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Platelets exert an essential role in vascular inflammation and thrombosis. Flavonoids are natural compounds employed for the clinical management of vascular disorders preventing capillary permeability, working as phlebotonics and improving the blood rheology, although their mechanism of action remains partially unknown. The effects of quercetin, rutin, diosmetin and diosmin were investigated in platelet activation utilizing blood from healthy and non-treated volunteers. The arrangement of the different activation states of platelets and GPIIb/IIIa receptor occupation was computed by flow cytometry working with calcium ionophore as pro-aggregant to provoke platelet activation and aggregation. The flavonoids studied demonstrated relevant antiplatelet activity through the blocked of GPIIb/IIIa receptors, the suppression of the platelet activation, as well as the pro-aggregate effect of calcium ionophore. Therefore, whichever of the active ingredients examined could be beneficious in the prevention of cardiovascular disease and this article also contributes to elucidate a new mechanism of action for these drugs.

Hyperbaric oxygen therapy (HBOT) consists of using of pure oxygen at increased pressure (in general, 2–3 atmospheres) leading to augmented oxygen levels in the blood (Hyperoxemia) and tissue (Hyperoxia). The increased pressure and oxygen bioavailability might be related to a plethora of applications, particularly in hypoxic regions, also exerting antimicrobial, immunomodulatory and angiogenic properties, among others. In this review, we will discuss in detail the physiological relevance of oxygen and the therapeutical basis of HBOT, collecting current indications and underlying mechanisms. Furthermore, potential areas of research will also be examined, including inflammatory and systemic maladies, COVID-19 and cancer. Finally, the adverse effects and contraindications associated with this therapy and future directions of research will be considered. Overall, we encourage further research in this field to extend the possible uses of this procedure. The inclusion of HBOT in future clinical research could be an additional support in the clinical management of multiple pathologies.

Actinomycin D (ActD) is an FDA-approved NCI oncology drug that specifically targets and downregulates stem cell transcription factors, which leads to a depletion of stem cells within the tumor bulk. Recently, our research group demonstrated the importance of IRS-4 in the development of liver cancer. In this study, we evaluated the protective effects of IRS-4 against ActD. For this study, three hepatocellular carcinoma cell lines (HepG2, Huh7, and Chang cells) were used to study the mechanism of actinomycin D. Most assays were carried out in the Hep G2 cell line, due to the high expression of stem cell biomarkers. We found that ActD caused HepG2 cell necroptosis characterized by DNA fragmentation, decreased mitochondrial membrane potential, cytochrome c depletion, and decreased the levels of reduced glutathione. However, we did not observe a clear increase in apoptosis markers such as annexin V presence, caspase 3 activation, or PARP fragmentation. ActD produced an activation of MAP kinases (ERK, p38, and JNK) and AKT. ActD-induced activation of AKT and MAP kinases produced an activation of the Rb-E2F cascade together with a blockage of cell cycle transitions, due to c-jun depletion. ActD led to the inhibition of pCdK1 and pH3 along with DNA fragmentation resulting in cell cycle arrest and the subsequent activation of p53-dependent cell death in the HepG2 cell line. Only JNK and AKT inhibitors were protective against the effects of ActD. N-Acetyl-L-cysteine also had a protective effect as it restored GSH levels. A likely mechanism for this is IRS-4 stimulating GCL-GSH and inhibiting the Brk-CHK1-p53 pathway. The assessment of the IRS-4 in cancer biopsies could be of interest to carry out a personalized treatment with ActD.

In cirrhosis, intestinal dysbiosis, intestinal barrier impairment, and systemic immune system abnormalities lead to gut bacterial translocation (GBT) and bacterial infection. However, intestinal immune system dysfunction and its contribution to barrier damage are poorly understood. This study correlates immune system dysregulation in the intestines of rats at different stages of CCl4 -induced cirrhosis with barrier function and pathogenic microbiota. The following variables were addressed in the small intestine: intraepithelial lymphocyte (IEL) and lamina propria lymphocyte (LPL) activation status and cytokine production (flow cytometry), cytokine mRNA and protein expression (quantitative real-time PCR and immunofluorescence), microbiota composition of ileum content (16S recombinant DNA massive sequencing), permeability (fecal albumin loss), and epithelial junctions (immunohistochemistry and immunofluorescence). The intestinal mucosa in rats with cirrhosis showed a proinflammatory pattern of immune dysregulation in IELs and LPLs, which featured the expansion of activated lymphocytes, switch to a T helper 1 (Th1) regulatory pattern, and Th17 reduction. In rats with cirrhosis with ascites, this state was associated with epithelial junction protein disruption, fecal albumin loss, and GBT. Direct correlations (P < 0.01) were observed between elevated interferon gamma (IFNγ)-expressing T cytotoxic LPLs and fecal albumin and between inflammatory taxa abundance and IFNγ-producing immune cells in the ileum. Bowel decontamination led to redistributed microbiota composition, reduced proinflammatory activation of mucosal immune cells, normalized fecal albumin levels, and diminished GBT; but there were no modifications in Th17 depletion. Conclusion: The intestinal mucosa of rats with cirrhosis acquires a proinflammatory profile of immune dysregulation that parallels the severity of cirrhosis; this impaired intestinal immune response is driven by gut dysbiosis and leads to disrupted barrier function, promoting GBT.

Major Depressive Disorder (MDD) is a growing disabling condition affecting around 280 million people worldwide. This complex entity is the result of the interplay between biological, psychological, and sociocultural factors, and compelling evidence suggests that MDD can be considered a disease that occurs as a consequence of an evolutionary mismatch and unhealthy lifestyle habits. In this context, diet is one of the core pillars of health, influencing multiple biological processes in the brain and the entire body. It seems that there is a bidirectional relationship between MDD and malnutrition, and depressed individuals often lack certain critical nutrients along with an aberrant dietary pattern. Thus, dietary interventions are one of the most promising tools to explore in the field of MDD, as there are a specific group of nutrients (i.e., omega 3, vitamins, polyphenols, and caffeine), foods (fish, nuts, seeds fruits, vegetables, coffee/tea, and fermented products) or dietary supplements (such as S-adenosylmethionine, acetyl carnitine, creatine, amino acids, etc.), which are being currently studied. Likewise, the entire nutritional context and the dietary pattern seem to be another potential area of study, and some strategies such as the Mediterranean diet have demonstrated some relevant benefits in patients with MDD; although, further efforts are still needed. In the present work, we will explore the state-of-the-art diet in the prevention and clinical support of MDD, focusing on the biological properties of its main nutrients, foods, and dietary patterns and their possible implications for these patients., Fondo de Investigacion de la Seguridad Social, Instituto de Salud Carlos III PI18/01726 PI19/00766, Programa de Actividades de I+D de la Comunidad de Madrid en Biomedicina B2017/BMD3804 B2020/MITICAD-CM

Chronic venous disease (CVD) is a multifactorial condition affecting an important percentage of the global population. It ranges from mild clinical signs, such as telangiectasias or reticular veins, to severe manifestations, such as venous ulcerations. However, varicose veins (VVs) are the most common manifestation of CVD. The explicit mechanisms of the disease are not well-understood. It seems that genetics and a plethora of environmental agents play an important role in the development and progression of CVD. The exposure to these factors leads to altered hemodynamics of the venous system, described as ambulatory venous hypertension, therefore promoting microcirculatory changes, inflammatory responses, hypoxia, venous wall remodeling, and epigenetic variations, even with important systemic implications. Thus, a proper clinical management of patients with CVD is essential to prevent potential harms of the disease, which also entails a significant loss of the quality of life in these individuals. Hence, the aim of the present review is to collect the current knowledge of CVD, including its epidemiology, etiology, and risk factors, but emphasizing the pathophysiology and medical care of these patients, including clinical manifestations, diagnosis, and treatments. Furthermore, future directions will also be covered in this work in order to provide potential fields to explore in the context of CVD.

Background: We have investigated the distribution of the Th1, Th2 and Th17 subsets in circulating CD4+ T lymphocytes and their naïve (TN), effector (TE), central (TCM) and effector memory (TEM) activation/differentiation stages in patients with major depressive disorder (MDD). Methods: Thirty MDD patients and 30 healthy controls were studied. The counts of circulating CD4+ T lymphocytes and their distribution on the TN, TE, TCM and TEM activation/differentiation stages were analyzed by polychromatic flow cytometry. The intracytoplasmic interferon gamma (IFNγ), interleukin (IL)-4, IL-17A and tumor necrosis factor alpha (TNF-alpha) and membrane CD28 expression were also measured. The serum IFNγ, IL-4, Il-17A and TNF-alpha were measured by Luminex, respectively. Results: MDD patients had normal counts of CD4+ T lymphocytes and of their TN, TCM and TEM subsets but increased number and percentage of TE CD4+ subset. CD4+ T lymphocytes had significantly enhanced percentage of cells that express IL-17 and TNF-alpha explained by the expansions found in the TN, TCM and, TEM and TCM, TEM and TE activation/differentiation stages, respectively. A selective increase in the percentages of TCM and TEM expressing IFNγ was also observed. We found a significant correlation between the percentages of CD4+ T lymphocytes expressing IFNγ and TNF-alpha in these patients. MDD patients showed increased serum levels of IL-17 and TNF-alpha, but normal IFNγ and IL-4 concentration. Limitations: the cross-sectional nature of the study could be considered a limitation. Conclusions: MDD patients have abnormal circulating CD4+ T lymphocytes with expansion of the IL-17 and TNF-alpha expressing cells as well as increased levels of circulating IL-17 and TNF-alpha.

Mitochondrial activation and the production of mitochondrial reactive oxygen species (mROS) are crucial for CD4

The most prevalent diseases of our time, non-communicable diseases (NCDs) (including obesity, type 2 diabetes, cardiovascular diseases and some types of cancer) are rising worldwide. All of them share the condition of an “inflammatory disorder”, with impaired immune functions frequently caused or accompanied by alterations in gut microbiota. These multifactorial maladies also have in common malnutrition related to physiopathology. In this context, diet is the greatest modulator of immune system–microbiota crosstalk, and much interest, and new challenges, are arising in the area of precision nutrition as a way towards treatment and prevention. It is a fact that the westernized diet (WD) is partly responsible for the increased prevalence of NCDs, negatively affecting both gut microbiota and the immune system. Conversely, other nutritional approaches, such as Mediterranean diet (MD), positively influence immune system and gut microbiota, and is proposed not only as a potential tool in the clinical management of different disease conditions, but also for prevention and health promotion globally. Thus, the purpose of this review is to determine the regulatory role of nutritional components of WD and MD in the gut microbiota and immune system interplay, in order to understand, and create awareness of, the influence of diet over both key components.

Our aim was to investigate the subset distribution and function of circulating monocytes, proinflammatory cytokine levels, gut barrier damage, and bacterial translocation in chronic spinal cord injury (SCI) patients. Thus, 56 SCI patients and 28 healthy donors were studied. The levels of circulating CD14+highCD16&minus, CD14+highCD16+, and CD14+lowCD16+ monocytes, membrane TLR2, TLR4, and TLR9, phagocytic activity, ROS generation, and intracytoplasmic TNF-&alpha, IL-1, IL-6, and IL-10 after lipopolysaccharide (LPS) stimulation were analyzed by polychromatic flow cytometry. Serum TNF-&alpha, IL-1, IL-6 and IL-10 levels were measured by Luminex and LPS-binding protein (LBP), intestinal fatty acid-binding protein (I-FABP) and zonulin by ELISA. SCI patients had normal monocyte counts and subset distribution. CD14+highCD16&minus, and CD14+highCD16+ monocytes exhibited decreased TLR4, normal TLR2 and increased TLR9 expression. CD14+highCD16&minus, monocytes had increased LPS-induced TNF-&alpha, but normal IL-1, IL-6, and IL-10 production. Monocytes exhibited defective phagocytosis but normal ROS production. Patients had enhanced serum TNF-&alpha, and IL-6 levels, normal IL-1 and IL-10 levels, and increased circulating LBP, I-FABP, and zonulin levels. Chronic SCI patients displayed impaired circulating monocyte function. These patients exhibited a systemic proinflammatory state characterized by enhanced serum TNF-&alpha, and IL-6 levels. These patients also had increased bacterial translocation and gut barrier damage.

This study aimed to create an individualized analysis model of the risk of intensive care unit (ICU) admission or death for coronavirus disease 2019 (COVID-19) patients as a tool for the rapid clinical management of hospitalized patients in order to achieve a resilience of medical resources. This is an observational, analytical, retrospective cohort study with longitudinal follow-up. Data were collected from the medical records of 3489 patients diagnosed with COVID-19 using RT-qPCR in the period of highest community transmission recorded in Europe to date: February&ndash, June 2020. The study was carried out in in two health areas of hospital care in the Madrid region: the central area of the Madrid capital (Hospitales de Madrid del Grupo HM Hospitales (CH-HM), n = 1931) and the metropolitan area of Madrid (Hospital Universitario Pr&iacute, ncipe de Asturias (MH-HUPA) n = 1558). By using a regression model, we observed how the different patient variables had unequal importance. Among all the analyzed variables, basal oxygen saturation was found to have the highest relative importance with a value of 20.3%, followed by age (17.7%), lymphocyte/leukocyte ratio (14.4%), CRP value (12.5%), comorbidities (12.5%), and leukocyte count (8.9%). Three levels of risk of ICU/death were established: low-risk level (&lt, 5%), medium-risk level (5&ndash, 20%), and high-risk level (&gt, 20%). At the high-risk level, 13% needed ICU admission, 29% died, and 37% had an ICU&ndash, death outcome. This predictive model allowed us to individualize the risk for worse outcome for hospitalized patients affected by COVID-19.

El estudiodel impacto de las innovaciones educativas sobre el aprendizaje producido en las asignaturas universitarias, requiere medir los efectos del cambio metodológico sobre diversas variables asociadas a la mejora del aprendizaje. En esta revisión mostramos e ilustramos cómo realizar una serie de medidas sobre algunas de estas variables: la implicación de los alumnos en la asignatura, el tiempo de estudio de los alumnos, el rendimiento académico (cuantificando las tasas de éxito y de fracaso), el aprendizaje (calificaciones medias obtenidas en las pruebas de evaluación del aprendizaje). El impacto sobre estas variables puede cuantificarse por el tamaño de efecto (g) que relaciona el nivel de cambio observado en la variable de estudio con respecto a su nivel de variación inicial estimado por su desviación estándar. Finalmente, desde la perspectiva del alumno es importante conocer cuál es su percepción y valoración sobre el cambio metodológico experimentado. En esta revisión aportamos ejemplos de casos que ilustran cómo pueden estudiarse los efectos del modelo de aula invertida sobre la implicación, el aprendizaje y la valoración de los alumnos sobre el cambio de metodología de enseñanza y el aprendizaje y mostramos los resultados que hemos obtenido. Utilizando estas metodologías demostramos que la puesta en práctica del aula inversa en asignaturas universitarias estimula el trabajo de los alumnos, aunque usualmente se requieren 2-3 años de práctica para vencer la resistencia estudiantil inicial y lograr su máximo impacto sobre el rendimiento académico, el aprendizaje y la apreciación de su valor por los alumnos.aula inversa, implicación estudiantil, rendimiento académico, tamaño de efecto, aprendizaje activo, implicación estudiantil, motivación, gamificación., The study of the impact of educational innovations on student learning in university courses requires measurements of the effects of the methodological changes on several variables associated to the enhancement of learning. In this review we show how to perform measurements of the involvement of the students in the subject, the student study time, academic performance (quantifying passing and failure rates), and the learning (changes in average grades on learning assessment exams). The impact on these variables is quantified by the effect size (g) that relates the level of change observed in the study variable with respect to its level of initial variation estimated by its standard deviation. Finally, from a student's perspective it is important to know what the students' perception and assessment are about the methodological change experienced. In this work, we provide some examples of how can be studied the effect of the implementation of the inverted classroom model on the involvement, learning and assessment of students on the change in teaching methodology and learning, and we show the results we have obtained. Using these methodologies we demonstrate that the implementation of the flipped classroom in university courses stimulates the work of the students, although usually it takes 2-3 years of practice to overcome student resistance, to achieve its maximum impact on academic performance, student learning, and the student’s appreciation of its value.

Chronic venous disease (CVeD) is defined as a set of disorders affecting the venous system mainly manifested in the form of varicose veins. CVeD is characterized by a sustained venous hypertension, leading to a plethora of functional and structural changes in the vein that may cause valve incompetence and pathologic reflux. In turn, venous reflux aggravates the venous hypertension and enhances the progression of CVeD into the most advanced stages. Previous studies have proposed that there are several alterations in the venous wall preceding the valve dysfunction and venous reflux. Besides, it has also been identified that young patients with CVeD present premature aging and changes in the venous wall composition that may be related to the presence of venous reflux. In this context, the aim of the present study is to examine the possible pathophysiological role of elastic fibers and their precursors in the venous wall of patients with reflux in comparison to those without reflux, considering the variable age in both groups (

Polymer-based composites are a group of biomaterials that exert synergic and combined activity. There are multiple reported uses of these composites in multiple biomedical areas, such as drug carriers, in wound dressings, and, more prominently, in tissue engineering and regenerative medicine. Bone grafting is a promising field in the use of polymeric composites, as this is the second most frequently transplanted organ in the United States. Advances in novel biomaterials, such as polymeric composites, will undoubtedly be of great aid in bone tissue engineering and regeneration. In this paper, a general view of bone structure and polymeric composites will be given, discussing the potential role of these components in bone tissue. Moreover, the most relevant jawbone and maxillofacial applications of polymeric composites will be revised in this article, collecting the main knowledge about this topic and emphasizing the need of further clinical studies in humans.

Patients with long-term, treated, rheumatoid arthritis (RA) show abnormalities in their circulating CD4+ T-lymphocytes, but whether this occurs in recently diagnosed na&iuml, ve patients to disease-modifying drugs (DMARDs) is under discussion. These patients show heterogeneous clinical response to methotrexate (MTX) treatment. We have examined the count of circulating CD4+ T-lymphocytes, and their na&iuml, ve (TN), central memory (TCM), effector memory (TEM) and effector (TE) subsets, CD28 expression and V&beta, TCR repertoire distribution by polychromatic flow cytometry in a population of 68 DMARD-na&iuml, ve recently diagnosed RA patients, before and after 3 and 6 months of MTX treatment. At pre-treatment baseline, patients showed an expansion of the counts of CD4+ TN, TEM, TE and TCM lymphocyte subsets, and of total CD4+CD28&minus, cells and of the TE subset with a different pattern of numbers in MTX responder and non-responders. The expansion of CD4+TEM lymphocytes showed a predictive value of MTX non-response. MTX treatment was associated to different modifications in the counts of the CD4+ subsets and of the V&beta, TCR repertoire family distribution and in the level of CD28 expression in responders and non-responders. In conclusion, the disturbance of CD4+ lymphocytes is already found in DMARD-na&iuml, ve RA patients with different patterns of alterations in MTX responders and non-responders.

BACKGROUND Twitter is an indicator of real-world performance, thus, is an appropriate arena to assess the social consideration and attitudes toward psychosis. OBJECTIVE The aim of this study was to perform a mixed-methods study of the content and key metrics of tweets referring to psychosis in comparison with tweets referring to control diseases (breast cancer, diabetes, Alzheimer, and human immunodeficiency virus). METHODS Each tweet’s content was rated as nonmedical (NM: testimonies, health care products, solidarity or awareness and misuse) or medical (M: included a reference to the illness’s diagnosis, treatment, prognosis, or prevention). NM tweets were classified as positive or pejorative. We assessed the appropriateness of the medical content. The number of retweets generated and the potential reach and impact of the hashtags analyzed was also investigated. RESULTS We analyzed a total of 15,443 tweets: 8055 classified as NM and 7287 as M. Psychosis-related tweets (PRT) had a significantly higher frequency of misuse 33.3% (212/636) vs 1.15% (853/7419; P CONCLUSIONS We show a reduced number and a different pattern of contents in tweets about psychosis compared with control diseases. PRT showed a predominance of nonmedical content with increased frequencies of misuse and pejorative tone. However, the medical content of PRT showed high scientific appropriateness aimed toward prevention.

Background: Twitter is an indicator of real-world performance, thus, is an appropriate arena to assess the social consideration and attitudes toward psychosis. Objective: The aim of this study was to perform a mixed-methods study of the content and key metrics of tweets referring to psychosis in comparison with tweets referring to control diseases (breast cancer, diabetes, Alzheimer, and human immunodeficiency virus). Methods: Each tweet’s content was rated as nonmedical (NM: testimonies, health care products, solidarity or awareness and misuse) or medical (M: included a reference to the illness’s diagnosis, treatment, prognosis, or prevention). NM tweets were classified as positive or pejorative. We assessed the appropriateness of the medical content. The number of retweets generated and the potential reach and impact of the hashtags analyzed was also investigated. Results: We analyzed a total of 15,443 tweets: 8055 classified as NM and 7287 as M. Psychosis-related tweets (PRT) had a significantly higher frequency of misuse 33.3% (212/636) vs 1.15% (853/7419; P

New evidence suggests that insulin receptor substrate 4 (IRS-4) may play an important role in the promotion of tumoral growth. In this investigation, we have evaluated the role of IRS-4 in a pilot study performed on patients with liver cancer. We used immunohistochemistry to examine IRS-4 expression in biopsies of tumoral tissue from a cohort of 31 patient suffering of hepatocellular carcinoma (HCC). We simultaneously analyzed the expression of the cancer biomarkers PCNA, Ki-67, and pH3 in the same tissue samples. The in vitro analysis was conducted by studying the behavior of HepG2 cells following IRS-4 overexpression/silencing. IRS-4 was expressed mainly in the nuclei of tumoral cells from HCC patients. In contrast, in healthy cells involved in portal triads, canaliculi, and parenchymal tissue, IRS-4 was observed in the cytosol and the membrane. Nuclear IRS-4 in the tumoral region was found in 69.9 ± 3.2%, whereas in the surrounding healthy hepatocytes, nuclear IRS-4 was rarely observed. The percentage of tumoral cells that exhibited nuclear PCNA and Ki-67 were 52.1 ± 7%, 6.1 ± 1.1% and 1.3 ± 0.2%, respectively. Furthermore, we observed a significant positive linear correlation between nuclear IRS-4 and PCNA (r = 0.989, p &lt, 0.001). However, when we correlated the nuclear expression of IRS-4 and Ki-67, we observed a significant positive curvilinear correlation (r = 0.758, 0.010). This allowed us to define two populations, (IRS-4 + Ki-67 ≤ 69%) and (IRS-4 + Ki-67 &gt, 70%). The population with lower levels of IRS-4 and Ki-67 had a higher risk of suffering from multifocal liver cancer (OR = 16.66, CI = 1.68–164.8 (95%), 0.05). Immunoblot analyses showed that IRS-4 in normal human liver biopsies was lower than in HepG2, Huh7, and Chang cells. Treatment of HepG2 with IGF-1 and EGF induced IRS-4 translocation to the nucleus. Regulation of IRS-4 levels via HepG2 transfection experiments revealed the protein’s role in proliferation, cell migration, and cell-collagen adhesion. Nuclear IRS-4 is increased in the tumoral region of HCC. IRS-4 and Ki-67 levels are significantly correlated with the presence of multifocal HCC. Moreover, upregulation of IRS-4 in HepG2 cells induced proliferation by a β-catenin/Rb/cyclin D mechanism, whereas downregulation of IRS-4 caused a loss in cellular polarity and in its adherence to collagen as well as a gain in migratory and invasive capacities, probably via an integrin α2 and focal adhesion cascade (FAK) mechanism.

Polyphenols are used as phlebotonic drugs, but their mechanism of action remains unknown. Since platelet activity and platelet-endothelial cell interactions are involved in the pathogenesis of cardiovascular disease, this work examines whether different flavonoid and coumarin drugs are able to inhibit platelet aggregation. This specific case of coumarins, the antiplatelet effect is not linked with a possible interaction over blood coagulation since this effect only dicoumarols have it. The antiplatelet capacity of polyphenols was assayed using peripheral blood platelets from healthy controls. The distribution of the different platelets subsets was quantified by flow cytometry, using the calcium ionophore as a pro-aggregant. The number of GPIIb/IIIa receptors occupied by the drugs was assayed by flow cytometry using two CD61 surface fluorescein antibodies. All the polyphenols tested inhibited platelet aggregation. A percentage antiplatelet activity of 88.91±7.98% was recorded for naringin, 48.43±8.84% for naringenin, 53.83±7.87% for esculetin, 54.65±6.91% for fraxetin, and 25.75±4.12% for coumarin. Naringin showed significantly greater percentage occupation of GPIIb/IIIa receptors than did naringenin (14.82±0.81% vs. 3.90±0.55%), and esculetin returned significantly higher values than fraxetin and coumarin (12.47±0.97 vs. 7.53±0.49 and 7.90±0.69 respectively). All drugs show important antiplatelet activity. Naringin was the best antiplatelet compound, showing the greatest antiplatelet activity and the highest percentage binding of GPIIb/IIIa receptors. However, any of the compounds used could be used in the prevention of cardiovascular disease.

Our purpose was to study the molecular basis of infliximab (IFX) effect on colon mucosa in a colitis model and to identify new biomarkers of mucosal healing.Healthy rats and rats which were subjected to experimental colitis induced by dextran sulfate sodium, with or without IFX treatment (in the short- and long-term), were studied along with forty-seven IBD patients. Colon mucosal integrity by periodic acid Schiff (PAS) staining, intestinal damage by immunohistochemistry (proliferating cell nuclear antigen, β-catenin, E-cadherin, phosphotyrosine, p-p38, allograft inflammatory factor-1 (AIF-1) and colonic mucosal apoptosis by TUNEL staining were evaluated in rats while serum and colon AIF-1 levels were determined in IBD patients.In rats with colitis, IFX reestablished the epithelial barrier integrity, recovered mucus production and decreased colon inflammation, as verified by reduced serum and colon AIF-1 levels; colon and serum AIF-1 levels were also lower in inactive IBD patients compare to active ones. P38 activation after IFX treatment tended to induce differentiation/proliferation of epithelial cells along the colonic crypt-villous axis.These findings support AIF-1 as a new biomarker of mucosal healing in experimental colitis and suggest that p38 activation is involved in the mucosal healing intracellular mechanism induced by IFX treatment.

CD4+T-lymphocytes are relevant in the pathogenesis of rheumatoid arthritis (RA), however, their potential involvement in early RA remains elusive. Methotrexate (MTX) is a commonly used disease-modifying antirheumatic drug (DMARD), but its mechanism has not been fully established. In 47 new-onset DMARD-na&iuml, ve RA patients, we investigated the pattern of IFN&gamma, IL-4 and IL-17A expression by na&iuml, ve (TN), central (TCM), effector memory (TEM) and effector (TE) CD4+ subsets, their STAT-1, STAT-6 and STAT-3 transcription factors phosphorylation, and the circulating levels of IFN&gamma, IL-4 and IL-17. We also studied the RA patients after 3 and 6 months of MTX treatment and according their clinical response. CD4+T-lymphocyte subsets and cytokine expression were measured using flow cytometry. New-onset DMARD-na&iuml, ve RA patients showed a significant expansion of IL-17A+, IFN&gamma, + and IL-17A+IFN&gamma, + CD4+T-lymphocyte subsets and increased intracellular STAT-1 and STAT-3 phosphorylation. Under basal conditions, nonresponder patients showed increased numbers of circulating IL-17A producing TN and TMC CD4+T-lymphocytes and IFN&gamma, producing TN, TCM, TEM CD4+T-lymphocytes with respect to responders. After 6 months, the numbers of CD4+IL-17A+TN remained significantly increased in nonresponders. In conclusion, CD4+T-lymphocytes in new-onset DMARD-na&iuml, ve RA patients show IL-17A and IFN&gamma, abnormalities in TN, indicating their relevant role in early disease pathogenesis. Different patterns of CD4+ modulation are identified in MTX responders and nonresponders.

Objective. The aim of this study was to summarize evidence on the effectiveness of therapeutic exercise in Fibromyalgia Syndrome. Design. Studies retrieved from the Cochrane Plus, PEDro, and Pubmed databases were systematically reviewed. Randomized controlled trials and meta-analyses involving adults with fibromyalgia were included. The primary outcomes considered in this systematic review were pain, global well-being, symptoms of depression, and health-related quality of life. Results. Effects were summarized using standardized mean differences with 95% confidence intervals using a random effects model. This study provides strong evidence that physical exercise reduces pain (−1.11 [95% CI] −1.52; −0.71; overall effect p<0.001), global well-being (−0.67 [95% CI] −0.89, −0.45; p<0.001), and symptoms of depression (−0.40 [95% CI] −0.55, −0.24; p<0.001) and that it improves both components of health-related quality of life (physical: 0.77 [95% CI] 0.47; 1.08; p<0.001; mental: 0.49 [95% CI] 0.27; 0.71; p<0.001). Conclusions. This study concludes that aerobic and muscle strengthening exercises are the most effective way of reducing pain and improving global well-being in people with fibromyalgia and that stretching and aerobic exercises increase health-related quality of life. In addition, combined exercise produces the biggest beneficial effect on symptoms of depression.

Background: T lymphocytes are involved in the pathogenesis of nonallergic asthma. The objective of this study was to characterize the subset distribution and pattern of chemokine receptor expression in circulating T lymphocyte subsets from nonallergic asthma patients. Methods: Forty stable nonallergic asthma patients and 16 sex- and age-matched healthy donors were studied. Twelve patients did not receive inhaled steroids (untreated patients), 16 received 50-500 μg b.i.d. of inhaled fluticasone propionate (FP) (standard-dose patients), and 12 received over 500 μg b.i.d. of inhaled FP (high-dose patients) for at least 12 months prior to the beginning of this study and were clinically well controlled. Flow cytometry was performed using a panel of monoclonal antibodies (4 colors). Results: Nonallergic asthma patients treated with high doses of inhaled FP showed a significant reduction in the percentages of CD3+ T lymphocytes compared to healthy controls. Untreated patients showed a significant increase in CCR6 expression in CD8+CD25+ and CD8+CD25+bright T cells compared to healthy controls. The results were similar for CXCR3 and CCR5 expression. In patients treated with standard doses of FP, CCR5 expression was significantly increased in CD3+ T lymphocytes relative to healthy controls. Conclusions: The different groups of clinically stable nonallergic asthmatic patients showed distinct patterns of alterations in subset distribution as well as CCR6, CXCR3, and CCR5 expression on circulating T lymphocytes.

espanolEl aula invertida es un modelo de ensenanza-aprendizaje en el que los alumnos tienen el primer contacto con la informacion a ser aprendida fuera de clase, mediante documentos (textos y videos) que el docente les hace llegar por medios electronicos. El tiempo de clase asi ahorrado se dedica a actividades de aula que consolidan la asimilacion de ese conocimiento y lo aplican a la resolucion de cuestiones, casos y problemas. Este modelo de aprendizaje reduce el tiempo de instruccion directa en clase y aumenta el dedicado al aprendizaje activo. Se transfi ere al alumno la responsabilidad de esforzarse inicialmente para alcanzar un nivel de comprension basico y comunicar sus difi cultades y dudas al docente. Asi, el docente recibe informacion sobre cuales son las difi cultades y necesidades de sus alumnos y podra adaptar las activida-des que realizara en el aula para resolver las dudas manifestadas por ellos. Denominamos a esta metodologia ‘aula inver-tida adaptativa’. El aula invertida logra un mayor grado de implicacion de los alumnos con su aprendizaje, mejoras en la valoracion de su percepcion sobre la docencia recibida y, sobre todo, mejoras en sus resultados academicos. En este ar-ticulo tambien se sopesan los benefi cios y los costes del cambio desde la metodologia expositiva tradicional al aula inver-tida adaptativa y, fi nalmente, se aportan recomendaciones para la puesta en practica del aula invertida en el contexto de una ensenanza tradicional de las ciencias sanitarias. EnglishFlipped classroom means that students have the fi rst exposure to new information to be learned outside the classroom by mean of electronic documents (texts and videos). Next, class time is devoted to class activities which reinforce the assimilation of that knowledge by applying it to answer questions and solving cases and problems. This learning model reduces the class time devoted to direct instruction and increases the time used in active learning. It transfers to the student the responsibility of initially striving to reach a basic understanding and communicate their diffi culties and doubts to the teacher. Thus, the teacher receives information about the diffi culties and needs of their students and can adapt the activities they will carry out in the classroom to solve the doubts expressed by their students. We named this teaching methodology as adaptive fl ipped classroom. The fl ipped classroom achieves a greater degree of involvement of students with their learning, improvements in academic results and in their assessment of the teaching received. In this report, the benefi ts and costs of the change are weighed from the traditional expositive methodology to the adaptive fl ipped classroom and, fi nally, recommendations are given for the implementation of the fl ipped classroom in the context of a traditional teaching of health sciences.

et al., Hepatoblastoma is a primary liver cancer that affects children, due to the sensitivity of this tumor to insulin-like growth factor 1 (IGF-1). In this paper we show that azathioprine (AZA) is capable of inhibiting IGF1-mediated signaling cascade in HepG2 cells. The efficiency of AZA on inhibition of proliferation differs in the evaluated cell lines as follows: HepG2 (an experimental model of hepatoblastoma) > Hep3B (derived from a hepatocellular carcinoma) > HuH6 (derived from a hepatoblastoma) ≫ HuH7 (derived from a hepatocellular carcinoma) = Chang Liver cells (a non-malignant cellular model). The effect of AZA in HepG2 cells has been proven to derive from activation of Ras/ERK/TSC2, leading to activation of mTOR/p70S6K in a sustained manner. p70S6K phosphorylates IRS-1 in serine 307 which leads to the uncoupling between IRS-1 and p85 (the regulatory subunit of PI3K) and therefore causing the lack of response of HepG2 to IGF-1. As a consequence, proliferation induced by IGF-1 is inhibited by AZA and autophagy increases leading to senescence of HepG2 cells. Our results suggest that AZA induces the autophagic process in HepG2 activating senescence, and driving to deceleration of cell cycle but not to apoptosis. However, when simultaneous to AZA treatment the autophagy was inhibited by bafilomycin A1 and the degradation of regulatory proteins of cell cycle (e.g. Rb, E2F, and cyclin D1) provoked apoptosis.In conclusion, AZA induces resistance in hepatoblastoma cells to IGF-1, which leads to autophagy activation, and causes apoptosis when it is combined with bafilomycin A1. We are presenting here a novel mechanism of action of azathioprine, which could be useful in treatment of IGF-1 dependent tumors, especially in its combination with other drugs., This work was supported by the Spanish Science and Education Ministry [Grant SAF2008-05355] and by the Comunidad de Madrid [Grants CCG10-UAH/BlO-5971, S2010/BMD-2423].

Background ZAP-70 upregulation in B chronic lymphocytic leukemia (B-CLL) cells is a recognized marker of poor prognosis in these patients; the biological basis of this differential clinical outcome nonetheless remains unknown. ZAP-70 overexpression is considered a surrogate marker of a B-CLL cell subset. To test whether the differential biological characteristics of these patients also include the T helper population, we studied naive, non-terminated memory (NTEM), terminated memory (TEM) and central memory (CM) cells, and cytokine expression by CD4 T lymphocytes from ZAP-70+ and ZAP-70− B-CLL patients. Methods Expression of CD3, CD8, CD45RA, CD27, and CD28 antigens and intracytoplasmic cytokine production (IFNγ, IL-2, IL-4, IL-10, and TNFα) were assessed simultaneously by nine-color flow-cytometry in peripheral blood lymphocytes from B-CLL patients. B cell ZAP-70 expression in B-CLL cells was also analyzed by flow cytometry. Results Compared to ZAP-70− B-CLL patients, ZAP-70+ B-CLL patients showed 1) significant reduction in the naive T helper subset and expansion of NTEM and TEM subsets, 2) a decrease in the percentage of activated CD4 T lymphocytes expressing IFNγ, TNFα, and IL-2, and 3) an increase in the percentage of CD4 T lymphocytes expressing IL-4 or IL-10. Conclusions In conclusion, in early stage B-CLL patients, ZAP-70 upregulation is associated with distinct patterns of activation/differentiation stage subset distribution and of cytokine expression in CD4 T lymphocytes. © 2013 International Clinical Cytometry Society

Depletion of circulating CD4(+) T-helper (Th) lymphocytes, especially naive Th cells, is common in cirrhosis. Little is known about the pathogenetic mechanisms involved in Th-cell depletion in cirrhosis. We investigated the mechanisms involved in circulating Th-cell lymphopenia in cirrhosis.Circulating naive and memory Th cells were analyzed by flow cytometry in 60 patients with cirrhosis and 40 sex- and age-matched healthy controls. Thymopoiesis, apoptosis, cell activation, and proliferation were assessed through CD31, annexin-V, HLA-DR and Ki-67 expression, respectively. Lipopolysaccharide (LPS)-binding protein (LBP) and spleen size were measured as indicators of bacterial translocation and splenic pooling, respectively.Compared to controls, patients showed reduced numbers of Th cells involving a greater depletion of the naive than memory Th-cell compartment (2.7- vs. 1.5-fold, respectively). Recent thymic emigrants were diminished (p0.01), and each patient had a lower number of CD31(+) naive Th cells than the matched-control. Spontaneous and induced apoptosis (Annexin-V(+)) of Th cells was increased in patients. Activated (HLA-DR(+)) and proliferating (Ki-67(+)) memory Th cells were increased in patients (p0.01), and they directly correlated with plasma LBP (p0.05) and negatively with naive Th cells (p0.01), respectively. Naive Th cells were inversely correlated (p0.01) with their frequencies of apoptosis and of activated memory Th cells, LBP, and spleen size. On multivariate analysis, defective thymic generation of naive Th cells, increased memory Th-cell activation, and splenomegaly were independently associated with Th-cell depletion.Th-cell immunodeficiency in cirrhosis is explained by a universal defect in thymopoiesis exacerbated by splenic pooling and activation-driven cell-death induced by bacterial translocation.

Vascular endothelium activation is a key pathogenic step in systemic inflammatory response syndrome (SIRS) that can be triggered by both microbial and sterile proinflammatory stimuli. The relevance of soluble adhesion molecules as clinical biomarkers to discriminate between infectious and non-infectious SIRS, and the individual patient prognosis, has not been established.We prospectively measured by sandwich ELISA, serum levels of soluble E-Selectin (sE-Selectin), soluble vascular cell adhesion molecule-1 (sVCAM-1), soluble intercellular adhesion molecule-1 (sICAM-1) and soluble intercellular adhesion molecule-2 (sICAM-2) at ICU admission and at days 3, 7, 14 and 28 in patients with sepsis and at days 3 and 7 in patients with non-infectious SIRS.At ICU admission, sE-Selectin, sVCAM-1 and sICAM-1 in patients with infectious SIRS were significantly higher than those found in patients with non-infectious SIRS. ROC analysis revealed that the AUC for infection identification was best for sICAM-1 (0.900±0.041; 95% CI 0.819-0.981; p0.0001). Moreover, multivariate analysis showed that 4 variables were significantly and independently associated with mortality at 28 days: male gender (OR 15.90; 95% CI, 2.54-99.32), MODS score (OR 5.60; 95% CI, 1.67-18.74), circulating sE-Selectin levels (OR 4.81; 95% CI, 1.34-17.19) and sVCAM-1 concentrations (OR 4.80; 95% CI, 1.34-17.14).Patients with SIRS secondary to infectious or non-infectious etiology show distinctive patterns of disturbance in serum soluble adhesion molecules. Serum ICAM-1 is a reliable biomarker for classifying patients with infectious SIRS from those with non-infectious SIRS. In addition, soluble E-Selectin is a prognostic biomarker with higher levels in patients with SIRS and fatal outcome.

Cirrhosis with ascites is associated with a high rate of gut bacterial translocation (GBT) and spontaneous bacterial infections of enteric origin. We addressed the activation state and role of intestinal dendritic cells (DCs) in experimental ascitic cirrhosis and their relationship with GBT. Cirrhosis with ascites was CCl4 induced in rats. To examine their activation state and functions, DCs (CD103+RT1B+CD3−CD45RA−) were isolated from the intestinal lamina propria and mesenteric lymph nodes (MLNs), and the following parameters were determined by flow cytometry: surface antigen expression; spontaneous or lipopolysaccharide-stimulated tumor necrosis factor alpha (TNF-α) production; and in vitro capacity to phagocytose latex beads and to migrate toward the chemokine (C-C motif) ligand 21. GBT was defined as the growth of bacteria in MLNs culture. Bacterial DNA (Bact-DNA) in MLNs was identified by polymerase chain reaction. In rats with Bact-DNA in MLNs without GBT, intestinal and MLNs CD103+-DCs showed features of activation, expansion of the proinflammatory CD4+-DC subpopulation, augmented TNF-α production, and increased phagocytic and migratory capacities. In contrast, in rats with GBT, CD103+-DCs showed the absence of an activated phenotype, lowered TNF-α production, and relatively deficient phagocytosis and migration capacities. The CD103+-DC of rats without Bact-DNA in MLNs or GBT were similar to controls. In cirrhotic rats, bowel decontamination with antibiotics eliminated Bact-DNA in MLNs and GBT, normalized the activation state and functions of CD103+-DCs, and increased their TNF-α production. Conclusion: In experimental cirrhosis with ascites, continuous pressure of gut bacteria shapes the phenotypic and functional profile of intestinal DCs to produce effects that range from their activation and enhanced functions to their exhaustion and tolerance. (HEPATOLOGY 2012;56:1861–1869)

BACKGROUND: Products cryopreserved with dimethyl sulfoxide (DMSO) in stem cell transplant (SCT) often cause many adverse effects during their infusion (major cardiovascular events, dyspnea … even death). These are especially frequent in pediatric patients. We tested if a fully automated and closed wash procedure (Sepax S-100, Biosafe) allowed us to maintain the absolute CD34+ cell number, cell viability, and engraftment potential, decreasing the untoward reactions. STUDY DESIGN AND METHODS: Forty-six washes of DMSO cryopreserved peripheral blood hematopoietic progenitor (HP) apheresis were studied. Blood aliquots were taken both after thawing and after washing to assess the total nucleated and CD34+ cell counts, as well as cell viability. The washed products were infused in 26 autologous SCTs (ASCTs). Results were compared with the 53 previous SCTs performed without DMSO removal. RESULTS: After washing there were no significant differences between the pre- and postwashing CD34+ cell counts (p = 0.08) or viability (p = 0.68). No significant differences were observed between washed and nonwashed infusions in relation to the day of the neutrophil (p = 0.46) and platelet (p = 0.26) engraftment. One adverse event, abdominal pain, occurred during the washed cells infusions. When compared with the 14 untoward reactions that took place during the nonwashed HP infusions, significance was reached (p = 0.00043). CONCLUSIONS: The automatic method described is effective in terms of CD34+ cell recovery and viability in ASCT. Moreover, Sepax decreased significantly the untoward reactions during the infusion.

Background: Mortality in patients with septic shock remains unacceptably high and the attempts to antagonize certain proinflammatory cytokines based on the results of animal model studies have failed to improve survival rates. The objective of this article is to examine the pro-/anti-inflammatory cytokine balance in patients with septic shock and its connection with mortality. Methods: Serum levels of proinflammatory cytokines (tumor necrosis factor-α [TNF-α], interleukin 1β [IL-1β], interferonγ [IFN-γ], and IL-6) and soluble cytokine antagonists (soluble TNF receptor I [sTNF-RI], sTNF-RII, and IL-1Ra) were determined on admission to the intensive care unit (ICU) and 3, 7, 14, and 28 days later in 52 patients with septic shock and in 36 healthy controls. Specific sandwich enzyme-linked immunosorbent assay (ELISA) was used for all determinations. Results: Serum levels of most of the pro- and anti-inflammatory molecules examined (TNF-α, IL-6, sTNF-RI, sTNF-RII, and IL-1 receptor agonist [IL-1Ra]) were significantly elevated on admission and during the 28-day observation period in patients when compared to controls. Notably, the anti-inflammatory mediators sTNF-RI, sTNF-RII, and IL-1Ra were better predictors of mortality. Receiver-operating characteristic (ROC) analysis revealed that sTNF-RI or sTNF-RII concentrations over 2767 or 4619 pg/mL, respectively, determined a high risk of death (sensitivity: 100%-100%, specificity: 57.1%-71.4%, area under the curve [AUC] 0.759-0.841, respectively), whereas IL-1Ra concentrations below 7033 pg/mL determined a high probability of survival (sensitivity: 60%, specificity: 100%, AUC 0.724). In addition, IFN-γ levels were significantly higher in survivors than in controls during the initial 2 weeks of observation. Conclusions: Our data show that serum cytokine disturbance patterns have prognostic significance in patients with septic shock admitted to the ICU. The pattern, defined by an early response to continuously elevated anti-inflammatory cytokine serum levels, is associated with an enhanced risk of a fatal outcome for patients.

Background Herpes zoster (HZ) and its complications can have a substantial impact on patients’ quality of life (QoL), particularly in immunocompromised patients. The vaccine efficacy (VE) of an adjuvanted recombinant zoster vaccine (RZV) was studied in a randomized, placebo-controlled, phase 3 study in adult hematopoietic stem cell transplant (HSCT) recipients (NCT01610414). The VE in preventing HZ cases was 68.2% (95% CI: 55.6%–77.5%). Herein we report the impact of the vaccine on patients’ quality of life (QoL) associated with HZ episodes. Methods HSCT recipients were randomized 1:1 to receive 2 doses of RZV or placebo, given 1–2 months apart and followed for the occurrence of HZ. QoL parameters were measured by the Short-Form health survey (SF-36) and Euro-Quality of Life-5 Dimension (EQ-5D) at baseline, 1 month and 1 year post-dose 2, as well as during suspected HZ episodes in conjunction with the Zoster Brief Pain Inventory (ZBPI). For confirmed HZ cases, QoL scores were compared between the vaccine and placebo groups. The RZV impact in reducing the ZBPI Burden of Illness and Burden of Interference scores was estimated in patients in the modified total vaccinated cohort (mTVC). The 2 scores were calculated from the area under the curve (Days 0 to 182) of the ZBPI Worst Pain and ZBPI Activities of Daily Living scores, respectively, assuming a score of 0 for patients who did not have a confirmed HZ episode. Results Both the ZBPI maximum Worst Pain and Average Pain scores were significantly lower in the vaccine than placebo group (Table 1), suggesting less burden in breakthrough HZ cases following RZV. Consequently, the HZ Burden of Illness and Burden of Interference VE estimates were higher than the HZ VE estimate. RZV showed statistically significantly better QoL scores than placebo one week following rash-onset among patients with confirmed HZ, i.e., SF-36 bodily pain, social functioning, role emotional, mental health and mental component scores, and the EQ-5D Utility Score. Conclusion In addition to reducing the risk of HZ and HZ complications, RZV significantly reduces the impact of HZ on patient’s QoL in those who develop breakthrough disease. Funding: GlaxoSmithKline Biologicals SA. Disclosures D. Curran, GlaxoSmithKline: Employee, GSK Shares and Salary. A. Bastidas, GSK: Employee, Salary. M. El Idrissi, GSK: Employee and Shareholder, Salary. S. Matthews, GSK group of companies: Consultant, Consulting fee. L. Oostvogels, GSK group of companies: Employee, Salary and stock and stock option. J. A. Young, GSK: Investigator, The University of Minnesota is reimbursed for contract costs associated with conducting clinical trials of vaccine. I receive no personal financial benefit.

Systemic activation of the inflammatory immune system contributes to the progression of cirrhosis with ascites. Immune cells become activated after interacting at the mesenteric lymph nodes (MLNs) with bacteria translocated from the gut, and thereafter reach the bloodstream through recirculation. It is unknown whether systemic activation of the immune system is present in pre-ascitic cirrhosis, in which gut bacterial translocation has not been described. The purpose of this study was to determine whether systemic activation of the immune system initiates in rats with compensated carbon tetrachloride (CCl4)-induced cirrhosis, and if so to establish the activation site of immune cells. We studied the activation status of immune cells in peripheral blood, MLNs, and hepatic lymph nodes (HLNs). Systemic inflammation was present in rats with cirrhosis, as shown by expansion (P < 0.01) of circulating total and inflammatory monocytes and recently activated CD134+ T helper (Th) cells. The same populations of cells were increased (P < 0.01) in MLNs and HLNs. Bacterial translocation was absent in rats with cirrhosis or control rats, but bacterial DNA fragments were present in the MLNs of 54% of rats with cirrhosis. The liver was the source of activated immune cells present in the blood, as shown by the direct correlation between activated Th cells in the blood and HLNs, but not in MLNs, and the normalization by gut decontamination with antibiotics of activated cells in MLNs, but not in the blood or HLNs. Conclusion: In experimental cirrhosis, systemic activation of the immune system occurs before ascites development and is driven by recirculation of cells activated in HLNs. In addition, in compensated cirrhosis, bacterial DNA fragments reach the MLNs, where they elicit a local inflammatory response. (HEPATOLOGY 2010;52:2086-2095)

In animal models, a defective Th1 response appears to be critical in the pathogenesis of brucellosis, but the Th1 response in human brucellosis patients remains partially undefined. Peripheral blood from 24 brucellosis patients was studied before and 45 days after antibiotherapy. Twenty-four sex- and age-matched healthy donors were analyzed in parallel. Significantly increased levels of interleukin 1β (IL-1β), IL-2, IL-4, IL-6, IL-12p40, gamma interferon (IFN-γ), and tumor necrosis factor alpha (TNF-α), but not of IL-10, in serum and/or significantly increased percentages of samples with detectable levels of these cytokines, measured by enzyme-linked immunosorbent assays (ELISA), were found for untreated brucellosis patients, but these levels were reduced and/or normalized after treatment. Flow cytometry studies showed that the intracytoplasmic expression of IFN-γ, IL-2, and TNF-α, but not that of IL-4, by phorbol myristate-activated CD4 + CD3 + and CD8 + CD3 + T lymphocytes was significantly increased in untreated brucellosis patients and was also partially normalized after antibiotherapy. The percentage of phagocytic cells, the mean phagocytic activity per cell, and the phagocytic indices for monocytes at baseline were defective and had only partially reverted at follow-up. T lymphocytes from untreated brucellosis patients are activated in vivo and show Th1 cytokine production polarization, with strikingly high serum IFN-γ levels. In spite of this Th1 environment, we found deficient effector phagocytic activity in peripheral blood monocytes.

Studies here respond to two long-standing questions: Are human “pre/pro-B” CD34+CD10−CD19+and “common lymphoid progenitor (CLP)/early-B” CD34+CD10+CD19−alternate precursors to “pro-B” CD34+CD19+CD10+cells, and do the pro-B cells that arise from these progenitors belong to the same or distinct B-cell development pathways? Using flow cytometry, gene expression profiling, and Ig VH-D-JHsequencing, we monitor the initial 10 generations of development of sorted cord blood CD34highLineage−pluripotential progenitors growing in bone marrow S17 stroma cocultures. We show that (i) multipotent progenitors (CD34+CD45RA+CD10−CD19−) directly generate an initial wave of Pax5+TdT−“unilineage” pre/pro-B cells and a later wave of “multilineage” CLP/early-B cells and (ii) the cells generated in these successive stages act as precursors for distinct pro-B cells through two independent layered pathways. Studies by others have tracked the origin of B-lineage leukemias in elderly mice to the mouse B-1a pre/pro-B lineage, which lacks the TdT activity that diversifies the VH-D-JHIg heavy chain joints found in the early-B or B-2 lineage. Here, we show a similar divergence in human B-cell development pathways between the Pax5+TdT−pre/pro-B differentiation pathway that gives rise to infant B-lineage leukemias and the early-B pathway.

Insulin receptor substrate-4 (IRS-4) transmits signals from the insulin-like growth factor receptor (IGF-IR) and the insulin receptor (IR) to the PI3K/AKT and the ERK1/2 pathways. IRS-4 expression increases dramatically after partial hepatectomy and plays an important role in HepG2 hepatoblastoma cell line proliferation/differentiation. In human hepatocarcinoma, IRS-4 overexpression has been associated with tumor development. Herein, we describe the mechanism whereby IRS-4 depletion induced by RNA interference (siRNA) sensitizes HepG2 cells to treatment with actinomycin D (Act D) and combined treatment with Act D plus tumor necrosis factor-alpha (TNF-alpha). Similar results have been obtained in HuH 7 and Chang cell lines. Act D therapy drove the cells to a mitochondrial-dependent apoptotic program involving cytochrome c release, caspase 3 activation, PARP fragmentation and DNA laddering. TNF-alpha amplifies the effect of Act D on HepG2 cell apoptosis increasing c-jun N-terminal kinase (JNK) activity, IkappaB-alpha proteolysis and glutathione depletion. IRS-4 depleted cells that were treated with Act D showed an increase in cytochrome c release and procaspase 3 and PARP proteolysis with respect to control cells. The mechanism involved in IRS-4 action is independent of Akt, IkappaB kinase and JNK. IRS-4 down regulation, however, decreased gamma-glutamylcysteine synthetase content and cell glutathione level in the presence of Act D plus TNF-alpha. These results suggest that IRS-4 protects HepG2 cells from oxidative stress induced by drug treatment.

Abnormal apoptosis has been reported in circulating T lymphocytes in patients with multiple sclerosis. The effects of 12 months of IFNbeta treatment in T and B lymphocyte spontaneous ex vivo apoptosis were studied in patients with MS. Peripheral blood mononuclear cells were obtained from 48 patients before and at 1, 6 and 12 months after treatment with IFNbeta. Spontaneous ex vivo apoptosis was quantified by four-color flow cytometry. A significant reduction and normalization of the percentage of apoptotic cells was found in all T lymphocyte subsets. B cell apoptosis values were unaffected by therapy; Relapses of the clinical activity of the disease were associated to transitory upturns of lymphocyte apoptosis. In conclusion, IFNbeta therapy progressively normalizes the increased ex vivo T lymphocyte apoptosis observed in MS. However, it is not clear if this reduction in spontaneous T lymphocyte apoptosis is due to direct effect of IFNbeta or secondary to decreased clinical and sub-clinical activity.

The peripheral blood lymphocytes of eight patients with metastatic renal cell carcinoma, and of eight healthy volunteers were analyzed by four-color flow cytometry to characterize the immunophenotypic alterations manifested, determine the prevalence of lymphocyte apoptosis, and detect evidence of the systemic effect of inhaled IL-2. The T, B and NK lymphocytes of untreated patients were found to have undergone profound changes characterized by an increase in susceptibility to both spontaneous and mitogen-induced ex vivo apoptosis, a modified distribution of the main lymphocyte populations in the peripheral blood, and alterations in activation status. An increase in the proportion of regulatory T cells was also seen in these patients. Treatment with inhaled IL-2, however, normalized the rate of apoptosis in all the lymphocyte subpopulations studied, as well as their distribution and activation status. These findings demonstrate that inhaled IL-2 has systemic immunomodulatory effects.

The aims of this study were to examine the changes in plasma concentrations of inflammatory cytokines induced by training and competition in professional cyclists. We report the serum concentrations of interleukin-6 (IL-6), tumour necrosis factor alpha (TNF-a), tumour necrosis factor receptors I and II (TNFR-I and -II) in a prospective, randomized, double-blind trial involving the administration of AM3 (Inmunoferon), an oral booster immunomodulator, or placebo to 16 professional cyclists (n = 8 in each group) for 65 consecutive days. Serum was collected just before treatment began (baseline), at the end of pre-competition training, before the mountain stage of the competition (60 days), 4 h after finishing this stage (62 days), and 18 h after the fifth and last day of competition (65 days). To determine the normal levels of cytokines and soluble TNF receptors, individual samples from 14 moderately trained healthy controls were studied. After 60 days of training, the serum concentrations of IL-6 did not differ significantly from those at the beginning of the study for either group of cyclists (placebo and AM3). A significant rise was seen in IL-6 concentrations in both the AM3 and placebo groups at 62 days, 4 h after finishing the mountain stage. The increase was significantly greater in the placebo group than in the AM3 group. At 65 days of treatment, 18 h after the fifth and last day of competition, IL-6 concentrations were similar to those recorded at the end of the training, but were significantly higher in the placebo group than in the AM3 group. At the end of training, serum TNFR-I concentrations in both groups of cyclists were significantly lower than at baseline. The concentrations of serum TNFR-I and -II both 4 h after finishing the mountain stage and 18 h after the fifth and last day of competition were significantly higher than those recorded after training in both groups. Professional cycling competition is associated with increases in serum IL-6 and TNFR-I and -II concentrations. Inmunoferon treatment reduced significantly the concentrations of IL-6 but not those of TNFR-I and -II.

In order to characterize the immunophenotype and the lymphocyte apoptosis in multiple sclerosis (MS) patients, the peripheral blood lymphocytes of 46 MS patients and 12 healthy volunteers were studied by flow cytometry. Immunophenotypic alterations included significant increases in T CD4+ lymphocytes and reductions in the percentages of CD3+ and CD8+ T cells. After 24 h of culture spontaneous apoptosis was increased in almost T lymphocyte subsets from MS patients and it was significantly higher in those patients who had suffered more than two relapses in the two previous years. The incidence of spontaneous apoptosis was not dependent on FasL-Fas interactions and correlated with the percentages of cells positive for active caspases but not with percentages of Fas+ cells. The increased susceptibility to apoptosis of peripheral blood T lymphocytes from MS patients is difficult to reconcile with the previously proposed role of a defective lymphocyte apoptosis in the pathophysiology of MS.

Background: The use of ratiometric cell enumeration methods emerges as a more accurate method of measurement of the occurrence of apoptosis in cell cultures. These new flow cytometry methods were used to quantify the impact of cell fragmentation and loss of lineage antigen (LAg) expression on measurement of apoptosis. Methods: Highly purified human lymphocyte populations were negatively sorted and cultured for 24 h. Apoptotic cells were identified using annexin V, 7-amino-actinomycin D and their LAgs were stained with antibodies. A new indicator, the apoptotic rate, was used to determine apoptosis occurrence and its validity compared with the widely accepted percentage of apoptotic cells (apoptotic index, AI). Results: Loss of LAg expression and cell fragmentation were observed under all conditions assayed and for all cell populations studied. Conclusions: Current methods for quantifying of apoptosis involving AI systematically underestimate apoptosis occurrence in all populations and conditions, especially among cells undergoing spontaneous apoptosis. © 2006 International Society for Analytical Cytology

Intestinal bacterial overgrowth and translocation, both common in cirrhosis with ascites, may lead to the activation of monocytes and lymphocytes, increased levels of proinflammatory cytokines, and enhanced synthesis of nitric oxide present in cirrhosis. Bacterial endotoxin promotes the synthesis of lipopolysaccharide (LPS)-binding protein (LBP), and forms a LPS-LBP complex that binds to CD14. This study was designed to evaluate LBP levels and their correlation to the immune response and the hemodynamic status in cirrhotic patients. Plasma LBP, endotoxin, soluble CD14 (sCD14), cytokines, renin, nitrites, and systemic vascular resistance were determined before and 4 weeks after norfloxacin or placebo in 102 cirrhotic patients and 30 controls. LBP was elevated in 42% of ascitic cirrhotic patients (15.7 ± 0.7 versus 6.06 ± 0.5 μg/mL, P < .01). In 60% of high LBP patients, endotoxin was within normal range. Among ascitic patients, those with high LBP showed greater (P < .05) levels of sCD14, tumor necrosis factor α (TNF-α), interleukin 6 (IL-6), nitrites + nitrates (NOx)/creatinine, and renin, and lower vascular resistance. In the cirrhotic patients with high LBP, norfloxacin normalized (P < .01) LBP (from 16.6 ± 0.5 to 5.82 ± 0.8 μ g/mL) and sCD14; reduced the level of cytokines, NOx/creatinine, and renin; and increased vascular resistance; but lacked effect in patients with normal LBP. Portal pressure was unchanged after norfloxacin in another group of 18 cirrhotic patients with high and 19 with normal LBP. In conclusion, the subset of ascitic cirrhotic patients with marked immune and hemodynamic derangement is identified by increased LBP levels. Amelioration of these abnormalities by norfloxacin suggests the involvement of enteric bacteria or their products in the triggering of the process. (HEPATOLOGY 2003;37:208-217.)

Background Late apoptotic cells divide into apoptotic bodies and are missed by current detection methods. This results in an artificially low apoptotic index (AI). Methods This study proposes a flow cytometry-based ratiometric method that uses an internal reference standard of microbeads combined with fluorescein-annexin V binding and 7-aminoactinomycin D to enumerate viable, necrotic, and early and late apoptotic cells within specific subsets of a heterogeneous culture. Results In the absence of cell growth, the number of apoptotic cells that undergo fragmentation into apoptotic bodies in culture can also be determined accurately by this method. This information can then be used to obtain the apoptotic rate (AR), a new indicator of apoptosis that calculates the proportion of cells that have undergone apoptosis with respect to the total number of seeded cells. The main limitation of the method is that the AR is only suitable for the study of apoptosis in noncycling cells. Conclusions This study reveals the superiority of the proposed method over the widely used Nicoletti method and current annexin-V binding methods. The AI did not reflect the true incidence of lymphocyte apoptosis, neither in response to lectins or phorbol esters, nor to serum deprivation. AR was more sensitive than AI, detecting apoptosis at lower concentrations of cell death inducers in all the subsets studied. Cytometry 48:185–193, 2002. © 2002 Wiley-Liss, Inc.

ResumenProfesores de inmunología de la Universidad de Alcalá hemos implementado en los últimos cinco años el modelo de aula inversa en tres asignaturas del grado de Biología Sanitaria. El porcentaje de alumnos que estudiaron los materiales instructivos (documentos y vídeos) antes de que los temas fuesen tratados en clase ha aumentado desde valores minoritarios en los primeros años a porcentajes cercanos al 100% en los últimos años. La calificación media obtenida por los alumnos en las pruebas de evaluación del aprendizaje ha mejorado en las tres asignaturas en más de una desviación estándar con respecto a la que se obtenía con metodologías expositivas tradicionales antes de la implantación del modelo de aprendizaje inverso. Los porcentajes de alumnos que fracasan han disminuido y han aumentado los porcentajes de alumnos que alcanzan un nivel de maestría (calificación ¿8 en una escala de diez). Los alumnos reconocen desarrollar competencias con las actividades realizadas en los seminarios. El grado de satisfacción de los alumnos también ha aumentado. Concluimos que con el modelo inverso los alumnos universitarios dedican más trabajo no presencial al estudio de sus asignaturas y a consecuencia de ello sus calificaciones en la pruebas de evaluación del aprendizaje mejoran significativamente AbstractImmunology teachers form Alcala’s University implementes flipped classroom model in the last five years into three immunology courses in sanitary biology university degree. The percentage of students that studied the instructive materials (documents and videos) before class has increased from minority values in the first years to percentages close to 100% in the last years. The mean grade obtained by the classes in the learning assessment exams has increased in the three courses in more than one standard deviation with respect to the original mean obtained in each course with traditional expositive methods before the implementation of the flipped learning model. The percentages of failed students have decreased and those of students that attain mastery levels (grades ¿80% of the maximum grade) have increased. The students recognize that the flipped model helps them to develop competencies in the activities developed in the seminars. The degree of satisfaction with the teaching has also increased We can conclude that university students thanks to the flipped model devote more time of work to the study of flipped courses and as a consequence of this increase in study they learn more and obtain increased grades in the learning assessment exams.

The results obtained thanks to the flipped classroom with ‘just Iiin time’ teaching method are reviewed. This method allowed to know what the students did not understand after trying to study the instructive materials assigned to them. To achieve that the students work before class methods of marketing and gamification were developed. Methods for the analysis of student responses were also developed. The JiTT method allowes the teacher know in advance those aspects that are most interesting or most dificult to understand for the students as well as their most urgent doubts. Finally, we have developed methods to use the urgent doubts of the students to generate formative feedback and activities to do in the classroom. In the method named “flipped learning forte” the teacher answer the urgent doubts of his students. In the method named ‘flip in colours’ the teacher classify the doubts by their posible utility in the classroom using a color code. With the combined application of these methods in university courses the failure rate of the students has decreased and the mean grade in the exams for the assessment of learning has increased in more than one standard deviation. The rate of students that attain the level of mastery has increased, as well as the student evaluations about the teachers of these courses. Finally, the reasons underlying the efficacy of the proposed flipped method are discussed.

Infliximab (IFX) is widely used in ulcerative colitis and in Crohn’s disease treatment. Both diseases are characterised by increased oxidative stress, which may affect albumin oxidation. In order to test this hypothesis, the effect of IFX on colitis induced by dextran sulphate sodium (DSS) in rats was evaluated by measuring the Disease Activity Index, biochemical parameters, serum albumin oxidation and colonic mucosa oxidation. Rats with colitis showed an increase in oxidised serum albumin levels and in the oxidation of colon mucous cells. Both decreased after IFX treatment. This suggests that oxidised albumin could be a useful biomarker for monitoring inflammatory bowel disease.

Sepsis is a systemic inflammatory response syndrome due to infection. The incidence rate is estimated to be up to 19 million cases worldwide per year and the number of cases is rising. Infection triggers a complex and prolonged host response, in which both the innate and adaptive immune response are involved. The disturbance of immune system cells plays a key role in the induction of abnormal levels of immunoregulatory molecules. Furthermore, the involvement of effector immune system cells also impairs the host response to the infective agents and tissue damage. Recently, postmortem studies of patients who died of sepsis have provided important insights into why septic patients die and showed an extensive depletion of CD4 and CD8 lymphocytes and they found that circulating blood cells showed similar findings. Thus, the knowledge of the characterization of circulating lymphocyte abnormalities is relevant for the understanding of the sepsis pathophysiology. In addition, monitoring the immune response in sepsis, including circulating lymphocyte subsets count, appears to be potential biomarker for predicting the clinical outcome of the patient. This paper analyzes the lymphocyte involvement and dysfunction found in patients with sepsis and new opportunities to prevent sepsis and guide therapeutic intervention have been revealed.

We have investigated both modifications in natural (innate) immunity caused by chronic obstructive pulmonary disease (COPD) and the effects of a glycophosphopeptical immunomodulator (Inmunoferón) treatment on COPD-associated immunoalterations. In a double-blinded clinical trial, 60 patients with COPD received glycophosphopeptical or placebo during 90 consecutive days at oral doses of 3 g/d. Fifty-six sex- and age-matched healthy control subjects were included as a reference group for immunologic parameters. Peripheral blood natural killer (PBNK) cell cytotoxic activity and phagocytic activity of peripheral monocytes/macrophages (Mo/Ma) and polymorphonuclear (PMN) cells were assessed at baseline and then again at the end of treatments. We found both PBNK activity and phagocytic activity to be significantly decreased in patients with COPD compared with levels in healthy volunteers. The treatment with glycophosphopeptical provoked significant stimulatory effects on PBNK cytotoxic activity. This stimulation was not mediated by an increase in CD3(-)CD56(+) NK cells. Further, glycophosphopeptical significantly increased the percentage of monocytes and PMNs that phagocytize Escherichia coli in vitro, as well as increased phagocytic indices. We conclude that peripheral blood cells of patients with COPD show clear defects in natural immunity that are partially rescued by glycophosphopeptical.

BACKGROUND Internal standards have been used in flow cytometry methods to enumerate lymphoid subsets and hemopoietic progenitor cells ex vivo. However, the currently available methods cannot be readily applied to the analysis of cultured cells because of the frequent occurrence of cell death during in vitro assays. METHODS This paper reports a new method for the enumeration of both viable and nonviable cells in culture. Cells were counted with the aid of an internal reference standard of microbeads, and live versus dead cell discrimination was performed using 7-amino-actinomycin D which allows the double staining of surface antigens. RESULTS The method is more precise, accurate and sensitive than either conventional light microscopy-based or automated cell counting. Additionally, it may be used to accurately measure the number of apoptotic cells in a culture. RESULTS Through the enumeration of surviving cells it is demonstrated that, when applied to the study of mitogen-activated T lymphocytes, current flow cytometry techniques (which do not use internal standards) for the study of the viability and apoptosis overestimate the fraction of viable cells and underestimate both the fraction of dead and apoptotic cells. CONCLUSIONS The new method overcomes these limitations and is of use in the in vitro study of cell growth and apoptosis.Cytometry 39:56–66, 2000 © 2000 Wiley-Liss, Inc.