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La malattia di Crohn(MC)è una malattia infiammatoria cronica dell’intestino che può colpire qualsiasi tratto del tubo digerente. Nonostante siano stati effettuati numerosi studi, la patogenesi della MC non è ancora chiara e non si dispone di trattamenti in grado di guarire stabilmente la malattia. Il sistema immunitario innato sembra giocare un ruolo fondamentale nella patogenesi: è stato osservato infatti un coinvolgimento di alcuni geni dell’immunità innata ed uno squilibrato rapporto tra sistema immune mucosale e microbiota intestinale. Dati clinici e genetici portano a pensare che alla base della patogenesi stiano un difettoso funzionamento dell’immunità innata mucosale e una risposta infiammatoria adattativa compensatoria. Lo sviluppo di modelli adeguati a studiare la complessità di questa malattia risulta difficile per la molteplicità di fattori coinvolti, genetici e ambientali. L’obiettivo di questa tesi è stato quello di sviluppare dei modelli che tenessero conto della complessità della patologia e che permettessero di evidenziare dei profili tipici di risposta a componenti batteriche. Il primo approccio ex vivo sviluppato ha analizzato le diverse pathway di attivazione in monociti circolanti, per evidenziare l’eventuale presenza di un difetto di riposta dell’immunità innata. Da questo test è emerso che i pazienti affetti da MC non sembrano presentare un difetto di risposta, bensì un’iperattivazione delle pathways innate, testimoniata da un’aumentata produzione monocitaria di TNFα. Questo stato infiammatorio, riscontrabile sia in condizione basale sia in seguito a stimolazione di alcuni recettori innati, non è riconducibile né al genotipo di NOD2 (analizzate le principali mutazioni associate a malattia) né all’attività di malattia dei pazienti presi in analisi, né alla presenza in circolo di componenti batteriche (segno di traslocazione batterica intestinale). Il secondo modello sviluppato ha analizzato direttamente la risposta immunologica mucosale nel tessuto intestinale, più pertinente alla patogenesi della malattia rispetto alle cellule presenti nel circolo periferico. Attraverso una cultura ex vivo di biopsie coliche in presenza o assenza di antibiotici, si è voluto valutare l’influenza del microbiota sulla secrezione citochinica. Con un’analisi logistica multivariata, è stato possibile dimostrare l’esistenza di un profilo citochinico (G-CSF, TNFα, IL4 e IL17) in grado di discriminare la MC dalla rettocolite ulcerosa. E’ stato quindi dimostrato che la MC e la rettocolite ulcerosa presentano differenti profili di risposta citochinica e che la flora batterica associata a mucosa è in grado di modulare la risposta mucosale. Con il terzo approccio è stata valutata la risposta cellulare di una linea epiteliale intestinale a diversi composti di origine microbica, utilizzando un sistema automatizzato che fornisce in tempo reale informazioni sulla crescita, adesione, morfologia e vitalità cellulare. Da questa analisi è emerso che solo il composto purificato di derivazione micobatterica è in grado di indurre un’alterazione temporanea della cinetica cellulare; questa variazione non è dovuta alla morte cellulare, ma probabilmente ad una variazione della permeabilità del simil-epitelio ricreato in vitro. Ulteriori test sono necessari per far chiarezza sull’effetto dato da questo composto sulla mucosa intestinale, visto il ruolo controverso del Mycobacterium Avium paratubercolis nella patogenesi della MC. In conclusione, in questi tre anni è stato possibile sviluppare diversi modelli per evidenziare specifici profili di risposta mucosale tipici della MC, su cui studiare l’effetto di nuove strategie terapeutiche correttive, basate sulla modulazione dell’interazione tra immunità mucosale e microbiota intestinale. I risultati ottenuti sono stati oggetto di due pubblicazioni sulla rivista internazionale “World Journal of Gastroenterology”.

Cholesterol metabolism is crucial for cells and, in particular, its biosynthesis in the central nervous system occurs in situ, and its deregulation involves morphological changes that cause functional variations and trigger programmed cell death. The pathogenesis of rare diseases, such as Mevalonate Kinase Deficiency or Smith–Lemli–Opitz Syndrome, arises due to enzymatic defects in the cholesterol metabolic pathways, resulting in a shortage of downstream products. The most severe clinical manifestations of these diseases appear as neurological defects. Expanding the knowledge of this biological mechanism will be useful for identifying potential targets and preventing neuronal damage. Several studies have demonstrated that deregulation of the cholesterol pathway induces mitochondrial dysfunction as the result of respiratory chain damage. We set out to determine whether mitochondrial damage may be prevented by using protective mitochondria-targeted compounds, such as MitoQ, in a neuronal cell line treated with a statin to induce a biochemical block of the cholesterol pathway. Evidence from the literature suggests that mitochondria play a crucial role in the apoptotic mechanism secondary to blocking the cholesterol pathway. Our study shows that MitoQ, administered as a preventive agent, could counteract the cell damage induced by statins in the early stages, but its protective role fades over time.

Background: Mevalonate Kinase Deficiency (MKD, OMIM #610377) is a rare autosomal recessive metabolic and inflammatory disease. In MKD, defective function of the enzyme mevalonate kinase, due to a mutation in the MVK gene, leads to the shortage of mevalonate- derived intermediates, which results in unbalanced prenylation of proteins and altered metabolism of sterols. These defects lead to a complex multisystem inflammatory and metabolic syndrome. Objective: Although biologic therapies aimed at blocking the inflammatory cytokine interleukin- 1 can significantly reduce inflammation, they cannot completely control the clinical symptoms that affect the nervous system. For this reason, MKD can still be considered an orphan drug disease. The availability of MKD models reproducing the MKD-systematic inflammation, is crucial to improve the knowledge on its pathogenesis, which is still unknown. New therapies are also required in order to improve pateints’ conditions and their quality of life. Methods: MKD-cellular models can be obtained by biochemical inhibition of mevalonatederived isoprenoids. Of note, these cells present an exaggerated response to inflammatory stimuli that can be reduced by treatment with zaragozic acid, an inhibitor of squalene synthase, thus increasing the availability of isoprenoids intermediates upstream the enzymatic block. Results: A similar action might be obtained by lapaquistat acetate (TAK-475, Takeda), a drug that underwent extensive clinical trials as a cholesterol lowering agent 10 years ago, with a good safety profile. Conclusions: Here we describe the preclinical evidence supporting the possible repositioning of TAK-475 from its originally intended use to the treatment of MKD and discuss its potential to modulate the mevalonate pathway in inflammatory diseases.

The purpose of this study is to determine if a preventive treatment with curcumin can protect intestinal epithelial cells from inflammatory damage induced by IFNγ. To achieve this goal we have used a human intestinal epithelial cell line (HT29) treated with IFNγ to undergo apoptotic changes that can reproduce the damage of intestinal epithelia exposed to inflammatory cytokines. In this model, we measured the effect of curcumin (curcuminoid from Curcuma Longa) added as a pre-treatment at different time intervals before stimulation with IFNγ. Curcumin administration to HT29 culture before the inflammatory stimulus IFNγ reduced the cell apoptosis rate. This effect gradually declined with the reduction of the curcumin pre-incubation time. This anti-apoptotic action by curcumin pre-treatment was paralleled by a reduction of secreted IL7 in the HT29 culture media, while there was no relevant change in the other cytokine levels. Even though curcumin pre-administration did not impact the activation of the NF-κB pathway, a slight effect on the phosphorylation of proteins in this inflammatory signaling pathway was observed. In conclusion, curcumin pre-treatment can protect intestinal cells from inflammatory damage. These results can be the basis for studying the preventive role of curcumin in inflammatory bowel diseases.

Deregulation of the cholesterol pathway is an anomaly observed in human diseases, many of which have in common neurological involvement and unknown pathogenesis. In this study we have used Mevalonate Kinase Deficiency (MKD) as a disease-model in order to investigate the link between the deregulation of the mevalonate pathway and the consequent neurodegeneration. The blocking of the mevalonate pathway in a neuronal cell line (Daoy), using statins or mevalonate, induced an increase in the expression of the inflammasome gene (NLRP3) and programmed cell death related to mitochondrial dysfunction. The morphology of the mitochondria changed, clearly showing the damage induced by oxidative stress and the decreased membrane potential associated with the alterations of the mitochondrial function. The co-administration of geranylgeraniol (GGOH) reduced the inflammatory marker and the damage of the mitochondria, maintaining its shape and components. Our data allow us to speculate about the mechanism by which isoprenoids are able to rescue the inflammatory marker in neuronal cells, independently from the block of the mevalonate pathway, and about the fact that cell death is mitochondria-related.

Chronic inflammation associated with autoim- mune activation is characteristic of rheumatic diseases from childhood to adulthood. In recent decades, signi cant improvements in the treatment of these types of disease have been achieved using disease modifying anti-rheumatic drugs (DMARDs), such as methotrexate (MTX) and, more recently, using biologic inhibitors. The recent introduction of kinase inhibitors (for example, tofacitinib; Tofa) further increases the available ARDs. However, there are patients that do not respond to any treatment strategies, for whom combination therapies are proposed. The data regarding the combined action of different drugs is lacking and the knowledge of the mechanisms of ARDs and their actions upon pathogenic lymphocytes, which are hypothesized to sustain disease, is poor. An in vitro model of in ammation was developed in the current study, in which stimulated and unstimulated lymphocytes were cultured together, but tracked separately, to investigate the action of MTX and Tofa on the two populations. By analysing lymphocyte proliferation and activation, and cytokine secretion in the culture supernatants, it was estab- lished that, due to the presence of activated cells, unstimulated cells underwent a bystander activation that was modulated by the ARDs. Additionally, varying administration schedules were demonstrated to affect lymphocytes differently in vitro, either directly or via bystander activation. Furthermore, MTX and Tofa exerted different effects; while MTX showed an antiproliferative effect, Tofa marginally effected activation, although only a slight antiproliferative action, which could be potentiated by sequential treatment with MTX. Thus, it was hypothesized that these differences may be exploited in sequential therapeutic strategies, to maximize the anti-rheu- matic effect. These ndings are notable and must be accounted for, as bystander-activated cells in vivo could contribute to the spread of autoimmune activation and disease progression.

Problem We have previously found that C1q is constitutively expressed by invading trophoblast and endothelial cells of decidua and contributes to vascular and tissue remodeling. Based on these findings, we sought to determine whether there were changes in the circulating level of C1q that may be used as a diagnostic and predictive marker of preeclampsia. Method of Study We measured the levels of C1q, C4, and complement activation products in serum or plasma of normal pregnant women and preeclamptic patients from different cohorts. Results We observed a marked decrease in the concentration of C1q associated with a reduced level of C4 in preeclamptic patients as compared to matched healthy pregnant woman but no significant difference in the circulating level of the activating products C5a and the soluble terminal complement complex sC5b-9. Analysis of serum samples collected at early phase of pregnancy from women who later developed preeclampsia failed to show a decrease in C1q level. Conclusion The results of the present investigation demonstrate that low levels of C1q and C4 are associated with preeclampsia but cannot be used as predictive markers.

To evaluate how mucosal bacteria impact on the spontaneous and muramyl dipeptide (MDP)-induced inflammation in Crohn’s disease (CD) and ulcerative colitis (UC). METHODS Colonic mucosal biopsies were collected from children with active or remissive CD, UC and controls. Twotissue samples were taken from inflamed mucosal segments (in patients with active disease) or fromnoninflamed mucosa [in patients in remission or in healthy controls (HC)]. Experiments were performed in the presence or absence of antibiotics, to assess whether the disease-associated microbiota can modulate the cytokine response ex vivo . For this purpose, each specimen was half-cut to compare spontaneous and MDP-induced inflammation in the presence of live bacteria (LB) or antibiotics. After 24 h of culture, an array of 17 cytokines was assessed in supernatants. Statistical analyses were performed to find significant differences in single cytokines or in patterns of cytokine response in the different groups. RESULTS We demonstrated that subjects with CD display aspontaneous production of inflammatory cytokines including granulocyte-colony stimulating factor (G-CSF), interleukin (IL) 6, IL8, IL10 and IL12, that was not significantly influenced by the addition of antibiotics. UC specimens also displayed a trend of increased spontaneous secretion of several cytokines, which however was not significant due to broader variability among patients. After the addition of antibiotics, spontaneous IL8 secretion was significantly higher in UC than in controls. In HC, a trend towards the weakening of spontaneous IL8 production was observed in the presence of live mucosal bacteria with respect to the presence of antibiotics. In contrast, in the presence of LB UC showed an increasing trend of spontaneous IL8 production, while MDP stimulation resulted in lower IL8 production in the presence of antibiotics. We also showed that subjects with CD seem to have a lowered production of IL8 in response to MDP in the presence of LB. Only with the addition of antibiotics, likely reducing the contribution of LB, multivariate statistical analysis could identify the combination of measures of G-CSF, tumor necrosis factor alpha, IL4 and IL17 as a good discriminator between CD and UC. CONCLUSION We showed that the presence of LB or antibiotics can significantly influence the inflammatory response ex vivo in inflammatory bowel diseases.

AIM: To evaluate the inflammatory state in Crohn's disease (CD) patients and correlate it with genetic background and microbial spreading. METHODS: By means of flow cytometry, production of tumor necrosis factor-alpha (TNF-α) was measured in peripheral blood monocytes from patients suffering from CD, ulcerative colitis (UC) and in healthy subjects after stimulation of the NOD2 and TLR pathways. CD patients were genotyped for the three most common NOD2 variants (R702W, G908R and L1007Pfs*2) and basal production of TNF-α was correlated to NOD2 genotype. Also, production of TNF-α was correlated to plasmatic levels of LPS Binding Protein (LBP), soluble (s) CD14 and to the activity state of the disease. RESULTS: The patients with CD were characterized by a significantly higher monocyte basal expression of TNF-α compared with healthy subjects and UC patients, and after stimulation with Pam3CSK4 (ligand of TLR2/1) and MDP-L18 (ligand of NOD2) this difference was maintained, while other microbial stimuli (LPS, ligand of TLR4 and PolyI:C, ligand of TLR3) induced massive activation in CD monocytes as well as in UC and in healthy control cells. There was no significant difference in the production of TNF-α between patients who carried CD-associated heterozygous or homozygous variants in NOD2 and patients with wild type NOD2 genotype. Although serum LBP levels have been shown to correlate positively with the state of activity of the disease, TNF-α production did not show a clear correlation with either LBP or sCD14 levels in plasma. Moreover, no clear correlation was seen between TNF-α production and activity indices in either CD or UC. CONCLUSION: Peripheral monocytes from CD express higher basal and stimulated TNF-α than controls, regardless of NOD2 genotype and without a clear correlation with disease activity.

Background: Aggressive course and resistance to treatments usually characterize very early onset inflammatory bowel disease (VEO-IBD). Some VEO-IBD cases are due to monogenic immune defects and can benefit from hematopoietic stem cell transplantation (HSCT). Case presentation: We describe a Caucasian male baby who presented in the first months of life macrophage activation syndrome, followed by intractable colitis, recurrent episodes of fever and mild splenomegaly. After several immunological, genetic and clinical investigations, subsequently a therapeutic attempt with colectomy, analysis of VEO-IBD-associated genes, revealed a causative mutation in XIAP. The genetic diagnosis of a primary immune deficiency allowed curing the boy with hematopoietic stem cell transplantation. Conclusion: Our report, together with novel findings from recent literature, should contribute to increase awareness of monogenic immune defects as a cause of VEO-IBD. Comprehensive genetic analysis can allow a prompt diagnosis, resulting in the choice of effective treatments and sparing useless and damaging procedures.

Transforming growth factor beta is a pleiotropic cytokine which plays a central role in the homeostasis of the immune system. A complex dysregulation of its signaling occurs in Loeys-Dietz syndrome, a monogenic disorder caused by mutations of transforming growth factor beta receptors type 1 or type 2, characterized by skeletal involvement, craniofacial abnormalities, and arterial tortuosity with a strong predisposition for aneurysm and dissection. In addition, several immunologic abnormalities have been described in these patients, including an increased risk of allergic disorders as well as eosinophilic gastrointestinal disorders. The occurrence of inflammatory bowel disorders has been also reported, but it is poorly documented. We describe two unrelated children with Loeys-Dietz syndrome affected by severe chronic inflammatory colitis appearing at an early age. The intestinal disease presented similar features in both patients, including a histopathological picture of non-eosinophilic chronic ulcerative colitis, striking elevation of inflammatory markers, and a distinctly severe clinical course leading to failure to thrive, with resistance to multiple immunosuppressive treatments. One of the patients also presented autoimmune thyroiditis. Our report confirms that chronic ulcerative colitis may be associated with Loeys-Dietz syndrome. This finding suggests that an alteration of transforming growth factor beta signaling may by itself predispose to inflammatory colitis in humans, and represent an invaluable model to understand inflammatory bowel diseases.

Nowadays India is undergoing an impressive economic growth accompanied by a very slow decline, almost stagnation, in malnutrition levels. In developing countries, studies on dietary patterns and their relationship with nutritional status are scarce. Over the years some nutritional studies have been performed to explore different types of food consumed in various Indian regions, among different social samples. The aim of the present paper is to review and describe trends in food and nutrition intake patterns in the different states of India. The review was carried out in PubMed, using the advanced research criteria: [food* OR ("meal pattern*") OR ("eating pattern*")] AND ("nutrient intake") AND India*. PubMed research gave back 84 results and out of these, 7 papers due to their focus on food intake and consumption levels in India have been included in this study. Food intake patterns showed that most of the Indians are vegetarians and that food items rich in micronutrients (pulses, other vegetables, fruits, nuts, oilseeds and animal foods) are generally consumed less frequently. Poor and monotonous cereals-based diet may promote inadequate nutrition intakes according to Recommended Daily Allowance (RDA) standards.

Introduction: Despite having a proven immunosuppressive potential in vitro, human mesenchymal stromal cells (MSCs) are reported to display variable efficacy in vivo and, in fact, their proven benefit in the clinical practice is still limited and controversial. Methods: The interplay between clinical grade MSCs and pre-activated donor lymphocytes or selected lymphocyte subsets was studied in vitro. The kinetics of MSC growth and viability was evaluated by adhesion-dependent changes of culture plate impedance and biochemically by a colorimetric assay. Activation of natural killer (NK) cells was assessed as well, using a flow cytometry assay. Results: A strong inhibition of MSC growth was rapidly induced by the addition of pre-activated lymphocytes but not of resting lymphocytes. Inhibition seems not to be attributable to a single cell population, as similar results can be obtained by depleting NK cells or by using either selected CD4+ or CD8+ lymphocytes. In addition, conditioned medium (CM) from activated lymphocytes was able to inhibit MSC growth in a dose-dependent manner. Furthermore, licensing with IFN-γ partially protected MSCs from pre-activated lymphocytes but not from their CM. These results suggest an inhibitory role of lymphocyte-activation-derived substances. However, the identification of a single molecule responsible for MSC inhibition remained elusive, even if preliminary experiments showed that ATP and, to a lesser extent, TNF-α might play a role. Conclusions: These results suggest that survival of MSCs can be affected by soluble mediators released by activated lymphocytes. Thus it can be hypothesized that MSC immunosuppressive action in vivo could be impaired by ongoing immune activation through the release of inflammatory mediators.

Background: The fetal and infant life are periods of rapid development, characterized by high susceptibility to exposures. Birth cohorts provide unique opportunities to study early-life exposures in association with child development and health, as well as, with longer follow-up, the early life origin of adult diseases. Piccolipiù is an Italian birth cohort recently set up to investigate the effects of environmental exposures, parental conditions and social factors acting during pre-natal and early post-natal life on infant and child health and development. We describe here its main characteristics. Methods/design: Piccolipiù is a prospective cohort of expected 3000 newborns, who will be recruiting in six maternity units of five Italian cities (Florence, Rome, Trieste, Turin and Viareggio) since October 2011. Mothers are contacted during pregnancy or at delivery and are offered to participate in the study. Upon acceptance, their newborns are recruited at birth and followed up until at least 18 years of age. At recruitment, the mothers donate a blood sample and complete a baseline questionnaire. Umbilical cord blood, pieces of umbilical cord and heel blood spots are also collected. Postnatal follow-up currently occurs at 6, 12, and 24 months of age using on-line or postal self administered questionnaire; further questionnaires and medical examinations are envisaged. Questionnaires collect information on several factors, including mother’s and/or child’s environmental exposures, anthropometric measures, reproductive factors, diet, supplements, medical history, cognitive development, mental health and socioeconomic factors. Health promotion materials are also offered to parents. Discussion: Piccolipiù will broaden our understanding of the contribution of early-life factors to infant and child health and development. Several hypotheses on the developmental origins of health can be tested or piloted using the data collected from the Piccolipiù cohort. By pooling these data with those collected by other existing birth cohorts it will be possible to validate previous findings and to study rare exposures and outcomes.