progression overestimate the accumulation of permanent disability by up to 30%. This could lead to spurious results in short-term clinical trials, and the issue may be magnified further in cohorts consisting predominantly of younger patients and patients with relapsing-remitting disease. Extension of the required confirmation period increases the persistence of progression events.

Cestodes change the isotopic signature of brine shrimp, Artemia, hosts: implications for aquatic food webs.

To reach the final host (greater flamingos), the cestode Flamingolepis liguloides alters the behaviour of its intermediate host, the brine shrimp, Artemia parthenogenetica, causing it to spend more time close to the water surface. During summer 2010, we showed that the prevalence of this cestode was consistently higher at the top of the water column in the Odiel salt pans in south-western Spain. We used stable nitrogen (N) and carbon (C) isotopic analysis to test the hypothesis that cestodes also alter resource use by Artemia. In early summer, we compared stable isotopes in infected hosts at the surface with those from uninfected hosts at the bottom of the water column. In late summer, we compared infected and uninfected Artemia from the bottom. δ(15)N was consistently enriched in infected individuals compared with uninfected hosts, especially in Artemia with multiple infections of F. liguloides (family Hymenolepididae) and those with mixed infections of F. liguloides and cestodes of the family Dilepididae. Infected individuals from the surface were enriched in δ(13)C compared with uninfected ones from the bottom, but the opposite was found when comparing uninfected and infected Artemia from the same depth. This may be caused by the increase in lipid concentration in infected Artemia. Isolated cysticercoids of F. liguloides were significantly enriched in δ(13)C compared with cysticercoids in infected hosts, but surprisingly were not enriched in N. Our findings illustrate the way cestodes can alter food webs and highlight the importance of considering the parasitic status of prey in studies of trophic ecology in saline wetlands.

Differential effects of monophosphoryl lipid A and cytokine cocktail as maturation stimuli of immunogenic and tolerogenic dendritic cells for immunotherapy.

Immunotherapy using monocyte-derived dendritic cells (MDDC) is increasingly being considered as alternative therapeutic approach in cancer, infectious diseases and also in autoimmunity when patients are not responsive to conventional treatments. In general, generation of MDDC from monocytes is induced in the presence of GM-CSF and IL-4, and a maturation stimulus is added to the culture to obtain mature DCs suitable for therapy. For DC maturation, different combinations of pro-inflammatory mediators and Toll-like receptor ligands have been tested, obtaining DCs that differ in their properties and the type of immune response they promote. Therefore, it is necessary to find an optimal cytokine environment for DC maturation to obtain a cellular product suitable for DC-based immunotherapeutic protocols. In this study, we have evaluated in vitro the effects of different maturation stimuli on the viability, phenotype, cytokine profile, stability and functionality of immunogenic and tolerogenic (1α,25-dihydroxyvitamin D(3)-treated) MDDC. Maturation was induced using the clinical grade TLR4-agonist: monophosphoryl lipid A (LA), compared to the traditional cytokine cocktail (CC; clinical grade TNF-α, IL-1ß, PGE2) and a combination of both. Our results showed the combination of CC+LA rendered a potent immunogenic DC population that induced the production of IFN-γ and IL-17 in allogeneic co-cultures, suggesting a Th17 polarization. Moreover, these immunogenic DCs showed a high surface expression of CD83, CD86, HLA-DR and secretion of IL-12p70. When aiming to induce tolerance, using LA to generate mature TolDC did not represent a clear advantage, and the stability and the suppressive capability exhibited by CC-matured TolDC may represent the best option. Altogether, these findings demonstrate the relevance of an appropriate maturation stimulus to rationally modulate the therapeutic potential of DCs in immunotherapy.