Correlazione dei livelli sierici di IL-21 con autoanticorpi IgA anti -tTG e danno mucosale in pazienti affetti da malattia celiaca

Numerose patologie pur sviluppandosi in assenza di una minaccia esterna come per esempio un\u2019infezione, esposizione a tossine o predisposizione per la formazione di tumori, tendono a sviluppare una condizione infiammatoria e un danno specifico a livello tissutale con conseguente attivazione del sistema immunitario. Tali condizioni mostrano l'attivazione del sistema immunitario innato e un eccesso di mediatori infiammatori, ma nessuna evidenza di una risposta immunitaria antigene-specifica. In alternativa, ci sono malattie caratterizzate da un'attivazione della risposta immunitaria adattativa con linfociti T e B che rispondono all'autoantigene in assenza di qualsiasi aggressione microbica o invasione tumorale rilevabile; queste patologie costituiscono la stragrande maggioranza delle malattie considerate di origine autoimmune. Attualmente sono state identificate circa 80 malattie di natura autoimmune che colpiscono il 5,7% della popolazione anche se dagli ultimi dati emersi in letteratura, la loro incidenza \ue8 in aumento. Si definisce patologia autoimmune, quella condizione in cui il danno tissutale \ue8 causato dalla reattivit\ue0 delle cellule T o degli anticorpi reattivi verso antigeni propri. In alcuni casi, si presume che una malattia sia di origine autoimmune solo perch\ue9 i linfociti B e T sono presenti nel tessuto colpito. I modelli animali per lo studio di queste patologie, sono stati estremamente utili per aiutare a comprendere sia i meccanismi che guidano l'insorgenza della malattia che quelli della patogenesi. Due sono gli elementi importanti per comprendere l\u2019alta frequenza delle malattie autoimmunitarie: in primo luogo, l'autoreattivit\ue0. Infatti, il repertorio dei linfociti immunocompetenti che fornisce l'immunit\ue0 protettiva \ue8 selezionato in base all'autoreattivit\ue0. La regolazione dell'autoreattivit\ue0 aiuta a modellare il sistema immunitario in modo che essa non diventi associabile al danno tissutale, questo richiede una vigilanza costante. Il sistema immunitario mantiene un equilibrio precario tra i due: una risposta troppo scarsa porta a un potenziale non riconoscimento del \u201cpericolo\u201d, mentre una risposta sovraespressa pu\uf2 potenzialmente portare all'autoreattivit\ue0. In secondo luogo, esiste una predisposizione genetica all'autoimmunit\ue0 e aspetti di questa predisposizione possono essere simili per molte malattie autoimmuni differenti. La malattia autoimmune quindi non richiede solo autoreattivit\ue0, ma pu\uf2 anche essere influenzata dalla vulnerabilit\ue0 genetica dell'organo bersaglio. Alcuni dei recenti studi sulle basi genetiche dell'autoimmunit\ue0 mostrano che i fattori genetici che regolano la vulnerabilit\ue0 di organi specifici sono distinti da quelli che regolano l'autoreattivit\ue0. Pertanto, gli individui possono condividere percorsi che promuovono l'autoreattivit\ue0, ma presenti con diverse malattie autoimmuni. La celiachia (CD) per esempio \ue8 una malattia sistemica immuno-mediata indotta dalla presenza del glutine in soggetti geneticamente predisposti, caratterizzata da una reazione autoimmune contro la transglutaminasi tissutale (tTG), un enzima che deamina la gliadina nei soggetti portatori degli alleli HLA-DQA1 e DQB1 e per gli eterodimeri DQ2 o DQ8. La celiachia si manifesta con una condizione di malassorbimento cronico: il tipico danno intestinale \ue8 caratterizzato dalla distruzione dei villi e dall\u2019iperplasia delle cripte, ma oggigiorno \ue8 possibile tenerlo sotto controllo con l\u2019eliminazione del glutine dalla dieta. A livello molecolare \ue8 stato dimostrato che l\u2019esposizione alla gliadina (una prolammina che insieme alla glutenina costituisce il glutine) induce una risposta infiammatoria che causa il danno dei villi che rivestono l\u2019intestino tenue (atrofia dei villi). Il potenziale paziente celiaco \ue8 caratterizzato da un\u2019alta espressione di IL-10 e da un aumento significativo del rapporto IL-10/IFN-y, seguito poi da una maggiore densit\ue0 di cellule regolatorie come CD4+CD25 +Foxp3 + T reg, che esercitano effetti soppressivi. Sebbene sia riconosciuto che la CD sia mediata da una risposta Th1, non \ue8 ancora chiaro il meccanismo per cui le cellule Th1 siano indotte e mantenute nell\u2019intestino dei pazienti. Le prove indicano che una interazione attiva e dinamica tra cellule immunitarie e non, ha un ruolo cruciale nell\u2019iniziare e nel modellare questo pathway patologico, e che le citochine siano i mediatori principali di questo dialogo incrociato. In particolare, l\u2019IL-21 pare avere un ruolo chiave in questo fenomeno: la sua espressione, infatti, \ue8 risultata maggiore nella mucosa intestinale di pazienti con CD attivo (sia pediatrici che adulti) rispetto a quella di pazienti CD trattati con una dieta priva di glutine e a quella di soggetti controllo. L\u2019IL-21 \ue8 una citochina fortemente coinvolta nei meccanismi dell\u2019infiammazione e in quelli dell\u2019autoimmunit\ue0, con relativo coinvolgimento nello sviluppo e mantenimento di specifiche classi dei linfociti T (CD4+ e CD8+) e dei linfociti B. L\u2019obiettivo di questo studio \ue8 stato quello di approfondire ed analizzare il possibile ruolo di questa citochina nello lo sviluppo della celiachia. In particolare ci siamo occupati di valutare dal punto di vista quantitativo la relazione tra i livelli sierici dell\u2019IL-21 e i titoli sierici degli autoanticorpi IgA anti-tTG con relativo confronto del danno tissutale che viene a manifestarsi a livello della mucosa duodenale in pazienti considerati attivi. In questo studio sono stati analizzati i sieri di 160 pazienti CD, 120 non trattati e 40 trattati (sottoposti a dieta priva di glutine), e quelli di 45 volontari sani, con funzione di controllo. I risultati hanno dimostrato che i pazienti CD alla diagnosi esprimevano concentrazioni di IL-21 significativamente pi\uf9 elevate rispetto ai pazienti CD sottoposti a dieta e ai controlli. Inoltre i dati ottenuti, hanno dimostrato una correlazione positiva tra i livelli di IL-21 e le concentrazioni di autoanticorpi IgA anti-tTG, indice di un possibile ruolo di questa citochina nell\u2019attivazione dei linfociti B. Infine, si \ue8 evidenziata una correlazione positiva dei livelli di IL-21 con il danno alla mucosa duodenale: questo ci ha permesso di ipotizzare la possibilit\ue0 di un forte effetto immunomodulatore dell\u2019IL-21 sulle funzioni dei linfociti T citotossici. Questo studio fornisce un'ulteriore prova dei dati emergenti sul potenziale ruolo dell'IL-21 nella patogenesi della CD, suggerendo il suo coinvolgimento nello sviluppo e nella progressione della CD.Numerous pathologies, while developing in the absence of an external threat such as infection, exposure to toxins or predisposition for the formation of tumors, tend to develop an inflammatory condition and specific damage to the tissue with consequent activation of the immune system. Such conditions show activation of the innate immune system and an excess of inflammatory mediators, but no evidence of an antigen-specific immune response. Alternatively, there are diseases characterized by an activation of the adaptive immune response with T and B lymphocytes responding to the autoantigen in the absence of any detectable microbial aggression or tumor invasion; these pathologies constitute the vast majority of diseases considered to be of autoimmune origin. Currently, about 80 autoimmune diseases have been identified that affect 5.7% of the population even if, according to the latest data in the literature, their incidence is increasing. Autoimmune disease is defined as a condition in which tissue damage is caused by the reactivity of T cells or reactive antibodies to their own antigens. In some cases, a disease is assumed to be of autoimmune origin only because B and T lymphocytes are present in the affected tissue. Animal models for the study of these diseases have been extremely useful in helping to understand both the mechanisms that drive the onset of the disease and those of pathogenesis. There are two important elements for understanding the high frequency of autoimmune diseases: first, self-reactivity. Indeed, the repertoire of immunocompetent lymphocytes providing protective immunity is selected on the basis of self-reactivity. Regulating self-reactivity helps shape the immune system so that it does not become associated with tissue damage, which requires constant vigilance. The immune system maintains a precarious balance between the two: too little response leads to a potential non-recognition of "danger", while an over-expressed response can potentially lead to self-reactivity. Second, there is a genetic predisposition to autoimmunity and aspects of this predisposition may be similar for many different autoimmune diseases. Autoimmune disease therefore not only requires self-reactivity, but can also be affected by the genetic vulnerability of the target organ. Some of the recent studies on the genetic basis of autoimmunity show that the genetic factors that regulate the vulnerability of specific organs are distinct from those that regulate self-reactivity. Therefore, individuals can share pathways that promote self-reactivity, but present with several autoimmune diseases. Celiac disease (CD), for example, is an immune-mediated systemic disease induced by the presence of gluten in genetically predisposed subjects, characterized by an autoimmune reaction against tissue transglutaminase (tTG), an enzyme that deaminates gliadin in subjects carrying the HLA- alleles. DQA1 and DQB1 and for the heterodimers DQ2 or DQ8. Celiac disease manifests itself with a condition of chronic malabsorption: typical intestinal damage is characterized by the destruction of the villi and hyperplasia of the crypts, but nowadays it is possible to keep it under control by eliminating gluten from the diet. At the molecular level, it has been shown that exposure to gliadin (a prolamine that together with glutenin makes up gluten) induces an inflammatory response that causes damage to the villi lining the small intestine (villous atrophy). The potential celiac patient is characterized by a high expression of IL-10 and a significant increase in the IL-10 / IFN-y ratio, followed by a higher density of regulatory cells such as CD4 + CD25 + Foxp3 + T reg, which exert suppressive effects. Although it is recognized that CD is mediated by a Th1 response, the mechanism by which Th1 cells are induced and maintained in the gut of patients is not yet clear. Evidence indicates that an active and dynamic interaction between immune and non-immune cells plays a crucial role in initiating and modeling this pathological pathway, and that cytokines are the main mediators of this cross dialogue. In particular, IL-21 seems to have a key role in this phenomenon: its expression, in fact, was greater in the intestinal mucosa of patients with active CD (both pediatric and adult) than that of CD patients treated with a diet. gluten-free and that of control subjects. IL-21 is a cytokine strongly involved in the mechanisms of inflammation and autoimmunity, with its involvement in the development and maintenance of specific classes of T lymphocytes (CD4 + and CD8 +) and B lymphocytes. The goal of this study was to investigate and analyze the possible role of this cytokine in the development of celiac disease. In particular, we have dealt with the quantitative evaluation of the relationship between the serum levels of IL-21 and the serum titers of the anti-tTG IgA autoantibodies with relative comparison of the tissue damage that occurs at the level of the duodenal mucosa in patients considered. active. In this study the sera of 160 CD patients, 120 untreated and 40 treated (on a gluten-free diet), and those of 45 healthy volunteers, with control function, were analyzed. The results demonstrated that CD patients at diagnosis expressed significantly higher IL-21 concentrations than dietary and control CD patients. Furthermore, the data obtained showed a positive correlation between the levels of IL-21 and the concentrations of IgA anti-tTG autoantibodies, indicating a possible role of this cytokine in the activation of B lymphocytes. Finally, a positive correlation was highlighted. IL-21 levels with damage to the duodenal mucosa: this allowed us to hypothesize the possibility of a strong immunomodulatory effect of IL-21 on the functions of cytotoxic T lymphocytes. This study provides further evidence of the emerging data on the potential role of IL-21 in the pathogenesis of CD, suggesting its involvement in the development and progression of CD

Regioselective Synthesis, Structural Characterization, and Antiproliferative Activity of Novel Tetra-Substituted Phenylaminopyrazole Derivatives

A small library of highly functionalized phenylaminopyrazoles, bearing different substituents at position 1, 3 and 4 of the pyrazole ring, was prepared by the one pot condensation of active methylene reagents, phenylisothiocyanate and substituted hydrazine (namely, methyl- and benzyl-hydrazine). The identified reaction conditions proved to be versatile and efficient. Fur-thermore, the evaluation of alternative stepwise protocols affected the chemo- and re-gio-selectivity outcome of the one-pot procedure. The chemical identity of two N-methyl pyrazole isomers, selected as prototypes of the whole series, was unambiguously identified by means of NMR and mass spectrometry studies. Additionally, semiempirical calculations provided a structural rationale for the different chromatographic behaviour of the two isomers. The prepared tetra-substituted phenylaminopyrazoles were tested in cell-based assays on a panel of cancer and normal cell lines. The tested compounds did not show any cytotoxic effect on the selected cell lines, thus supporting their pharmaceutical potentials

Anti-hypothalamus autoantibodies in anorexia nervosa: a possible new mechanism in neuro-physiological derangement?

Purpose: Anorexia nervosa (AN) is a serious and complex mental disorder affecting mainly young adult women. AN patients are characterized by low body weight in combination with self-induced starvation, intense fear of gaining weight, and distortion of body image. AN is a multifactorial disease, linked by recent evidence to a dysregulation of the immune system. Methods: In this pilot study, 22 blood serums from AN patients were tested for the presence of autoantibodies against primate hypothalamic periventricular neurons by immunofluorescence and by a home-made ELISA assay. Cellular fluorescence suggests the presence of autoantibodies which are able to recognize these neurons (both to body cell and fiber levels). By means of ELISA, these autoantibodies are quantitatively evaluated. In addition, orexigenic and anorexigenic molecules were measured by ELISA. As control, 18 blood serums from healthy age matched woman were analysed. Results: All AN patients showed a reactivity against hypothalamic neurons both by immunofluorescence and ELISA. In addition, ghrelin, pro-opiomelanocortin (POMC), and agouti-related peptide (AGRP) were significantly higher than in control serums (p < 0.0001). In contrast, leptin was significantly lower in AN patients than controls (p < 0.0001). Conclusions: Immunoreaction and ELISA assays on AN blood serum suggest the presence of autoantibodies AN related. However, it is not easy to determine the action of these antibodies in vivo: they could interact with specific ligands expressed by hypothalamic cells preventing their physiological role, however, it is also possible that they could induce an aspecific stimulation in the target cells leading to an increased secretion of anorexigenic molecules. Further studies are needed to fully understand the involvement of the immune system in AN pathogenesis

Two calix[4]pyrroles as potential therapeutics for castration-resistant prostate cancer

Macrocyclic compounds meso-(p-acetamidophenyl)-calix[4]pyrrole and meso-(m-acetamidophenyl)-calix[4]pyrrole have previously been reported to exhibit cytotoxic properties towards lung cancer cells. Here, we report pre-clinical in vitro and in vivo studies showing that these calixpyrrole derivatives can inhibit cell growth in both PC3 and DU145 prostatic cancer cell lines. We explored the impact of these compounds on programmed cell death, as well as their ability to inhibit cellular invasion. In this study we have demonstrated the safety of these macrocyclic compounds by cytotoxicity tests on ex-vivo human peripheral blood mononuclear cells (PBMCs), and by in vivo subcutaneous administration. Preliminary in vivo tests demonstrated no hepato-, no nephro- and no genotoxicity in Balb/c mice compared to controls treated with cisplatin. These findings suggest these calixpyrroles might be novel therapeutic tools for the treatment of prostate cancer and of particular interest for the treatment of androgen-independent castration-resistant prostate cancer

Anticancer effects of the Novel Pyrazolyl-Urea GeGe-3

In a screen of over 200 novel pyrazole compounds, ethyl 1-(2-hydroxypentyl)-5-(3-(3-(trifluoromethyl) phenyl)ureido)-1H-pyrazole-4-carboxylate (named GeGe-3) has emerged as a potential anticancer compound. GeGe-3 displays potent anti-angiogenic properties through the presumptive targeting of the protein kinase DMPK1 and the Ca2+-binding protein calreticulin. We furtherexplored the anticancer potential of GeGe-3 on a range of established cancer cell lines, including PC3(prostate adenocarcinoma), SKMEL-28 (cutaneous melanoma), SKOV-3 (ovarian adenocarcinoma),Hep-G2 (hepatocellular carcinoma), MDA-MB231, SKBR3, MCF7 (breast adenocarcinoma), A549(lung carcinoma), and HeLa (cervix epithelioid carcinoma). At concentrations in the range of 10 µM,GeGe-3 significantly restricted cell proliferation and metabolism. GeGe-3 also reduced PC3 cellmigration in a standard wound closure and trans-well assay. Together, these results confirm theanticancer potential of GeGe-3 and underline the need for more detailed pre-clinical investigationsinto its molecular targets and mechanisms of action

Fraisinib: a calixpyrrole derivative reducing A549 cell-derived NSCLC tumor in vivo acts as a ligand of the glycine-tRNA synthase, a new molecular target in oncology

Background and purpose: Lung cancer is the leading cause of death in both men and women, constituting a major public health problem worldwide. Non-small-cell lung cancer accounts for 85%–90% of all lung cancers. We propose a compound that successfully fights tumor growth in vivo by targeting the enzyme GARS1.Experimental approach: We present an in-depth investigation of the mechanism through which Fraisinib [meso-(p-acetamidophenyl)-calix(4)pyrrole] affects the human lung adenocarcinoma A549 cell line. In a xenografted model of non-small-cell lung cancer, Fraisinib was found to reduce tumor mass volume without affecting the vital parameters or body weight of mice. Through a computational approach, we uncovered that glycyl-tRNA synthetase is its molecular target. Differential proteomics analysis further confirmed that pathways regulated by Fraisinib are consistent with glycyl-tRNA synthetase inhibition.Key results: Fraisinib displays a strong anti-tumoral potential coupled with limited toxicity in mice. Glycyl-tRNA synthetase has been identified and validated as a protein target of this compound. By inhibiting GARS1, Fraisinib modulates different key biological processes involved in tumoral growth, aggressiveness, and invasiveness.Conclusion and implications: The overall results indicate that Fraisinib is a powerful inhibitor of non-small-cell lung cancer growth by exerting its action on the enzyme GARS1 while displaying marginal toxicity in animal models. Together with the proven ability of this compound to cross the blood–brain barrier, we can assess that Fraisinib can kill two birds with one stone: targeting the primary tumor and its metastases “in one shot.” Taken together, we suggest that inhibiting GARS1 expression and/or GARS1 enzymatic activity may be innovative molecular targets for cancer treatment

Serum IL-21 levels from celiac disease patients correlates with anti-tTG IgA autoantibodies and mucosal damage

Objectives: Coeliac disease is a multifactorial disorder influenced by environmental, genetic and immunological factors. Interleukin (IL)-21 has been linked to an increase disease risk and the serum level of IL-21 seems to be increased in CD compared to a healthy control population. Methods: Sera were collected from 160 CD patients, 120 untreated and 40 following a gluten-free diet, and form 45 healthy subjects. Serum IL-21 was evaluated by specific ELISA tests. Results: Our data show that patients with untreated CD display IL-21 concentrations significantly higher than both treated-CD patients (following a gluten-free diet) and controls. In addition, serum IL-21 correlates with serum titres of anti-tTG IgA autoantibodies. Finally, our results show a correlation of this cytokine with duodenal mucosal damage. Conclusions: A role of gluten, as antigen with stimulatory function on IL-21 production, seems to be confirmed by the longitudinal analyses showing that the gluten-free diet decreases to a nearly undetectable amount this cytokine. In addition, the finding of a positive correlation between the serum amount of IL-21 and the grade of duodenal mucosa damage suggests a strong immunomodulatory effect of this cytokine on cytotoxic T lymphocyte functions. This study provides an extra evidence to emerging data on the potential role IL-21 in CD pathogenesis, suggesting its involvement in the development and progression of CD. Significance statement: In untreated CD, serum IL-21 shows higher levels compared with treated CD and healthy subjects. Serum amounts of IL-21 correlate with anti-tTG IgA autoantibodies and with duodenal mucosa damage

Cyclic diacyl thioureas enhance activity of corrector Lumacaftor on F508del‐CFTR

: Cystic fibrosis is a genetic disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) protein. In the search of novel series of CFTR modulators, a library of mono and diacyl thioureas were prepared by sequential synthesis. When tested alone, the obtained compounds 5 and 6 poorly affected F508del-CFTR conductance but, in combination with Lumacaftor, selected derivatives showed the ability to increase the activity of the approved modulator. Analogue 6 i displayed the most marked enhancing effect and acylthioureas 6 d and 6 f were also able to improve efficacy of Lumacaftor. All compounds proved to be non-cytotoxic against different cancer cell lines. Good pharmacokinetic properties were predicted for derivatives 5 and 6, thus supporting the value of these compounds for the development of novel modulators potentially useful for cystic fibrosis