Hedgehog Signalling Modulates Immune Response and Protects against Experimental Autoimmune Encephalomyelitis.

The Hedgehog (Hh) pathway is essential for the embryonic development and homeostatic maintenance of many adult tissues and organs. It has also been associated with some functions of the innate and adaptive immune system. However, its involvement in the immune response has not been well determined. Here we study the role of Hh signalling in the modulation of the immune response by using the Ptch-1-LacZ+/- mouse model (hereinafter referred to as ptch+/-), in which the hemizygous inactivation of Patched-1, the Hh receptor gene, causes the constitutive activation of Hh response genes. The in vitro TCR stimulation of spleen and lymph node (LN) T cells showed increased levels of Th2 cytokines (IL-4 and IL-10) in ptch+/-cells compared to control cells from wild-type (wt) littermates, suggesting that the Th2 phenotype is favoured by Hh pathway activation. In addition, CD4+ cells secreted less IL-17, and the establishment of the Th1 phenotype was impaired in ptch+/- mice. Consistently, in response to an inflammatory challenge by the induction of experimental autoimmune encephalomyelitis (EAE), ptch+/- mice showed milder clinical scores and more minor spinal cord damage than wt mice. These results demonstrate a role for the Hh/ptch pathway in immune response modulation and highlight the usefulness of the ptch+/- mouse model for the study of T-cell-mediated diseases and for the search for new therapeutic strategies in inflammatory diseases.

Impact of the SARS-CoV-2 (COVID19) pandemic on the morbidity and mortality of high risk patients undergoing surgery: a non-inferiority retrospective observational study.

BACKGROUND: During the COVID-19 crisis it was necessary to generate a specific care network and reconvert operating rooms to attend emergency and high-acuity patients undergoing complex surgery. The aim of this study is to classify postoperative complications and mortality and to assess the impact that the COVID-19 pandemic may have had on the results. METHODS: this is a non-inferiority retrospective observational study. Two different groups of surgical patients were created: Pre-pandemic COVID and Pandemic COVID. Severity of illness was rated according to the Diagnosis-related Groups (DRG) score. Comparisons were made between groups and between DRG severity score-matched samples. Non-inferiority was set at up to 10 % difference for grade III to V complications according to the Clavien-Dindo classification, and up to 2 % difference in mortality. RESULTS: A total of 1649 patients in the PreCOVID group and 763 patients in the COVID group were analysed; 371 patients were matched for DRG severity score 3-4 (236 preCOVID and 135 COVID). No differences were found in relation to re-operation (22.5 % vs. 21.5 %) or late admission to critical care unit (5.1 % vs. 4.5 %). Clavien grade III to V complications occurred in 107 patients (45.3 %) in the PreCOVID group and in 56 patients (41.5 %) in the COVID group, and mortality was 12.7 % and 12.6 %, respectively. During the pandemic, 3 % of patients tested positive for Covid-19 on PCR: 12 patients undergoing elective surgery and 11 emergency surgery; there were 5 deaths, 3 of which were due to respiratory failure following Covid-19-induced pneumonia. CONCLUSIONS: Although this study has some limitations, it has shown the non-inferiority of surgical outcomes during the COVID pandemic, and indicates that resuming elective surgery is safe. TRIAL REGISTRATION: Clinicaltrials.gov identifier: NCT04780594 .

Combined use of pharmacophoric models together with drug metabolism and genotoxicity "in silico" studies in the hit finding process.

In this study we propose a virtual screening strategy based on the generation of a pharmacophore hypothesis, followed by an in silico evaluation of some ADME-TOX properties with the aim to apply it to the hit finding process and, specifically, to characterize new chemical entities with potential to control inflammatory processes mediated by T lymphocytes such as multiple sclerosis, systemic lupus erithematosus or rheumatoid arthritis. As a result, three compounds with completely novel scaffolds were selected as final hits for future hit-to-lead optimization due to their anti-inflammatory profile. The biological results showed that the selected compounds increased the intracellular cAMP levels and inhibited cell proliferation in T lymphocytes. Moreover, two of these compounds were able to increase the production of IL-4, an immunoregulatory cytokine involved in the selective deviation of T helper (Th) immune response Th type 2 (Th2), which has been proved to have anti-inflammatory properties in several animal models for autoimmune pathologies as multiple sclerosis or rheumatoid arthritis. Thus our pharmacological strategy has shown to be useful to find molecules with biological activity to control immune responses involved in many inflammatory disorders. Such promising data suggested that this in silico strategy might be useful as hit finding process for future drug development.

Nuts and Metabolic Syndrome: Reducing the Burden of Metabolic Syndrome in Menopause.

Menopause imposes a dramatic fall in estrogens, which is followed by an increase in the proportion of fat. The rising androgen/estrogen ratio along the menopause transition favors the accumulation of central fat, which contributes to insulin resistance and a series of concatenated effects, leading to a higher incidence of metabolic syndrome. The modulatory effect of diet on the metabolic syndrome phenotype has been shown for the Mediterranean diet, and nuts are key determinants of these health benefits. This review of the impact of nuts on the risk factors of the metabolic syndrome cluster examined studies-prioritizing meta-analyses and systemic reviews-to summarize the potential benefits of nut ingestion on the risk of metabolic syndrome associated with menopause. Nuts have a general composition profile that includes macronutrients, with a high proportion of unsaturated fat, bioactive compounds, and fiber. The mechanisms set in motion by nuts have shown different levels of efficacy against the disturbances associated with metabolic syndrome, but a beneficial impact on lipids and carbohydrate metabolism, and a potential, but minimal reduction in blood pressure and fat accumulation have been found.

The RANKL/RANK system in female reproductive organ tumors: A preclinical and clinical overview.

The receptor activator of nuclear factor-κB (RANK) and its ligand RANKL are members of the tumor necrosis factor (TNF) super-family of cytokines with a role in progestogen-associated malignancies in breast. Basic and clinical data support the participation of the cytokine pathway in metastatic disease and as poor prognosis markers. The value of RANK/RANKL as prognostic indicators in endometrial and ovarian tumors, as well as the data suggesting a potential role of RANK/RANKL in hormone dependent tumorigenesis in the endometrium, have been described. The D-CARE study could not confirm benefit in the modulation of RANKL in breast cancer.

Beta interferon restricts the inflammatory potential of CD4+ cells through the boost of the Th2 phenotype, the inhibition of Th17 response and the prevalence of naturally occurring T regulatory cells.

Beta-interferon (IFN-beta) is a valuable therapy for multiple sclerosis (MS) which is also effective in the animal model of experimental autoimmune encephalomyelitis (EAE). However, the accurate mechanisms to explain its anti-inflammatory activity in the disease are not fully revealed. Available data support that T lymphocytes are among the main cell targets of IFN-beta. We have found that in vitro anti-CD3 stimulation of uncommitted murine naïve T cells under IFN-beta treatment results in skewing the T cell differentiation process towards the T2 phenotype, in a prevention from apoptosis of naturally occurring CD4+ T regulatory cells (nTreg) in correlation with an increase in Bcl-XL expression, and in a decrease of IL-17 expression. Elimination of nTreg from the primary culture of naïve CD4+ cells abolished the down-regulation of IL-17 driven by IFN-beta, what suggests the interaction between Th17 and nTreg subsets. Experiments in EAE induced in SJL mice, showed in vivo evidence for the accumulation of spleen CD4+CD25+GITR+Foxp3+ cells after IFN-beta treatment. On the other hand, treated animals showed a striking decrease of IL-17 expression by peripheral CD4+ cells (Th17) and MBP-specific spinal cord cells. Both the in vivo and in vitro results point out new targets through which IFN-beta could exert its therapeutic action.

Beta-interferon unbalances the peripheral T cell proinflammatory response in experimental autoimmune encephalomyelitis.

Interferon beta (IFNbeta) is a widespread therapy for multiple sclerosis (MS). We have analyzed some critical features of the T cell activation process in lymph nodes after IFNbeta treatment of experimental autoimmune encephalomyelitis (EAE) in SJL mice. Prevention of clinical signs and drastic reduction of perivascular infiltrates in the central nervous system (CNS) were accompanied by alterations in nuclear DNA binding activity levels of NFkappaB and Stat6 transcription factors in lymph node cells (LNC). A decrease of active NFkappaB subunits in treated animals correlated with lower levels of the cytoplasmic phosphorylated form of IkappaBalpha. Results also showed that nuclear DNA binding activity of Stat6 was increased by IFNbeta treatment, as were the cytoplasmic levels of phosphorilated Stat6 (P-Stat6). These high levels of P-Stat6 in IFNbeta-treated animals were accompanied by an increase of IL-4 expression levels measured by real time PCR. In vitro experiments with the IL-4 producing clone D10.G4.1 indicates that the IFNbeta-mediated IL-4 induction is not an effect exclusive to MBP-reactive cells, and suggest that it could be mediated by mRNA stability enlargement. On the other hand, IFNbeta treatment of EAE produced no significant changes in peripheral IFNgamma expression and a striking decrease of IL-17. These findings suggest that the inhibition of NFkappaB activity, the increase of IL-4 expression and its signaling transduction, and the decrease of IL-17 may cooperate to some of the antiinflammatory effects of IFNbeta on EAE.

The activity of interleukin-4 receptor alpha-chain promoter is regulated by a GT box element.

Interleukin-4 receptor (IL-4R) is the cell surface complex through which interleukin-4 (IL-4) signals exert its critical biological effects. The alpha-chain of IL-4R is responsible for the high affinity binding of IL-4. In this report, is characterized, the 5' untranslated flanking region of murine IL-4Ralpha gene in the Th2 clone D10.G4.1. We have analyzed a DNA fragment spanning from -995 to +84 relative to the transcription start point. Mutagenesis analysis shows that, neither the previously described Stat6 (-395) nor the NFAT (-266) and NFkappaB (+25) sequences localized here, are involved in the IL-4Ralpha promoter activity. Reporter assays demonstrate that maximum transcriptional activity is achieved by the -89 to +84 sequence and this activity is independent of a TATA-like box located at -25. We have identified a GT box located at -45 as the critical element for the IL-4Ralpha promoter activity. Experiments in SL2 cells, which lack endogenous Sp proteins, show that IL-4Ralpha minimal promoter is transactivated by proteins of Sp family.

Manejo con cirugía conservadora de tumor mucinoso infiltrante ovárico coexistente con tumor mucinoso borderline contralateral en gestante de 8 semanas / Conservative surgery for management of invasive mucinous ovarian tumor with concurrent borderline mucinous tumor on contralateral ovary in a woman in 8th week of pregnancy

INTRODUCCIÓN: Aproximadamente un 4% de mujeres embarazadas presentan tumoraciones anexiales en el primer trimestre de gestación, siendo la mayoría quistes anexiales simples. Solo el 1,5% persisten tras el primer trimestre y en torno al 0,9% son malignos. CASO CLÍNICO: Paciente de 31 años que consultó en urgencias por sangrado y amenorrea de 5 semanas. La ecografía transvaginal evidenció un saco gestacional intraútero y una tumoración anexial izquierda compleja de 68 mm, multilocular, sólida, de ecogenicidad mixta, con septos gruesos y un área sólida vascularizada de 40 mm, sospechosa de neoplasia de ovario. Se realizó manejo quirúrgico conservador mediante anexectomía bilateral laparoscópica en la octava semana de gestación. El diagnóstico fue de adenocarcinoma mucinoso infiltrante bien diferenciado en ovario derecho, coexistente con tumor mucinoso borderline en ovario izquierdo (etapa IC FIGO). El embarazo cursó con normalidad, a excepción de feto pequeño para la edad gestacional en la semana 39. Se finalizó la gestación en la semana 40 mediante parto vaginal. Debido al deseo de la paciente, la cirugía se realizó en el primer trimestre de embarazo en lugar de en el segundo como se recomienda. La cirugía se completó meses después del parto, con histerectomía y apendicectomía laparoscópica. DISCUSIÓN: El cáncer de ovario es el segundo tumor ginecológico más diagnosticado durante el embarazo. Suele diagnosticarse en el primer trimestre debido al cribado ecográfico gestacional. Ocurre en mujeres con baja paridad y en sus últimos años de edad reproductiva. Esto podría verse reflejado en un aumento de su incidencia debido al retraso de la edad materna al primer embarazo. Normalmente se diagnostica en etapa I, siendo entonces la supervivencia superior al 90% a los 5 años. En etapas iniciales en mujeres embarazadas, la laparoscopia es tan válida como la laparotomía.

INTRODUCTION: Approximately 4% of pregnant women present adnexal tumors in the first trimester of gestation, the majority being simple adnexal cysts. Only 1.5% persist after the first trimester and around 0.9% are malignant. CASE REPORT: 31-year-old patient who came to the emergency room because of spotting and 5-week amenorrhea. A transvaginal ultrasound revealed an intrauterine gestational sac and a 69 mm complex multiocular-solid left adnexal tumor, with thick septa and a solid vascularized area of 40 mm, with suspicion of ovarian neoplasia. Conservative surgical management was performed through laparoscopic bilateral salpingo-oophorectomy in eighth week of gestation. The diagnosis was a well-differentiated infiltrating mucinous adenocarcinoma in the right ovary, and a coexisting borderline mucinous tumor in the left ovary (FIGO stage IC). The pregnancy proceeded normally except for a small for gestational age fetus at week 39 and pregnancy was completed at week 40 by vaginal delivery. Due to the patients wishes, the surgery was performed in the 1st trimester of pregnancy instead in the 2nd as is recommended. Final surgery was completed after giving birth, with laparoscopic hysterectomy and appendectomy. DISCUSSION: Ovarian cancer is the second most diagnosed gynecological tumor during pregnancy. It is usually diagnosed during the first trimester with gestational ultrasound screening. It occurs in women with low parity and in their later years of reproductive age. This could be reflected in an increase in its incidence due to the delay in the age of first pregnancy. Normally it is diagnosed in stage I, when the survival rate is superior to 90% after 5 years. In early stages, laparoscopic surgery in pregnant women is as valid as laparotomy.

Restrained Th17 response and myeloid cell infiltration into the central nervous system by human decidua-derived mesenchymal stem cells during experimental autoimmune encephalomyelitis.

BACKGROUND: Multiple sclerosis is a widespread inflammatory demyelinating disease. Several immunomodulatory therapies are available, including interferon-ß, glatiramer acetate, natalizumab, fingolimod, and mitoxantrone. Although useful to delay disease progression, they do not provide a definitive cure and are associated with some undesirable side-effects. Accordingly, the search for new therapeutic methods constitutes an active investigation field. The use of mesenchymal stem cells (MSCs) to modify the disease course is currently the subject of intense interest. Decidua-derived MSCs (DMSCs) are a cell population obtained from human placental extraembryonic membranes able to differentiate into the three germ layers. This study explores the therapeutic potential of DMSCs. METHODS: We used the experimental autoimmune encephalomyelitis (EAE) animal model to evaluate the effect of DMSCs on clinical signs of the disease and on the presence of inflammatory infiltrates in the central nervous system. We also compared the inflammatory profile of spleen T cells from DMSC-treated mice with that of EAE control animals, and the influence of DMSCs on the in vitro definition of the Th17 phenotype. Furthermore, we analyzed the effects on the presence of some critical cell types in central nervous system infiltrates. RESULTS: Preventive intraperitoneal injection of DMSCs resulted in a significant delay of external signs of EAE. In addition, treatment of animals already presenting with moderate symptoms resulted in mild EAE with reduced disease scores. Besides decreased inflammatory infiltration, diminished percentages of CD4(+)IL17(+), CD11b(+)Ly6G(+) and CD11b(+)Ly6C(+) cells were found in infiltrates of treated animals. Early immune response was mitigated, with spleen cells of DMSC-treated mice displaying low proliferative response to antigen, decreased production of interleukin (IL)-17, and increased production of the anti-inflammatory cytokines IL-4 and IL-10. Moreover, lower RORγT and higher GATA-3 expression levels were detected in DMSC-treated mice. DMSCs also showed a detrimental influence on the in vitro definition of the Th17 phenotype. CONCLUSIONS: DMSCs modulated the clinical course of EAE, modified the frequency and cell composition of the central nervous system infiltrates during the disease, and mediated an impairment of Th17 phenotype establishment in favor of the Th2 subtype. These results suggest that DMSCs might provide a new cell-based therapy for the control of multiple sclerosis.

Rolipram impairs NF-kappaB activity and MMP-9 expression in experimental autoimmune encephalomyelitis.

Rolipram suppresses experimental autoimmune encephalomyelitis (EAE) and diminishes cell infiltration of the central nervous system (CNS). In Lewis rats with EAE, rolipram reduced matrix metalloproteinase-9 (MMP-9) gene expression in lymph node cells (LNCs) and spinal cord, decreased basal levels of nuclear (p50/p65) NF-kappaB in LNCs from treated rats, and impaired CD3 mediated NF-kappaB translocation. Rolipram reduced the luciferase activity directed by the NF-kappaB binding site of the MMP-9 gene in lymphocytes. It also diminished NF-kappaB activity and the ability of a myelin basic protein (MBP) specific cell line to migrate across artificial basement membranes. IL-2 induced MMP-9 proteolytic activity was only slightly reduced indicating that additional factors contribute to inhibit cell migration mediated by rolipram.

Synthesis, structural analysis, and biological evaluation of thioxoquinazoline derivatives as phosphodiesterase 7 inhibitors.

PDE7 inhibitors regulate pro-inflammatory and immune T-cell functions, and are a potentially novel class of drugs especially useful in the treatment of a wide variety of immune and inflammatory disorders. Starting from our lead family of thioxoquinazolines, we designed, synthesized, and characterized a novel series of thioxoquinazoline derivatives. Many of these compounds showed inhibitory potencies at sub-micromolar levels against the catalytic domain of PDE7A1 and at the micromolar level against PDE4D2. Cell-based studies showed that these compounds not only increased intracellular cAMP levels, but also had interesting anti-inflammatory properties within a therapeutic window. The in silico data predict that these compounds are capable of the crossing the blood-brain barrier. The X-ray crystal structure of the PDE7A1 catalytic domain in complex with compound 15 at a resolution of 2.4 A demonstrated that hydrophobic interactions at the active site pocket are a key feature. This structure, together with molecular modeling, provides insight into the selectivity of the PDE inhibitors and a template for the discovery of new PDE7 or PDE7/PDE4 dual inhibitors.

Autocrine IL-4 gene regulation at late phases of TCR activation in differentiated Th2 cells.

IL-4 is a multifunctional cytokine whose secretion displays important immunomodulatory functions. Its expression is regulated at the level of transcription, and one of the main factors involved is NFAT. The IL-4-induced transcription factor Stat6 is required for the development of naive T cells into Th2 phenotype, capable of secreting IL-4. However, IL-4 production by differentiated Th2 cells is IL-4 independent; thus, it remains unclear whether Stat6 plays any role in the IL-4 expression by mature Th2 cells. We have analyzed in the Th2 clone D10.G4.1 the nuclear proteins able to bind the regulatory element P1 of the IL-4 promoter. Gel-shift assays show NFAT1 as the most abundant nuclear protein that binds to P1 after ionomycin plus PMA activation, whereas Stat6 accounts for the bulk of the P1 binding in the presence of exogenous IL-4. Reporter experiments agree with an inhibitory effect of Stat6 on the NFAT1-induced transcriptional activity directed by the P1 element. CD3 signaling leads to an early induction of NFAT1-P1 complexes correlating with a strong induction of the IL-4 gene. In later phases of CD3 activation, P1 is also bound by Stat6 and a fall in the IL-4 mRNA levels takes place. These two late events during CD3 activation were found to be sensible in experiments conducted with an anti-IL-4 Ab. These results suggest that IL-4 endogenously produced by Th2 cells under TCR triggering modulates its own expression through Stat6.

The Th17 lineage: Answers to some immunological questions

En los últimos años se han estudiado exhaustivamente las funciones ylas rutas de desarrollo del subtipo de células T helper especializado en la producción de IL-17 (Th17). Este linaje celular de células efectoras desempeñaun papel decisivo tanto en la respuesta inmune a agentes infecciosos, comoen inmunopatologías. Al igual que para los subtipos Th1 y Th2, la definiciónde Th17 está dirigida por citocinas y factores de transcripción específicos. Lacombinación de TGF-β e IL-6, y los factores de transcripción RORγt, RORαy Stat3 son esenciales para comprometer el subtipo Th17. IL-23 juega un papelclave en la estabilización del fenotipo y de la actividad patogénica de célulasproductoras de IL-17. La citocina IL-21 producida por células Th17 participaen un mecanismo de retroalimentación para favorecer el desarrollo de células productoras de IL-17, mientras que las citocinas IL-27, IL-4, IFN-γ, IL-25e IL-2 limitan el fenotipo Th17. Las células T reguladoras CD4+CD25+Foxp3+(Treg) siguen una ruta de desarrollo divergente al establecimiento de las células IL-17, aunque ambas alternativas son gobernadas por TGF-β, el cual dirige el destino de células CD4+naïve hacia uno u otro de estos subtipos celulares mutuamente excluyentes dependiendo de la presencia de IL-6. Además, datos recientes indican que células Treg ya establecidas pueden modificar su programa genético para convertirse en células Th17. En esta revisiónse resumen y analizan los datos disponibles actualmente acerca de la biología de las células Th17 (AU)

In recent years the function and developmental pathway for the Thelper subset specialized in IL-17 production (Th17) have been exhaustively studied. This lineage of effector cells plays a decisive role in the immune response to infectious agents, as well as in immunopathologies. Similar to the Th1 and Th2 subsets, the Th17 definition is orchestrated by specific cytokines and transcription factors. A combination of TGF-β plus IL-6, and the transcription factors RORγt, RORα and Stat3 are essential forTh17 commitment. IL-23 plays a key role in the stabilization of the phenotype and in the promotion of the pathogenic activity of IL-17-producercells. The IL-21 cytokine produced by Th17 cells participates in a feedbackmechanism to favour this phenotype, while IL-27, IL-4, IFN-γ, IL-25 andIL-2 cytokines limit the Th17 response. CD4+CD25+Foxp3+regulator cells(Treg) follow a development pathway divergent to Th17 establishment,although both alternatives are governed by TGF-β that directs the fateof naïve CD4+cells to each of these mutually exclusive T cell subsets depending on the presence of IL-6. Furthermore, recent data indicate that preestablished Treg cells can switch its genetic program to become IL-17-producer cells. In this review we summarize and discuss the current available data about the biology of Th17 cells (AU)