Morbidity burden of imported chronic schistosomiasis among West African migrants.

BACKGROUND: Past exposure to schistosomiasis is frequent among migrants from endemic countries, and chronic untreated infection may lead to long-term morbidities. METHODS: We carried out a prospective population-based cross-sectional study among migrants from endemic Sub-Saharan countries living in Barcelona, Spain. Participants had not been previously diagnosed or treated for schistosomiasis. Clinical signs and symptoms were scrutinised through a systematic revision of electronic medical records and an on-site standardised questionnaire, and blood and urine samples were screened for Schistosoma. FINDINGS: We recruited 522 eligible participants, 74.3% males, mean age 42.7 years (SD=11.5, range 18-76), Overall, 46.4% were from Senegal and 23.6% from Gambia. They had lived in the European Union for a median of 16 years (IQR 10-21). The prevalence of a Schistosoma-positive serology was 35.8%. S. haematobium eggs were observed in urine samples in 6 (1.2%) participants. The most prevalent symptoms among Schistosoma-positive participants were chronic abdominal pain (68.8%, OR=1.79; 95%CI 1.2-2.6), eosinophilia (44.9%, OR=2.69; 95%CI 1.8-4.0) and specific symptoms associated with urinary schistosomiasis, like self-reported episodes of haematuria (37.2%; OR=2.47; 95%CI 1.6-3.8), dysuria (47.9%, OR=1.84; 95%CI=1.3-2.7) and current renal insufficiency (13.4%; OR=2.35; 95%CI=1.3-4.3). We found a significant prevalence of gender-specific genital signs and symptoms among females (mainly menstrual disorders) and males (erectile dysfunction and pelvic pain). Individuals typically presented with a multitude of interconnected symptoms, most commonly chronic abdominal pain, which are often disregarded. CONCLUSIONS: Despite the lack of urine parasite identification, the high incidence of clinical signs and symptoms strongly correlated with a positive schistosomiasis serology suggests the existence of a heavy clinical burden among long-term West African migrants living for years/decades in the study region. More research is urgently required to determine whether these symptoms are the result of long-term sequelae or a persistent active Schistosoma infection.

A Zebrafish-Based Platform for High-Throughput Epilepsy Modeling and Drug Screening in F0.

The zebrafish model has emerged as a reference tool for phenotypic drug screening. An increasing number of molecules have been brought from bench to bedside thanks to zebrafish-based assays over the last decade. The high homology between the zebrafish and the human genomes facilitates the generation of zebrafish lines carrying loss-of-function mutations in disease-relevant genes; nonetheless, even using this alternative model, the establishment of isogenic mutant lines requires a long generation time and an elevated number of animals. In this study, we developed a zebrafish-based high-throughput platform for the generation of F0 knock-out (KO) models and the screening of neuroactive compounds. We show that the simultaneous inactivation of a reporter gene (tyrosinase) and a second gene of interest allows the phenotypic selection of F0 somatic mutants (crispants) carrying the highest rates of mutations in both loci. As a proof of principle, we targeted genes associated with neurodevelopmental disorders and we efficiently generated de facto F0 mutants in seven genes involved in childhood epilepsy. We employed a high-throughput multiparametric behavioral analysis to characterize the response of these KO models to an epileptogenic stimulus, making it possible to employ kinematic parameters to identify seizure-like events. The combination of these co-injection, screening and phenotyping methods allowed us to generate crispants recapitulating epilepsy features and to test the efficacy of compounds already during the first days post fertilization. Since the strategy can be applied to a wide range of indications, this study paves the ground for high-throughput drug discovery and promotes the use of zebrafish in personalized medicine and neurotoxicity assessment.

Male genitourinary schistosomiasis-related symptoms among long-term Western African migrants in Spain: a prospective population-based screening study.

BACKGROUND: Schistosomiasis is highly endemic in sub-Saharan Africa and frequently imported to Europe. Male urogenital manifestations are often neglected. We aimed to ascertain the prevalence of genitourinary clinical signs and symptoms among long-term African migrants in a non-endemic European country using a serology test. METHODS: We carried out a prospective, community-based cross-sectional study of adult male migrants from sub-Saharan Africa living in Spain. Schistosoma serology tests and microscopic urine examinations were carried out, and clinical data were obtained from an electronic medical record search and a structured questionnaire. RESULTS: We included 388 adult males, mean age 43.5 years [Standard Deviation (SD) = 12.0, range: 18-76]. The median time since migration to the European Union was 17 [Interquartile range (IQR): 11-21] years. The most frequent country of origin was Senegal (N = 179, 46.1%). Of the 338, 147 (37.6%) tested positive for Schistosoma. Parasite eggs were present in the urine of only 1.3%. Nine genitourinary clinical items were significantly associated with positive Schistosoma serology results: pelvic pain (45.2%; OR = 1.57, 95% CI: 1.0-2.4), pain on ejaculation (14.5%; OR = 1.85, 95% CI: 1.0-3.5), dyspareunia (12.4%; OR = 2.45, 95% CI: 1.2-5.2), erectile dysfunction (9.5%; OR = 3.10, 95% CI: 1.3-7.6), self-reported episodes of infertility (32.1%; OR = 1.69, 95% CI: 1.0-2.8), haematuria (55.2%; OR = 2.37, 95% CI: 1.5-3.6), dysuria (52.1%; OR = 2.01, 95% CI: 1.3-3.1), undiagnosed syndromic STIs (5.4%), and orchitis (20.7%; OR = 1.81, 95% CI: 1.0-3.1). Clinical signs tended to cluster. CONCLUSIONS: Urogenital clinical signs and symptoms are prevalent among male African long-term migrants with a positive Schistosoma serology results. Genital involvement can be frequent even among those with long periods of non-residence in their sub-Saharan African countries of origin. Further research is needed to develop diagnostic tools and validate therapeutic approaches to chronic schistosomiasis.

A single-arm study design with non-inferiority and superiority time-to-event endpoints: a tool for proof-of-concept and de-intensification strategies in breast cancer.

De-escalation trials in oncology evaluate therapies that aim to improve the quality of life of patients with low-risk cancer by avoiding overtreatment. Non-inferiority randomized trials are commonly used to investigate de-intensified regimens with similar efficacy to that of standard regimens but with fewer adverse effects (ESMO evidence tier A). In cases where it is not feasible to recruit the number of patients needed for a randomized trial, single-arm prospective studies with a hypothesis of non-inferiority can be conducted as an alternative. Single-arm studies are also commonly used to evaluate novel treatment strategies (ESMO evidence tier B). A single-arm design that includes both non-inferiority and superiority primary objectives will enable the ranking of clinical activity and other parameters such as safety, pharmacokinetics, and pharmacodynamics data. Here, we describe the statistical principles and procedures to support such a strategy. The non-inferiority margin is calculated using the fixed margin method. Sample size and statistical analyses are based on the maximum likelihood method for exponential distributions. We present example analyses in metastatic and adjuvant settings to illustrate the usefulness of our methodology. We also explain its implementation with nonparametric methods. Single-arm designs with non-inferiority and superiority analyses are optimal for proof-of-concept and de-escalation studies in oncology.

Schistosomiasis screening in non-endemic countries from a cost perspective: Knowledge gaps and research priorities. The case of African long-term residents in a Metropolitan Area, Spain.

BACKGROUND: Imported schistosomiasis is an emerging issue in European countries as a result of growing global migration from schistosomiasis-endemic countries, mainly in sub-Saharan Africa. Undetected infection may lead to serious long-term complications with an associated high cost for public healthcare systems especially among long-term migrants. OBJECTIVE: To evaluate from a health economics perspective the introduction of schistosomiasis screening programs in non-endemic countries with high prevalence of long-term migrants. METHODOLOGY: We calculated the costs associated with three approaches-presumptive treatment, test-and-treat and watchful waiting-under different scenarios of prevalence, treatment efficacy and the cost of care resulting from long-term morbidity. Costs were estimated for our study area, in which there are reported to reside 74,000 individuals who have been exposed to the infection. Additionally, we methodically reviewed the potential factors that could affect the cost/benefit ratio of a schistosomiasis screening program and need therefore to be ascertained. RESULTS: Assuming a 24% prevalence of schistosomiasis in the exposed population and 100% treatment efficacy, the estimated associated cost per infected person of a watchful waiting strategy would be €2,424, that of a presumptive treatment strategy would be €970 and that of a test-and-treat strategy would be €360. The difference in averted costs between test-and-treat and watchful waiting strategies ranges from nearly €60 million in scenarios of high prevalence and treatment efficacy, to a neutral costs ratio when these parameters are halved. However, there are important gaps in our understanding of issues such as the efficacy of treatment in infected long-term residents, the natural history of schistosomiasis in long-term migrants and the feasibility of screening programs. CONCLUSION: Our results support the roll-out of a schistosomiasis screening program based on a test-and-treat strategy from a health economics perspective under the most likely projected scenarios, but important knowledge gaps should be addressed for a more accurate estimations among long-term migrants.

Recomendaciones sobre el uso de la telemedicina aplicada a la patología nutricional / Recommendations on the use of telemedicine as applied to nutritional pathology

Introducción: la telemedicina puede mejorar la calidad asistencial y el uso de recursos. La pandemia de COVID-19 ha hecho necesaria su implementación en la práctica habitual. Por ello, un grupo de endocrinólogos de la Comunidad Valenciana, Murcia y Baleares creó un comité para su desarrollo. Objetivos: establecer unas recomendaciones para mejorar la calidad de la consulta de patología nutricional y diseñar unos indicadores para su gestión. Metodología: se siguió la metodología Delphi con participación de 13 profesionales con el fin de alcanzar el mayor consenso. Para ello se circuló un cuestionario de 16 puntos en 3 rondas: en la primera se estableció el grado de consenso; en la segunda, los participantes tuvieron acceso a los resultados de la primera y respondieron a las cuestiones planteadas. Se consideró que había acuerdo si el consenso era ≥ 75 % de los participantes, y que existía acuerdo fuerte si este era ≥ 90 %. Además, se estableció la temática de los indicadores de calidad. En la tercera se desarrollaron dichos indicadores. Resultados: tras 3 rondas y una reunión de revisión se establecieron los sobre aspectos organizativos 5 recomendaciones con acuerdo fuerte; sobre los aspectos administrativos, 6 recomendaciones con acuerdo fuerte y 4 con acuerdo. Se seleccionaron 8 indicadores de calidad que se desarrollaron en formato de fichas. Conclusiones: este documento recopila una serie de recomendaciones sobre cuestiones, necesidades y requisitos a tener en cuenta para una consulta telemática de calidad al paciente con patología nutricional. Así mismo, se han desarrollado unos indicadores para mejorar la calidad asistencial (AU)

Introduction: telemedicine can improve the standards of clinical care and use of resources. The COVID-19 pandemic has required its implementation in routine practice. For this reason, a group of endocrinologists from Valencia, Murcia and the Balearic Islands created a committee for its development. Objectives: to establish recommendations in order to improve the quality of consultation in nutritional disorders, and to create indicators for its management. Methodology: the Delphi methodology was followed with the participation of 13 professionals in order to reach the widest consensus. A 16-item questionnaire was distributed within 3 rounds: in the first round, the degree of consensus was established; in the second round, the participants responded to the posed questions after having access to the first-round results. Agreement was considered if ≥ 75 % participants reached consensus, while strong agreement was considered if consensus was reached by ≥ 90 %. In addition, quality indicators were established. In a third round, these indicators were developed. Results: after 3 rounds and a revision 5 recommendations with strong agreement were established based on organizational aspects. Regarding administrative aspects, 6 recommendations with strong agreement were established while 4 recommendations reached the level of agreement. Eight quality indicators were selected and developed. Conclusions this document compiles a list of recommendations about needs and requirements to be taken into account for a quality telematic consultation in patients with nutritional disorders. In addition, health care quality indicators have been created and developed (AU)

Atypical Extracellular Action Potentials from Posteromedial Hypothalamus in Anesthetized Humans.

BACKGROUND: We obtained microelectrode recordings from four patients with intractable aggressivity who underwent surgery at posteromedial hypothalamus under general anaesthesia. We described two general types of extracellular action potentials (EAPs): typical/canonical and atypical. METHODS: We analysed 337 units and 67 traces, which were characterized by the mean action potential (mAP). For the first phase, depolarization and repolarization, we computed amplitudes (VFP, VDep and VRep) and durations (dFP, dDep and dRep), maximum and minimum values of the first derivative (dVmax, dVmin), and amplitude and duration ratios. RESULTS: Most of the canonical mAPs were positive (81.1%). EAPs with atypical mean action potentials (amAPs) were recorded in 42/337 cases. Only 35.6% of mAPs showed 2 phases. We identified the following types: N1P1N2 (38.3%), P1N1 (35.9%), amAP (12.5%), P1P2N1 (12.2%), N1P1 (4.7%), P1N1P2 (4.1%) and N1N2P1 (3.2%). We can define the properties of canonical forms as those units with (i) at least two opposite phases; (ii) VDep∈[1.2,2.7]×|VRep| and strongly related by this function VRep=-0.56⁢(±0.01)⁢VDep-1.83⁢(±0.79); (iii) a very strong relationship between dVmax and dVmin, given by the equation d⁢Vmin=-0.91⁢(±0.03)⁢d⁢Vmax-0.37⁢(±0.12), both of which were included in the depolarization phase; (iv) related with VDep by the equation d⁢Vmax=0.08⁢(±0.001)⁢VDep-0.28⁢(±0.14); and (v) dDep⁢~⁢0.38⁢dRep. However, the first phase does not pertain to the same dynamic process responsible for depolarization and repolarization. CONCLUSIONS: Atypical units are described here for the first time and are true EAPs that differ strikingly from canonical forms. To date, they have been observed only in the hypothalamus, but future research is needed to assess their existence in other brain structures.

[Recommendations on the use of telemedicine as applied to nutritional pathology]. / Recomendaciones sobre el uso de la telemedicina aplicada a la patología nutricional.

Introduction: Introduction: telemedicine can improve the standards of clinical care and use of resources. The COVID-19 pandemic has required its implementation in routine practice. For this reason, a group of endocrinologists from Valencia, Murcia and the Balearic Islands created a committee for its development. Objectives: to establish recommendations in order to improve the quality of consultation in nutritional disorders, and to create indicators for its management. Methodology: the Delphi methodology was followed with the participation of 13 professionals in order to reach the widest consensus. A 16-item questionnaire was distributed within 3 rounds: in the first round, the degree of consensus was established; in the second round, the participants responded to the posed questions after having access to the first-round results. Agreement was considered if ≥ 75 % participants reached consensus, while strong agreement was considered if consensus was reached by ≥ 90 %. In addition, quality indicators were established. In a third round, these indicators were developed. Results: after 3 rounds and a revision 5 recommendations with strong agreement were established based on organizational aspects. Regarding administrative aspects, 6 recommendations with strong agreement were established while 4 recommendations reached the level of agreement. Eight quality indicators were selected and developed. Conclusions: this document compiles a list of recommendations about needs and requirements to be taken into account for a quality telematic consultation in patients with nutritional disorders. In addition, health care quality indicators have been created and developed.

Introducción: Introducción: la telemedicina puede mejorar la calidad asistencial y el uso de recursos. La pandemia de COVID-19 ha hecho necesaria su implementación en la práctica habitual. Por ello, un grupo de endocrinólogos de la Comunidad Valenciana, Murcia y Baleares creó un comité para su desarrollo. Objetivos: establecer unas recomendaciones para mejorar la calidad de la consulta de patología nutricional y diseñar unos indicadores para su gestión. Metodología: se siguió la metodología Delphi con participación de 13 profesionales con el fin de alcanzar el mayor consenso. Para ello se circuló un cuestionario de 16 puntos en 3 rondas: en la primera se estableció el grado de consenso; en la segunda, los participantes tuvieron acceso a los resultados de la primera y respondieron a las cuestiones planteadas. Se consideró que había acuerdo si el consenso era ≥ 75 % de los participantes, y que existía acuerdo fuerte si este era ≥ 90 %. Además, se estableció la temática de los indicadores de calidad. En la tercera se desarrollaron dichos indicadores. Resultados: tras 3 rondas y una reunión de revisión se establecieron los sobre aspectos organizativos 5 recomendaciones con acuerdo fuerte; sobre los aspectos administrativos, 6 recomendaciones con acuerdo fuerte y 4 con acuerdo. Se seleccionaron 8 indicadores de calidad que se desarrollaron en formato de fichas. Conclusiones: este documento recopila una serie de recomendaciones sobre cuestiones, necesidades y requisitos a tener en cuenta para una consulta telemática de calidad al paciente con patología nutricional. Así mismo, se han desarrollado unos indicadores para mejorar la calidad asistencial.

Neurophysiological Characterization of Posteromedial Hypothalamus in Anaesthetized Patients.

Deep brain stimulation (DBS) requires a precise localization, which is especially difficult at the hypothalamus, because it is usually performed in anesthetized patients. We aimed to characterize the neurophysiological properties posteromedial hypothalamus (PMH), identified by the best neurophysiological response to electrical stimulation. We obtained microelectrode recordings from four patients with intractable aggressivity operated under general anesthesia. We pooled data from 1.5 mm at PMH, 1.5 mm upper (uPMH) and 1.5 mm lower (lPMH). We analyzed 178 units, characterized by the mean action potential (mAP). Only 11% were negative. We identified the next types of units: P1N1 (30.9%), N1P1N2 (29.8%), P1P2N1 (16.3%), N1P1 and N1N2P1 (6.2%) and P1N1P2 (5.0%). Besides, atypical action potentials (amAP) were recorded in 11.8%. PMH was highly different in cell composition from uPMH and lPMH, exhibiting also a higher percentage of amAP. Different kinds of cells shared similar features for the three hypothalamic regions. Although features for discharge pattern did not show region specificity, the probability mass function of inter-spike interval were different for all the three regions. Comparison of the same kind of mAP with thalamic neurons previously published demonstrate that most of cells are different for derivatives, amplitude and/or duration of repolarization and depolarization phases and also for the first phase, demonstrating a highly specificity for both brain centers. Therefore, the different properties described for PMH can be used to positively refine targeting, even under general anesthesia. Besides, we describe by first time the presence of atypical extracellular action potentials.

Specific EEG Encephalopathy Pattern in SARS-CoV-2 Patients.

We used quantified electroencephalography (qEEG) to define the features of encephalopathy in patients released from the intensive care unit after severe illness from COVID-19. Artifact-free 120-300 s epoch lengths were visually identified and divided into 1 s windows with 10% overlap. Differential channels were grouped by frontal, parieto-occipital, and temporal lobes. For every channel and window, the power spectrum was calculated and used to compute the area for delta (0-4 Hz), theta (4-8 Hz), alpha (8-13 Hz), and beta (13-30 Hz) bands. Furthermore, Shannon's spectral entropy (SSE) and synchronization by Pearson's correlation coefficient () were computed; cases of patients diagnosed with either infectious toxic encephalopathy (ENC) or post-cardiorespiratory arrest (CRA) encephalopathy were used for comparison. Visual inspection of EEGs of COVID patients showed a near-physiological pattern with scarce anomalies. The distribution of EEG bands was different for the three groups, with COVID midway between distributions of ENC and CRA; specifically, temporal lobes showed different distribution for EEG bands in COVID patients. Besides, SSE was higher and hemispheric connectivity lower for COVID. We objectively identified some numerical EEG features in severely ill COVID patients that can allow positive diagnosis of this encephalopathy.

Quantified EEG for the Characterization of Epileptic Seizures versus Periodic Activity in Critically Ill Patients.

Epileptic seizures (ES) are frequent in critically ill patients and their detection and treatment are mandatory. However, sometimes it is quite difficult to discriminate between ES and non-epileptic bursts of periodic activity (BPA). Our aim was to characterize ES and BPA by means of quantified electroencephalography (qEEG). Records containing either ES or BPA were visually identified and divided into 1 s windows that were 10% overlapped. Differential channels were grouped by frontal, parieto-occipital and temporal lobes. For every channel and window, the power spectrum was calculated and the area for delta (0-4 Hz), theta (4-8 Hz), alpha (8-13 Hz), and beta (13-30 Hz) bands and spectral entropy (Se) were computed. Mean values of percentage changes normalized to previous basal activity and standardized mean difference (SMD) for every lobe were computed. We have observed that BPA are characterized by a selective increment of delta activity and decrease in Se along the scalp. Focal seizures (FS) always propagated and were similar to generalized seizures (GS). In both cases, although delta and theta bands increased, the faster bands (alpha and beta) showed the highest increments (more than 4 times) without modifications in Se. We have defined the numerical features of ES and BPA, which can facilitate its clinical identification.

Dynamics and heterogeneity of brain damage in multiple sclerosis.

Multiple Sclerosis (MS) is an autoimmune disease driving inflammatory and degenerative processes that damage the central nervous system (CNS). However, it is not well understood how these events interact and evolve to evoke such a highly dynamic and heterogeneous disease. We established a hypothesis whereby the variability in the course of MS is driven by the very same pathogenic mechanisms responsible for the disease, the autoimmune attack on the CNS that leads to chronic inflammation, neuroaxonal degeneration and remyelination. We propose that each of these processes acts more or less severely and at different times in each of the clinical subgroups. To test this hypothesis, we developed a mathematical model that was constrained by experimental data (the expanded disability status scale [EDSS] time series) obtained from a retrospective longitudinal cohort of 66 MS patients with a long-term follow-up (up to 20 years). Moreover, we validated this model in a second prospective cohort of 120 MS patients with a three-year follow-up, for which EDSS data and brain volume time series were available. The clinical heterogeneity in the datasets was reduced by grouping the EDSS time series using an unsupervised clustering analysis. We found that by adjusting certain parameters, albeit within their biological range, the mathematical model reproduced the different disease courses, supporting the dynamic CNS damage hypothesis to explain MS heterogeneity. Our analysis suggests that the irreversible axon degeneration produced in the early stages of progressive MS is mainly due to the higher rate of myelinated axon degeneration, coupled to the lower capacity for remyelination. However, and in agreement with recent pathological studies, degeneration of chronically demyelinated axons is not a key feature that distinguishes this phenotype. Moreover, the model reveals that lower rates of axon degeneration and more rapid remyelination make relapsing MS more resilient than the progressive subtype. Therefore, our results support the hypothesis of a common pathogenesis for the different MS subtypes, even in the presence of genetic and environmental heterogeneity. Hence, MS can be considered as a single disease in which specific dynamics can provoke a variety of clinical outcomes in different patient groups. These results have important implications for the design of therapeutic interventions for MS at different stages of the disease.

Impact of Zygosity on Bimodal Phenotype Distributions.

Allele number, or zygosity, is a clear determinant of gene expression in diploid cells. However, the relationship between the number of copies of a gene and its expression can be hard to anticipate, especially when the gene in question is embedded in a regulatory circuit that contains feedback. Here, we study this question making use of the natural genetic variability of human populations, which allows us to compare the expression profiles of a receptor protein in natural killer cells among donors infected with human cytomegalovirus with one or two copies of the allele. Crucially, the distribution of gene expression in many of the donors is bimodal, which indicates the presence of a positive feedback loop somewhere in the regulatory environment of the gene. Three separate gene-circuit models differing in the location of the positive feedback loop with respect to the gene can all reproduce the homozygous data. However, when the resulting fitted models are applied to the hemizygous donors, one model (the one with the positive feedback located at the level of gene transcription) is superior in describing the experimentally observed gene-expression profile. In that way, our work shows that zygosity can help us relate the structure and function of gene regulatory networks.

The analysis of semantic networks in multiple sclerosis identifies preferential damage of long-range connectivity.

OBJECTIVE: To analyze the characteristics of semantic networks derived from fluency tests in patients with multiple sclerosis (MS). METHODS: We built semantic networks by applying co-occurrence statistics to the data from verbal fluency tests performed on patients with MS (n=36) and matched controls (n=200), assessing the differences in network topology. RESULTS: As expected, the semantic networks from both patients and controls showed 'small-world' properties. Topological analysis of these semantic networks indicated that there were fewer nodes (words) and links (defined by significant co-occurrence of words) in those derived from MS patients. The average connectivity was not significantly affected, while the local connectivity (clustering coefficient) is preserved. Quantifiers of the cohesiveness of the network, which reflect long distance connectivity, such as assortativity and maximum centrality coefficients, differed significantly between MS patients and controls. CONCLUSIONS: The analysis of semantic networks reveals quantitative differences in MS patients and identifies preferential damage of long-range connectivity. The analysis of semantic networks may be useful in clinical practice for the assessment of cognitive impairment or recovery after damage.

Microtubule stabilization with paclitaxel does not protect against infarction in isolated rat hearts.

NEW FINDINGS: What is the central question of this study? The microtubule network is disrupted during myocardial ischaemia-reperfusion injury. It was suggested that prevention of microtubule disruption with paclitaxel might reduce cardiac infarct size; however, the effects on infarction have not been studied. What is the main finding and its importance? Paclitaxel caused a reduction in microtubule disruption and cardiomyocyte hypercontracture during ischaemia-reperfusion. However, it induced a greater increase in cytosolic calcium, which may explain the lack of effect against infarction that we have seen in isolated rat hearts. The large increase in perfusion pressure induced by paclitaxel in this model may have clinical implications, because detrimental effects of the drug were reported after its clinical application. Microtubules play a major role in the transmission of mechanical forces within the myocardium and in maintenance of organelle function. However, this intracellular network is disrupted during myocardial ischaemia-reperfusion. We assessed the effects of prevention of microtubule disruption with paclitaxel on ischaemia-reperfusion injury in isolated rat cardiomyocytes and hearts. Isolated rat cardiomyocytes were submitted to normoxia (1 h) or 45 min of simulated ischaemia (pH 6.4, 0% O2 , 37 °C) and reoxygenation, without or with treatment with the microtubule stabilizer, paclitaxel (10(-5) M), or the inhibitor of microtubule polymerization, colchicine (5 × 10(-6) M). Simulated ischaemia leads to microtubule disruption before the onset of ischaemic contracture. Paclitaxel attenuated both microtubule disruption and the incidence of hypercontracture, whereas treatment with colchicine mimicked the effects of simulated ischaemia and reoxygenation. In isolated normoxic rat hearts, treatment with paclitaxel induced concentration-dependent decreases in heart rate and left ventricular developed pressure and increases in perfusion pressure. Despite protection against hypercontracture, paclitaxel pretreatment did not modify infarct size (60.37 ± 2.27% in control hearts versus 58.75 ± 10.25, 55.44 ± 10.32 and 50.06 ± 10.14% after treatment with 10(-6) , 3 × 10(-6) and 10(-5) m of paclitaxel) after 60 min of global ischaemia and reperfusion in isolated rat hearts. Lack of protection was correlated with a higher increase in cytosolic calcium levels during simulated ischaemia in cardiomyocytes treated with paclitaxel (2.32 ± 0.15 versus 1.13 ± 0.16 a.u. in the presence or absence of 10(-6) m paclitaxel, respectively, P < 0.05), but not with changes in aortic reactivity. In conclusion, microtubule stabilization with paclitaxel reduces hypercontracture in isolated rat cardiomyocytes but does not protect against infarction in isolated rat hearts.

Transient oscillatory dynamics of interferon beta signaling in macrophages.

BACKGROUND: Interferon-beta (IFN-beta) activates the immune response through the type I IFN signaling pathway. IFN-beta is important in the response to pathogen infections and is used as a therapy for Multiple Sclerosis. The mechanisms of self-regulation and control of this pathway allow precise and environment-dependent response of the cells in different conditions. Here we analyzed type I IFN signaling in response to IFN-beta in the macrophage cell line RAW 264.7 by RT-PCR, ELISA and xMAP assays. The experimental results were interpreted by means of a theoretical model of the pathway. RESULTS: Phosphorylation of the STAT1 protein (pSTAT1) and mRNA levels of the pSTAT1 inhibitor SOCS1 displayed an attenuated oscillatory behavior after IFN-beta activation. In turn, mRNA levels of the interferon regulatory factor IRF1 grew rapidly in the first 50-90 minutes after stimulation until a maximum value, and started to decrease slowly around 200-250 min. The analysis of our kinetic model identified a significant role of the negative feedback from SOCS1 in driving the observed damped oscillatory dynamics, and of the positive feedback from IRF1 in increasing STAT1 basal levels. Our study shows that the system works as a biological damped relaxation oscillator based on a phosphorylation-dephosphorylation network centered on STAT1. Moreover, a bifurcation analysis identified translocation of pSTAT1 dimers to the nucleus as a critical step for regulating the dynamics of type I IFN pathway in the first steps, which may be important in defining the response to IFN-beta therapy. CONCLUSIONS: The immunomodulatory effect of IFN-beta signaling in macrophages takes the form of transient oscillatory dynamics of the JAK-STAT pathway, whose specific relaxation properties determine the lifetime of the cellular response to the cytokine.

Dynamic cross-regulation of antigen-specific effector and regulatory T cell subpopulations and microglia in brain autoimmunity.

BACKGROUND: Multiple Sclerosis (MS) is considered a T-cell-mediated autoimmune disease with a prototypical oscillatory behavior, as evidenced by the presence of clinical relapses. Understanding the dynamics of immune cells governing the course of MS, therefore, has many implications for immunotherapy. Here, we used flow cytometry to analyze the time-dependent behavior of antigen-specific effector (T(eff)) and regulatory (T(reg)) T cells and microglia in mice model of MS, Experimental Autoimmune Encephalomyelitis (EAE), and compared the observations with a mathematical cross-regulation model of T-cell dynamics in autoimmune disease. RESULTS: We found that T(eff) and T(reg) cells specific to myelin olygodendrocyte glycoprotein (MOG) developed coupled oscillatory dynamics with a 4- to 5-day period and decreasing amplitude that was always higher for the T(eff) populations, in agreement with the mathematical model. Microglia activation followed the oscillations of MOG-specific T(eff) cells in the secondary lymphoid organs, but they were activated before MOG-specific T-cell peaks in the CNS. Finally, we assessed the role of B-cell depletion induced by anti-CD20 therapy in the dynamics of T cells in an EAE model with more severe disease after therapy. We observed that B-cell depletion decreases T(eff) expansion, although its oscillatory behavior persists. However, the effect of B cell depletion was more significant in the T(reg) population within the CNS, which matched with activation of microglia and worsening of the disease. Mathematical modeling of T-cell cross-regulation after anti-CD20 therapy suggests that B-cell depletion may influence the dynamics of T cells by fine-tuning their activation. CONCLUSIONS: The oscillatory dynamics of T-cells have an intrinsic origin in the physiological regulation of the adaptive immune response, which influences both disease phenotype and response to immunotherapy.

Activation of cGMP/protein kinase G pathway in postconditioned myocardium depends on reduced oxidative stress and preserved endothelial nitric oxide synthase coupling.

BACKGROUND: The cGMP/protein kinase G (PKG) pathway is involved in the cardioprotective effects of postconditioning (PoCo). Although PKG signaling in PoCo has been proposed to depend on the activation of the phosphatidylinositol 3-kinase (PI3K)/Akt cascade, recent data bring into question a causal role of reperfusion injury signaling kinase (RISK) in PoCo protection. We hypothesized that PoCo increases PKG activity by reducing oxidative stress-induced endothelial nitric oxide synthase (NOS) uncoupling at the onset of reperfusion. METHODS AND RESULTS: Isolated rat hearts were submitted to 40 minutes of ischemia and reperfusion with and without a PoCo protocol. PoCo reduced infarct size by 48% and cGMP depletion. Blockade of cGMP synthesis (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one) and inhibition of PKG (KT5823) or NOS (l-NAME) abolished protection, but inhibition of PI3K/Akt cascade (LY294002) did not (n=5 to 7 per group). Phosphorylation of the RISK pathway was higher in PoCo hearts. However, this difference is due to increased cell death in control hearts because in hearts reperfused with the contractile inhibitor blebbistatin, a drug effective in preventing cell death at the onset of reperfusion, RISK phosphorylation increased during reperfusion without differences between control and PoCo groups. In these hearts, PoCo reduced the production of superoxide (O2(-)) and protein nitrotyrosylation and increased nitrate/nitrite levels in parallel with a significant decrease in the oxidation of tetrahydrobiopterin (BH4) and in the monomeric form of endothelial NOS. CONCLUSIONS: These results demonstrate that PoCo activates the cGMP/PKG pathway via a mechanism independent of the PI3K/Akt cascade and dependent on the reduction of O2(-) production at the onset of reperfusion, resulting in attenuated oxidation of BH4 and reduced NOS uncoupling.