Dissertations / Theses on the topic 'Trattamenti di malattie infiammatorie'

In 1969 Heaton el al. documented an increase of 4-5 times in the prevalence of cholelithiasis (GD) in patients with Crohn s disease (CD). These data were later confirmed by several Authors, but the mechanisms involved are controversal. Only three studies reported the prevalence of gallstones disease in ulcerative colitis (UC) and they gave controversial results as well. The aim of our study was to look at the prevalence of cholelithiasis in a consecutive series of patiens with CD and UC and to evaluate the possible risk factors involved. These results represent preliminary data for the first study about the incidence of GD and IBD. We included 412 patients with CD and 183 with UC, performed an abdominal US and evaluated several parameters as possible risk factors. We calculated the OR and the 95% CI by the regression logistic models after adjustment for the considered variables. The prevalence of GD was 9% in CD and 7 % in UC. Female sex and a high BMI did not correlate with an increase of GD. Older age was a risk factor for CD but not for UC. Previous bowel resections represent a risk factor for both CD an UC and, in particular, ileal resection was strongly associated with an increase of GD in CD. A number of hospitalizations > 3 and a cumulative number of days of hospitalization > 40 resulted to be a risk factor for CD and UC.

IL-33 is a novel member of the IL-1 family and ligand for the IL-1 receptor-related protein, ST2. Recent evidence suggests that the IL-33/ST2 axis plays a critical role in several autoimmune and inflammatory disorders; however, its role in inflammatory bowel disease (IBD) has not been clearly defined. We characterized IL-33 and ST2 expression and modulation following conventional anti-TNF therapy in Crohn’s disease and ulcerative colitis (UC) patients, and investigated the role of IL-33 in SAMP1/YitFc (SAMP) mice, a mixed Th1/Th2 model of IBD. Our results showed a specific increase of mucosal IL-33 in active UC, localized primarily to intestinal epithelial cells (IEC) and colonic inflammatory infiltrates. Importantly, increased expression of full-length IL-33, representing the most bioactive form, was detected in UC epithelium, while elevated levels of cleaved IL-33 were present in IBD serum. ST2 isoforms were differentially modulated in UC epithelium and sST2, a soluble decoy receptor with anti-inflammatory properties, was also elevated in IBD serum. Infliximab (anti-TNF) treatment of UC decreased circulating IL-33 and increased sST2, while stimulation of HT-29 IEC confirmed IL-33 and sST2 regulation by TNF. Similarly, IL-33 significantly increased and correlated with disease severity, and potently induced IL-5, IL-6 and IL-17 from mucosal immune cells in SAMP mice. Taken together, the IL-33/ST2 system plays an important role in IBD and experimental colitis, is modulated by anti-TNF therapy, and may represent a specific biomarker for active UC.

IL-33 is a novel member of the IL-1 family and ligand for the IL-1 receptor-related protein, ST2. Recent evidence suggests that the IL-33/ST2 axis plays a critical role in several autoimmune and inflammatory disorders; however, its role in inflammatory bowel disease (IBD) has not been clearly defined. We characterized IL-33 and ST2 expression and modulation following conventional anti-TNF therapy in Crohn’s disease and ulcerative colitis (UC) patients, and investigated the role of IL-33 in SAMP1/YitFc (SAMP) mice, a mixed Th1/Th2 model of IBD. Our results showed a specific increase of mucosal IL-33 in active UC, localized primarily to intestinal epithelial cells (IEC) and colonic inflammatory infiltrates. Importantly, increased expression of full-length IL-33, representing the most bioactive form, was detected in UC epithelium, while elevated levels of cleaved IL-33 were present in IBD serum. ST2 isoforms were differentially modulated in UC epithelium and sST2, a soluble decoy receptor with anti-inflammatory properties, was also elevated in IBD serum. Infliximab (anti-TNF) treatment of UC decreased circulating IL-33 and increased sST2, while stimulation of HT-29 IEC confirmed IL-33 and sST2 regulation by TNF. Similarly, IL-33 significantly increased and correlated with disease severity, and potently induced IL-5, IL-6 and IL-17 from mucosal immune cells in SAMP mice. Taken together, the IL-33/ST2 system plays an important role in IBD and experimental colitis, is modulated by anti-TNF therapy, and may represent a specific biomarker for active UC.

Background: Se l’uso crescente di farmaci immunomodulatori (ISS) e delle terapie biologiche può aumentare il rischio di neoplasia nelle malattie infiammatorie intestinali (IBD), è indefinito. Obiettivo: In uno studio di coorte monocentrico si vuole stimare la frequenza e l’istotipo di neoplasia in una popolazione di 1057 pazienti sotto regolare controllo medico. La frequenza di neoplasia è stimata anche in relazione ad un uso precedente di ISS e/o anti-TNFs . Materiali e metodi: Studio retrospettivo effettuato su dati registrati nel database del nostro centro di riferimento per le malattie infiammatorie intestinali. I pazienti esaminati sono stati controllati in un arco di tempo tra il 1984 ed il 2009. Sono stati valutati i seguenti dati: tipo di IBD / sede di malattia / durata di malattia, presenza di intervento chirurgico, fumo, uso di ISS e/o anti-TNFs e durata della somministrazione, presenza di neoplasia. Analisi statistica: Wilcoxon’test,t test e test del chi quadrato. Risultati: La popolazione studiata include 1057 pazienti IBD (512 F 545 M): 555 con malattia di Cren (CD), 502 con Colite Ulcerosa (UC). La neoplasia è presente in 73/1057 IBD (6.9%; 39M 34F) di cui: 47/555 CD (8.5%), 26/502 UC (5.2%). Sede della neoplasia / istotipo: mammario 14 (10 CD, 4 UC); intestinale 7 (3 CD, 4 UC), cutaneo 7 (5 CD, 2 UC), linfoma 6 (5 CD, 1 UC), canale anale 2 (2 CD), altre sedi 37. Fra i 73 pazienti, con IBD e neoplasia, usano ISS 20 pazienti (27.3%), anti-TNFs 10 pazienti (13.6%), ISS ed anti-TNFs 7 pazienti (9.5%). L’età mediana alla diagnosi di neoplasia è di 52.4 anni (range 21 -81) di cui: UC 52 anni (range 27 -81), CD 62 anni (range 13 -83). La relazione tra caratteristiche cliniche e neoplasia è stata stimata nei 1057 pazienti IBD: età mediana 52.4 anni ( range 13 -83) 555 CD, 502 UC; durata di IBD: UC 7 anni ( range1-86) versus CD 12 anni ( range 1-76); p = < 0.01. Trattamenti farmacologici precedenti: ISS in 275/1057 IBD (26%), 201/555 CD (36.2%), 74/502 UC (14.7%); Anti-TNFs in 179/1057 IBD (16.9%), 146/555 CD (26.3%), 33/502 UC (6.6%); ISS e concomitante anti-TNFs in 125/1057 IBD (11.8%), 104/555 CD (18.7%), 21/502 UC (4.2%). La neoplasia è stata diagnosticata in 73/1057 pazienti IBD (6.9%; 39M 34F): 47/555 CD (8.5%), 26/502 UC (5.2%; p=ns). Fra i 73 pazienti, con IBD e neoplasia, l’uso di ISS è stato effettuato in 20/73 pazienti (27.3%) (età 51 anni versus i 55 anni dei 53 pazienti con neoplasia e nessun uso di ISS; p=ns), suddiviso per tipo IBD 12/20 CD (0.6%), 8/20 UC (0.4%). Fra i 73 pazienti, con IBD e neoplasia, la somministrazione di anti-TNFs è stata eseguita in 10/73 pazienti (0.14%) (età 53 anni versus i 55 anni dei 63 pazienti con neoplasia e nessun trattamento anti-TNFs; p=ns), suddiviso per tipo IBD 9 / 47 CD (19.1%), 1/26 UC (3.8%). Fra i 73 pazienti, con IBD e neoplasia, l’uso concomitante di ISS e di anti-TNFs è stato riscontrato in 7/73 pazienti (9.5%) (età 51 anni versus i 54 anni dei 50 pazienti senza trattamenti ISS e anti-TNFs; p=ns), suddiviso per tipo IBD 7/47 CD (14.8%), 0/26 UC (0%). Sede della neoplasia / istotipo: mammario 11 (11 CD); intestinale 4 (2 CD), cutaneo 4 (3 CD), linfoma 3 (3 CD), canale anale 1 (CD), altri sedi 18. Conclusioni. Nella nostra popolazione di 1057 IBD pazienti, è stata riscontrata una frequenza alta di neoplasia, con un trend più alto ma non significativo nei pazienti con malattia di Crohn trattati con ISS, mentre la somministrazione di anti-TNFs sembra non aumentare significativamente il rischio di neoplasia.
Background. Whether the growing use of immunomodulatory drugs (ISS) and biologic therapies may increase the cancer risk in Inflammatory Bowel Disease (IBD) is undefined. Aims. In a single-center, cohort study we aimed to assess the frequency and histotype of cancer in a population of 1057 patients (pts) under regular follow up. The frequency of cancer was also assessed in relation to previous ISS and/or anti-TNFs use. Materials and methods. In a cross-sectional study, clinical records from all IBD pts. In follow up from 1984-2009 in our tertiary IBD referral center were reviewed. Data recorded: IBD type/ site/duration, surgery, smoking habits, follow up length, ISS and/or anti-TNFs use. Statistical analysis: Wilcoxon test,t test and χ test. Results. The study population included 1057 IBD pts (512 F 545 M): 555 Crohn’s Disease (CD), 502 Ulcerative Colitis (UC). Cancer was diagnosed in 73/1057 IBD (6.9%; 39M 34F): 47/555 CD (8.5%), 26/502 UC (5.2%). Cancer site/histotype: breast 14 (10 CD, 4 UC); colon 7 (3 CD, 4 UC), skin 7 (5 CD, 2 UC), lymphoma 6 (5 CD, 1 UC), anal canal 2 (2 CD), other cancers 37. Among the 73 pts with IBD and cancer, ISS use reported by 20 (27.3%), anti-TNFs by 10 (13.6%), both ISS and anti-TNFs by 7 pts (9.5%). Age at the diagnosis of cancer: IBD 52.4 yrs (range 21-81), UC 52 (range 27-81), CD 62 (range 13-83). The relation between clinical features and cancer was assessed in 1057 IBD pts (52.4 age range 13-83): 555 CD, 502 UC (IBD duration: UC 7 yrs, range 1-86 vs CD 12 yrs, range 1-76; p=< 0.01). Previous treatments: ISS in 275/1057 IBD (26%), 201/555 CD (36.2%), 74/502 UC (14.7%); Anti-TNFs in 179/1057 IBD (16.9%), 146/555 CD (26.3%), 33/502 UC (6.6%); Concomitant ISS and anti-TNFs in 125/1057 IBD (11.8%), 104/555 CD (18.7%), 21/502 UC (4.2%). Cancer was diagnosed in 73/1057 (6.9%; 39M 34F) IBD pts: 47/555 CD (8.5%), 26/502 UC (5.2%; p=ns). Among the 73 pts with IBD and cancer, ISS use reported by 20/73 (27.3%)(age 51 vs 55 yrs in the 53 pts with cancer and no ISS use; p=ns), including 12/20 CD (0.6%), 8/20 UC (0.4%). Among the 73 pts with IBD and cancer, anti-TNFs use reported by 10/73 IBD (0.14%) (age 53 vs 55 yrs in the 63 pts with cancer and no anti-TNFs; p=ns), including 9/ 47 CD (19.1%), 1/26 UC (3.8%). Among the 73 pts with IBD and cancer, both ISS and anti-TNFs use reported by 7/73 (9.5%)(age 51, vs 54 yrs in the50 pts with no concomitant ISS and anti-TNFs; p=ns), including 7/47 CD (14.8%), 0/26 UC (0%). Cancer site/histotype: breast 11 (11 CD); colon 4 (2 CD), skin 4 (3 CD), lymphoma 3 (3 CD), anal canal 1 (CD), other cancers 18. Conclusions. In our cohort of 1057 IBD pts, a high frequency of cancer was observed, with a not significantly higher trend in CD pts treated with ISS, while anti-TNFs appeared not to significantly increase the cancer risk.

INTRODUCTION: A relationship between inflammatory response and coagulation is suggested by many observations. In particular, pro-inflammatory cytokines, such as TNFalpha, promote the activation of coagulation and reduce the production of anticoagulant molecules. It is known that inflammatory bowel diseases show a prothrombotic state and a condition of hypercoagulability. Aim of our study was to evaluate whether anti-TNFalpha therapy induces changes in the levels of coagulation activation markers in IBD patients. MATERIALS AND METHODS: We analyzed 48 plasma samples obtained before and 1 hour after 24 infliximab infusions (5 mg/kg) in 9 IBD patients (5 men and 4 women; mean age: 47.6+17.6 years; 4 Crohn's disease, 4 Ulcerative Colitis,1 Indeterminate Colitis). F1+2 and D-dymer levels were measured in each sample using ELISA methods.The data were statistically analyzed by means of Wilcoxon matched paired test. RESULTS: Median F1+2 levels were markdely reduced 1 hour after anti-TNFα infusion (median pre-infusion levels were 247.0 pmol/L and median post-infusion levels were 185.3 pmol/L) (p<0.002). Median D-dymer levels were also significantly reduced, from 485.2 ng/mL to 427.6 ng/mL (p< 0.001). These modifications were more evident in patients naive for infliximab therapy (p<0.02 for F1+2 and p<0.02 for D-dymer) and in Crohn's disease compared with Ulcerative Colitis patients (p=0.01 for F1+2 and p<0.007 for D-dymer).CONCLUSIONS: Infusion of infliximab significantly reduces the activation of coagulation cascade in IBD patients. This effect is early enough to suggest a direct effect of infliximab on the coagulation cascade and a possible new anti-inflammatory mechanism of action of this molecule.

INTRODUCTION: A relationship between inflammatory response and coagulation is suggested by many observations. In particular, pro-inflammatory cytokines, such as TNFalpha, promote the activation of coagulation and reduce the production of anticoagulant molecules. It is known that inflammatory bowel diseases show a prothrombotic state and a condition of hypercoagulability. Aim of our study was to evaluate whether anti-TNFalpha therapy induces changes in the levels of coagulation activation markers in IBD patients. MATERIALS AND METHODS: We analyzed 48 plasma samples obtained before and 1 hour after 24 infliximab infusions (5 mg/kg) in 9 IBD patients (5 men and 4 women; mean age: 47.6+17.6 years; 4 Crohn's disease, 4 Ulcerative Colitis,1 Indeterminate Colitis). F1+2 and D-dymer levels were measured in each sample using ELISA methods.The data were statistically analyzed by means of Wilcoxon matched paired test. RESULTS: Median F1+2 levels were markdely reduced 1 hour after anti-TNFα infusion (median pre-infusion levels were 247.0 pmol/L and median post-infusion levels were 185.3 pmol/L) (p<0.002). Median D-dymer levels were also significantly reduced, from 485.2 ng/mL to 427.6 ng/mL (p< 0.001). These modifications were more evident in patients naive for infliximab therapy (p<0.02 for F1+2 and p<0.02 for D-dymer) and in Crohn's disease compared with Ulcerative Colitis patients (p=0.01 for F1+2 and p<0.007 for D-dymer).CONCLUSIONS: Infusion of infliximab significantly reduces the activation of coagulation cascade in IBD patients. This effect is early enough to suggest a direct effect of infliximab on the coagulation cascade and a possible new anti-inflammatory mechanism of action of this molecule.

Background: Several lines of evidence showed that inflammation is associated with changes in the expression of tachykinins both in human and animal models. Tachykinins, including substance P (SP), are small peptides expressed in the extrinsic primary afferent nerve fibres and enteric neurons of the gut: they exert their action through three distinct receptors, termed NK1, NK2 and NK3. SP modulates intestinal motility and enteric secretion, acting preferentially through the NK1 receptor. SP neural network and NK1 receptor expression are increased in patients with inflammatory bowel disease, and similar changes were observed in experimental models of inflammation. The 2,4 Dinitrobenzene Sulphonic Acid (DNBS) model of colitis is useful to study innate immunity, non-specific inflammation and wound healing; it has been suggested that the transmural inflammation seen in this model resembles that found in Crohn’s disease and can therefore be used to study what cells and mediators are involved in this type of inflammation. Aim: To test the possible protective effect of the NK1 receptor antagonist SSR140333 on: 1) acute model of intestinal inflammation; 2) reactivation of DNBS-induced colitis in rats. Methods: Acute colitis was induced in male SD rats by intrarectal administration of DNBS (15 mg/rat in 50% ethanol). Reactivation of colitis was induced by intrarectal injections of DNBS on day 28 (7.5 mg/rat in 35% ethanol). Animals were sacrificed on day 6 (acute colitis) and 29 (reactivation of colitis). SSR140333 (10 mg/kg) was administered orally starting from the day before the induction of colitis for 7 days (acute colitis) or seven days before the reactivation of colitis. Colonic damage was assessed by means of macroscopic and microscopic scores, myeloperoxidase activity (MPO) and TNF-α tissue levels. Enzyme immunoassay was used to measure colonic substance P levels. Statistical analysis was performed using analysis of variance (one-way or two-way, as appropriate) with the Bonferroni’s correction for multiple comparisons. Results: DNBS administration impaired body weight gain and markedly increased all inflammatory parameters (p<0.01). Treatment with SSR140333 10 mg/kg significantly counteracted the impairment in body weight gain, decreased macroscopic and histological scores and reduced colonic myeloperoxidase activity (p<0.01). Drug treatment counteracted TNF-α tissue levels and colonic SP concentrations (acute model). Similar results were obtained administering the NK1 receptor antagonist SSR140333 (3 and 10 mg/kg) for 5 days, starting the day after the induction of colitis. Intrarectal administration of DNBS four weeks after the first DNBS administration resulted in reactivation of colitis, with increases in macroscopic and histological damage scores and increase in MPO activity. Preventive treatment with SSR140333 10 mg/kg decreased macroscopic damage score, significantly reduced microscopic damage score but did not affect MPO activity. Conclusions: Treatment with SSR140333 significantly reduced intestinal damage in acute model of intestinal inflammation in rats. The NK1 receptor antagonist SSR140333 was also able to prevent relapse in experimental colitis. These results support the hypothesis of SP involvement in intestinal inflammation and indicate that NK receptor antagonists may have a therapeutic potential in inflammatory bowel disease.

Background: Several lines of evidence showed that inflammation is associated with changes in the expression of tachykinins both in human and animal models. Tachykinins, including substance P (SP), are small peptides expressed in the extrinsic primary afferent nerve fibres and enteric neurons of the gut: they exert their action through three distinct receptors, termed NK1, NK2 and NK3. SP modulates intestinal motility and enteric secretion, acting preferentially through the NK1 receptor. SP neural network and NK1 receptor expression are increased in patients with inflammatory bowel disease, and similar changes were observed in experimental models of inflammation. The 2,4 Dinitrobenzene Sulphonic Acid (DNBS) model of colitis is useful to study innate immunity, non-specific inflammation and wound healing; it has been suggested that the transmural inflammation seen in this model resembles that found in Crohn’s disease and can therefore be used to study what cells and mediators are involved in this type of inflammation. Aim: To test the possible protective effect of the NK1 receptor antagonist SSR140333 on: 1) acute model of intestinal inflammation; 2) reactivation of DNBS-induced colitis in rats. Methods: Acute colitis was induced in male SD rats by intrarectal administration of DNBS (15 mg/rat in 50% ethanol). Reactivation of colitis was induced by intrarectal injections of DNBS on day 28 (7.5 mg/rat in 35% ethanol). Animals were sacrificed on day 6 (acute colitis) and 29 (reactivation of colitis). SSR140333 (10 mg/kg) was administered orally starting from the day before the induction of colitis for 7 days (acute colitis) or seven days before the reactivation of colitis. Colonic damage was assessed by means of macroscopic and microscopic scores, myeloperoxidase activity (MPO) and TNF-α tissue levels. Enzyme immunoassay was used to measure colonic substance P levels. Statistical analysis was performed using analysis of variance (one-way or two-way, as appropriate) with the Bonferroni’s correction for multiple comparisons. Results: DNBS administration impaired body weight gain and markedly increased all inflammatory parameters (p<0.01). Treatment with SSR140333 10 mg/kg significantly counteracted the impairment in body weight gain, decreased macroscopic and histological scores and reduced colonic myeloperoxidase activity (p<0.01). Drug treatment counteracted TNF-α tissue levels and colonic SP concentrations (acute model). Similar results were obtained administering the NK1 receptor antagonist SSR140333 (3 and 10 mg/kg) for 5 days, starting the day after the induction of colitis. Intrarectal administration of DNBS four weeks after the first DNBS administration resulted in reactivation of colitis, with increases in macroscopic and histological damage scores and increase in MPO activity. Preventive treatment with SSR140333 10 mg/kg decreased macroscopic damage score, significantly reduced microscopic damage score but did not affect MPO activity. Conclusions: Treatment with SSR140333 significantly reduced intestinal damage in acute model of intestinal inflammation in rats. The NK1 receptor antagonist SSR140333 was also able to prevent relapse in experimental colitis. These results support the hypothesis of SP involvement in intestinal inflammation and indicate that NK receptor antagonists may have a therapeutic potential in inflammatory bowel disease.

Background: Antibodies against tumor necrosis factor represent an effective therapy for patients with inflammatory bowel disease. Despite their successful results, the exact mechanism by which infliximab suppresses intestinal inflammation is still a matter of debate. In this study, we used a translational approach to identify the key mechanisms associated with resolution of mucosal inflammation induced by infliximab. Methods: A total of 16 patients with active inflammatory bowel disease (9 with Crohn’s disease and 7 with ulcerative colitis) and 13 controls were enrolled in the study. Patients received infliximab infusions at 0, 2, and 6 weeks. At enrollment and at week 6, patients underwent flexible sigmoidoscopy, and biopsies were taken from the sigmoid colon. RNA was extracted, and mucosal expression of 96 immune-related genes was evaluated by qRT-PCR and confirmed by immunofluorescence microscopy on tissue. Correlation between infliximab-induced gene expression modulation and endoscopic response to therapy was calculated. Lamina propria mononuclear cell apoptosis induced by infliximab was evaluated on tissue sections by the terminal deoxynucleotidyl transferase dUTP nick end labeling assay. Results: We found that infliximab-induced downregulation of macrophage and Th17 pathway genes was significantly associated with both endoscopic response to the therapy and achievement of mucosal healing. Importantly, the observed reduction of lamina propria CD68+ macrophages was associated with an increased rate of macrophage apoptosis. Conclusions: The 2 mechanisms associated with infliximab-induced resolution of intestinal inflammation are the reduction of lamina propria infiltrating CD68+ macrophages and the downregulation of interleukin 17A. Moreover, the data suggest that infliximab-induced macrophage apoptosis may represent a key mechanism for the therapeutic success of anti–tumor necrosis factor antibodies.

Background: International guidelines recommend pulmonary rehabilitation for COPD patients in all stages of the disease, in particular for those patients who experience exercise-related restrictions in daily physical activities. The success of Pulmonary Rehabilitation programs resides in the integration between exercise prescription , the choice of methods and patients' compliance with home training. Several methods that can be applied to improve exercise performance in patients with COPD. One of the crucial issue for the patients is the understanding of the correct exercise intensity especially for the development of cardio-respiratory fitness (general exercise training). If the choice of method affects the area respiratory muscle training (inspiratory muscles training-IMT), normocapnic hyperventilation seems effective in improving exercise endurance in healthy subjects but few data are available for COPD patients. My PhD program consist in two studies with the common aim to evaluate the efficacy of different methods of training to improve exercise capacity and Quality of Life. Study N°1: The first study aimed to compare 2 methods of home exercise training (based on walking) titled “A simple method for home exercise training in COPD patients: 1-year study” Methods: 47 COPD were recruited and underwent respiratory function, exercise capacity evaluation with six minutes walking test (6MWT) and treadmill tests. Physical Activity was monitored by multisensor Armband. Patients were randomly assigned to 2 different home training methods and assessed again after 6 and 12 months. Group A1) speed walking marked by a metronome; Group A2) covering a known distance in a fixed time. Results: Thirty-six patients completed the study (77% of the enrolled patients). All subjects showed a significant improvement in 6MWT after 1 year but the improvement was higher in A1 than in A2 (p<0.05). Physical Activity levels were significantly higher at T12 vs baseline only in group A1(p<0.05). Conclusions: The use of a metronome to keep the rate of walking during the home exercise training improves the understanding of exercise intensity allowing the patients to follow the exercise prescription and to get better results. Study N°2: The second study aimed to assess the effects of 4 weeks of normocapnic hyperventilation (NH) by means of Spirotiger® titled “Inspiratory muscle training (IMT) with normocapnic hyperventilation (NH) improves respiratory muscle strength, exercise performance and ventilatory pattern in COPD patients”. Methods: 21 COPD were recruited. Respiratory function tests (FEV1, FVC, Pimax), QoL (St George's Questionnarie), 6MWT and endurance exercise performed at 75-80% of peak-work rate measured during an incremental test to the limit of tolerance (tLIM). 7 of 21 patients were instrumented with a portable inductive plethysmografhy (Lifeshirt System) to evaluate breathing pattern during tLIM. After 1 month of weekly supervised training, the patients trained at home for 4 weeks: 10 min twice a day at a breathing rate 12-24/min with a tidal volume (TV) equal to 50% of CV. Results: 6 patients dropped out (poor compliance). IMT significantly improved Pimax, QoL, exercise capacity (Tab 1). Ventilatory pattern after IMT is characterized by a significantly higher TV with no change in VE (Tab 2). Table1 FEV1(%) FVC(%) Pimax(KPa) QoL(tot) tLIM(min) 6MWt(m) preIMT 55,216,9 82,322,8 8,93 22,716,6 6,43,4 43674,5 postIMT 57,615,8 82,724,1 9,72,8* 17,512,2* 10,37,4* 466,279,7* Table2 SpO2mean(%) VE(L/min) TV(L/min) Br(b/min) preIMT 912,2 28,616,1 0,80,4 334,2 postIMT 92,31,5* 2916,4 0,90,4* 30,86,5 *p<0,05. Conclusions: After a short IMT with NH, COPD patients show a higher exercise capacity and an intriguing change in ventilatory pattern which improves oxygen saturation.

The α7 nicotinic receptor is involved in the cholinergic anti-inflammatory pathway, the mechanism through which the nervous system influences leukocytes inflammatory responses. Vagus nerve releases the neurotransmitter acetylcholine which binds to the α7 nicotinic receptors on the surface of macrophages inhibiting pro-inflammatory mediators release, such as TNFα and ILβ. Intestinal Bowel diseases (IBD), which comprise Crohn’s disease (CD) and ulcerative colitis (UC), are chronic immune mediated diseases characterized by a deregulated immune response to commensal flora in a genetically susceptible host. Epidemiologic studies revealed the dual effect of smoke on IBD patients: smoke ameliorates CU, by suppressing macrophages and lymphocytes activity, but worsen the symptoms and the histologic damage in CD patients. This thesis aimed to study the mechanism underlying the nicotine anti-inflammatory effect on CU patients (but not in CD patients), verifying the hypothesis that different expression levels of nicotinic receptor in IBD patients are responsible for its antithetic effects on diseases progress. Our main purpose was to verify first nicotinic receptor levels on mucosal macrophages during inflammation, and consequently whether macrophages’ sensibility to the anti-inflammatory pathway, could be influenced by the integrity of the enteric nervous system (ENS). Expression levels and functionality of α7nAchR in UC and CD patients in clinical remission or mild activity was compared to control subjects (HV, healthy volunteers or patients in screening for colonic cancer) in peripheral blood-derived macrophages and intestinal macrophages isolated from colon-sigma biopsies. In blood-derived macrophages α7nAchR levels were comparable between the three groups both at mRNA, quantified by Real Time-PCR, and protein, quantified by α-bungarotoxin-Alexa Fluor 488 binding, levels. On the contrary, the nicotinic receptors were more expressed in intestinal mucosal macrophages in UC patients than in healthy subjects and CD patients. Moreover, nicotine, the exogenous ligand of α7nAchR, significantly reduced LPS-induced TNFα synthesis in mucosal macrophages from UC but not MC and HV. To determine the mechanism responsible for the altered expression of α7nAchR in CD patients, we studied the cholinergic anti-inflammatory pathway in murine models of ENS neuropathy, since structural and functional damages in enteric neurons is well established in IBD patients. Our neuropathy models comprise TLR2 deficient mice (TLR2-/-) and mice infected by Herpes Simplex Virus type-1 (HSV-1). We quantified α7nAchR expression and inflammatory activation markers (F4/80 and caspase-1 activation) of intestinal macrophages in basal conditions and during early and late phases of experimental colitis induced by DSS. Mucosal macrophages showed no significant differences in α7nAchR expression in WT and TLR2-/- mice, while HSV-1 induced neuropathy caused a significant increase of α7nAchR levels. However, after three days of DSS administration, a significant increase of α7nAchR occurred in WT mice, but not in mice with enteric neuropathy. In parallel, in mucosal macrophages of WT mice, but not in mice with ENS neuropathy, we observed the activation of caspase-1 and surface F4/80 overexpression. Moreover, only in WT mice, nicotine reduced caspase-1 activation induced by LPS+ATP in mucosal macrophages. Furthermore, in vivo nicotine administration reduced the gravity of colitis in WT mice, but was ineffective in TLR2-/- mice. Finally, by correcting the integrity of ENS of TLR2-/- mice by administration in vivo of glial-derived neurotrophic factor (GDNF), caspase-1 activation in mucosal macrophages during colitis was normalized. All together our data suggest that for the cholinergic anti-inflammatory pathway to have an optimal action, it is required an increased expression of α7nAchR in mucosal macrophages in response to an inflammatory stimulus. Lack of α7nAchR up-regulation in mucosal macrophages, such as in MC patients, causes the loss of nicotine anti-inflammatory effects. The presence of a neuropathy might contribute to the inadequate expression of α7nAchR in mucosal macrophages during inflammatory processes, thus paving the way to amplified mucosal damage. Mediators that directly regulate α7nAchR expression in mucosal macrophages are now under investigation.
Il recettore nicotinico α7 è coinvolto nel sistema colinergico anti-infiammatorio, il meccanismo attraverso il quale il sistema nervoso regola la risposta infiammatoria dei leucociti. Il nervo vago rilascia acetilcolina che lega i recettori nicotinici α7 (α7nAChR) presenti nella superficie dei macrofagi, inibendo il rilascio di mediatori della risposta pro-infiammatoria, quali TNFα e IL1β. Le malattie infiammatorie croniche (in inglese IBD, Intestinal Bowel Disease) sono malattie idiopatiche caratterizzate da flogosi cronica che comprendono malattia di Crohn (MC) e colite ulcerosa (CU). Nell’uomo, studi epidemiologici hanno dimostrato che il fumo ha un effetto soppressivo su macrofagi e linfociti migliorando il decorso della CU mentre aggrava il quadro istologico della MC. I meccanismi responsabili di questa dicotomia non sono attualmente noti. Questo lavoro di tesi si è proposto di studiare i meccanismi alla base dell’attività anti-infiammatoria espletata dalla nicotina nei pazienti affetti da CU ma non da MC verificando l’ipotesi che diversi livelli di espressione dei recettori nicotinici nei pazienti con CU ed MC possano giustificare il diverso effetto della nicotina sul decorso della malattia. Si è quindi verificato se l’espressione dei recettori nicotinici nei macrofagi mucosali in corso di infiammazione, e quindi la loro sensibilità al riflesso anti-infiammatorio colinergico, potesse essere influenzata dal sistema nervoso enterico. I livelli di espressione e la funzionalità del recettore nicotinico α7nAChR in pazienti affetti da CU e MC in remissione clinica o in fase di attività lieve rispetto a soggetti di controllo (VS, volontari sani o soggetti in screening per cancro colico) sono stati studiati in macrofagi differenziati da monociti ottenuti da sangue periferico ed in macrofagi intestinali isolati da biopsie di colon-sigma. Nei macrofagi derivati dal sangue periferico, il recettore nicotinico α7nAChR è risultato paragonabile tra i tre gruppi sia a livello di mRNA, quantificato mediante Real Time-PCR, che di proteina, quantificata determinando il legame di α-bungarotossina-Alexa Fluor 488. Al contrario, il recettore nicotinico è risultato invece maggiormente espresso nei macrofagi della mucosa intestinale dei soggetti con CU rispetto ai soggetti sani o con MC. Inoltre la nicotina, ligando esogeno di α7nAChR, ha ridotto in maniera significativa l’espressione di TNFα stimolata da LPS nei macrofagi mucosali di CU ma non di MC. Al fine di determinare il meccanismo responsabile dell’alterata espressione del α7nAChR nella MC, il sistema colinergico anti-infiammatorio è stato studiato in diversi modelli murini caratterizzati dalla presenza di una neuropatia del sistema nervoso enterico, poiché è nota la presenza di danni strutturali e funzionali ai neuroni enterici nei pazienti con MC. In particolare sono stati utilizzati topi deficienti del recettore TLR2 (TLR2-/-) o topi con infezione nel sistema nervoso enterico da Herpes Virus simplex di tipo 1 (HSV-1). In questi animali è stata determinata, in condizioni basali e durante le fasi precoci e tardive di una colite sperimentale da DSS, l’espressione di α7nAChR nei macrofagi intestinali e il grado di attivazione pro-infiammatoria di queste cellule. I macrofagi della mucosa colica hanno evidenziato che in condizioni basali non vi è una significativa differenza tra topi WT e topi TLR2-/- nei livelli di espressione del recettore α7nAChR nei macrofagi intestinali, mentre nella neuropatia nei topi inoculati con HSV-1 si registra un significativo aumento del recettore α7nAChR. Tuttavia, a seguito della somministrazione di DSS per tre giorni si è osservato un significativo aumento del recettore α7nAChR in topi WT, ma non nei topi portatori di neuropatia enterica, TLR2-/- e infettati per via orogastrica con HSV-1. Parallelamente l’attivazione dei macrofagi mucosali è stata determinata quantificando l’espressione del marcatore di superficie F4/80 e l’attivazione della caspasi-1. Nei macrofagi della mucosa colica di topi WT ma non in topi portatori di neuropatia del SNE si osserva l’attivazione della caspasi-1 e la sovra-espressione di F4/80 durante le fasi iniziali della colite indotta da DSS. Inoltre, solo nei macrofagi ottenuti da topi WT la nicotina è in grado di ridurre l’attivazione della caspasi-1 indotta da LPS+ATP. La somministrazione in vivo di nicotina riduce la gravità della colite nei topi WT ma risulta inefficace nei topi TLR2-/-. Infine, abbiamo quindi verificato che ristabilendo l’integrità del SNE mediante la somministrazione di fattore neurotrofico derivante dalla glia (GDNF) in vivo a topi TLR2-/-, viene ripristinata una normale attivazione della caspasi-1 nei macrofagi mucosali in corso di colite. In conclusione, un’ottimale azione del sistema colinergico anti-infiammatorio richiede l’aumentata espressione di α7nAChR nei macrofagi mucosali in risposta ad un processo flogistico. Tuttavia, in presenza di neuropatia (i.e. virale o trofica) l’aumentata espressione di α7nAChR nei macrofagi mucosali può risultare insufficiente portando eventualmente ad un danno mucosale amplificato. I mediatori che direttamente regolano l’espressione di α7nAChR nei macrofagi mucosali sono attualmente oggetto di studio.

Le cellule dendritiche mieloidi (DCs) e i macrofagi evolvono da un precursore comune. Comunque, è ancora poco chiaro quale siano i fattori che controllano il differenziamento dei monociti in DC o macrofagi. Noi abbiamo riportato che la densità di superficie della subunità α del recettore del GM-CSF (GM-CSFR) in monociti del sangue periferico umano varia tra i diversi donatori. Sebbene poi nessuna correlazione sia stata trovata tra l’entità di espressione del GM-CSFR e la capacità di monociti di differenziare in DC in presenza di GM-CSF e IL-4, l’espressione del GM-CSFR è stata vista però influenzare fortemente la generazione di cellule CD1a+ DC-like in assenza di IL-4. Le cellule CD1a+ generate in presenza del solo GM-CSF sono risultate esprimere le molecole CD40, CD80, MHC I and II, DC-SIGN, MR, CCR5 e mantenere in modo parziale il CD14. Queste stesse cellule sono state anche trovate capaci di indurre spontaneamente l’espansione di linfociti T CD4+ and CD8+ allogenici rilascianti IFN-gamma, e migrare in risposta alla CCL4 e alla CCL19. Dopo stimolazione con vari ligandi dei TLR, infine, esse sono state trovate acquisire caratteristiche fenotipiche di DC mature. La loro capacità allostimolatoria, invece, non è risultata ulteriormente incrementare a causa della produzione endogena di IL-10. Infatti, una più alta risposta proliferativa di cellule T e produzione di IL-12 da parte delle stesse DC-like attivate è stata osservata solo in seguito al blocco dell’IL-10 endogena indotta da LPS. Al contrario, l’IL-10 endogena non è stata vista avere effetti sulla secrezione di IL-23. Questi risulatati sottolineano l’importanza dell’espressione del GM-CSFR in monociti per la generazione di DC indotta da citochine e guardano al GM-CSF come ad un diretto protagonista nella generazione di DC funzionalmente distinte.
Myeloid dendritic cells (DCs) and macrophages evolve from a common precursor. However, factors controlling monocyte differentiation toward DC or macrophage are poorly defined. We report that surface density of GM-CSF receptor (GM-CSFR) α subunit in human peripheral blood monocytes varies among donors. Although no correlation was found between the extent of GM-CSFR and monocyte differentiation into DC driven by GM-CSF and IL-4, GM-CSFR expression strongly influenced the generation of CD1a+ dendritic-like cells in the absence of IL-4. CD1a+ cells generated in the presence of GM-CSF express CD40, CD80, MHC I and II, DC-SIGN, MR, CCR5, and partially retain CD14 expression. Interestingly, they spontaneously induce the expansion of CD4+ and CD8+ allogeneic T lymphocytes producing IFN-gamma, and migrate toward CCL4 and CCL19. Upon stimulation with TLR ligands, they acquire the phenotypic features of mature DCs. In contrast, the allostimulatory capacity is not further increased upon LPS activation. However, by blocking LPS-induced IL-10, a higher T cell proliferative response and IL-12 production was observed. Interestingly, IL-23 secretion was not affected by endogenous IL-10. These results highlight the importance of GM-CSFR expression in monocytes for cytokine-induced DC generation and point to GM-CSF as a direct player in the generation of functionally distinct DCs.

Clostridium difficile is a Gram positive bacterium rarely present in normal human gut flora that under certain conditions of intestinal dysbiosis, in patients treated with broad-spectrum antibiotics, in hospitalized patients, in immunocompromised subjects and elderly, can cause disease of variable severity referred to as Clostridium Difficile Associated Diarrohea (CDAD). Although in the past Clostridium difficile has been indicated as a possible causal factor in the development of inflammatory bowel disease (also known as IBD), nowadays it is more likely to believe that the IBD may be a risk factor for Clostridium difficile infection (CDI). CDI in patients with IBD is of increasing importance because the frequency with which it occurs is growing over time, but also because it seems to have a negative impact on health outcomes and because the symptoms induced CDI are indistinguishable from that of an exacerbation of IBD: it is therefore essential to establish an early diagnosis in order to start the most suitable treatment of the case. The aim of this study was to describe the frequency of the CDI in healthy subjects, subjects not affected by IBD hospitalized with suspected CDAD and patients with IBD, characterize strains of Clostridium difficile isolated from IBD patients (sensitivity to antibiotics, types of toxins, adhesion to the intestinal epithelium), to identify risk factors for CDI in IBD patients (characteristics of the subject, illness, concomitant therapy) and to assess the impact of CDI on the course of IBD, both in symptomatic and asymptomatic carriers. From January 2010, stool samples from IBD outpatients and inpatients were collected and analyzed at the Gastroenterology unit of the University Hospital of Padua (both in the acute phase of disease and in remission), from patients admitted to the same unit without IBD but with symptoms and medical treatment suggestive of CDAD and from a control group of healthy subjects matched for age and sex. From the first evaluation (and the collection of the first stool sample), patients with IBD were evaluated at least every six months or in case of relapse or hospitalization for two years. On each sample an anaerobic culture was performed, followed by specific PCR to identify any colonies of Clostridium difficile. Each strain was then characterized by: - Toxins production - Antibiotics sensitivity - Adhesion to Caco-2 cells - Presence or absence of the tcdC gene in bacterial DNA Clinical data were collected from patients with IBD to identify any risk factors for CDI. Patients with IBD have a higher frequency of colonization by Clostridium difficile than the control group: in healthy subjects CDI was detected in 0/55 subjects, in hospitalized IBD patients was found in 5/55 patients (9%) and in IBD outpatients was detected in 9/195 subjects (4.6%). The production profile of the toxins appears to be different in IBD and non-IBD patients with antibiotic-associated diarrhea, confirming the hypothesis of community-acquired strains rather than hospital-acquired. Antibiotics sensitivity tests performed on strains isolated from patients with CDAD and patients with IBD showed that all strains are sensitive to metronidazole and vancomycin and markedly resistant to ciprofloxacin. Strains of Clostridium difficile isolated from patients with active IBD, in remission and from patients with CDAD have shown a different, albeit small, ability to adhere to monolayers of human intestinal epithelial cells (Caco-2), suggesting that strains from active IBD patients have a greater ability to colonize than those from patients in remission. The tcdC gene was identified in 8% of toxigenic strains isolated from IBD patients (active and in remission) and in 25% of those isolated from patients with CDAD, but the genome had deletions of varying extent, indicating a potential increased virulence of identified strains. The statistical analysis did not identify any risk factor associated with CDI in IBD. In the prospective study, CDI has not been identified as a risk factor for clinical or endoscopic relapse or for the need for surgical treatment, demonstrating instead, unexpectedly, to have a protective role against disease flare.
Il Clostridium difficile è un batterio Gram positivo raramente presente nella normale flora intestinale umana che in particolari condizioni di disbiosi intestinale, in pazienti trattati con antibiotici ad ampio spettro, in pazienti ospedalizzati, in soggetti immunocompromessi e in persone anziane, può causare patologie di variabile gravità indicate complessivamente come Clostridium difficile Associated Diarrohea (CDAD). Sebbene in passato il Clostridium difficile sia stato indicato come possibile concausa dello sviluppo delle malattie infiammatorie croniche intestinali (note anche come inflammatory bowel disease, IBD), oggi si è più propensi a ritenere che le IBD possano essere un fattore di rischio per l’infezione da Clostridium difficile (CDI). La CDI nei pazienti affetti da IBD riveste una sempre maggiore importanza, sia perché la frequenza con cui si presenta stà crescendo nel tempo, sià perché sembra determinare un impatto negativo sugli outcome di salute, ma anche perché la sintomatologia indotta dalla CDI è indistinguibile da quella di una riacutizzazione della IBD: è quindi fondamentale una diagnosi tempestiva per instaurare le terapie più idonee al trattamento del caso. Lo scopo dello studio è descrivere la frequenza della CDI in soggetti sani, soggetti non affetti da IBD ospedalizzati con sospetto di CDAD e soggetti affetti da IBD, caratterizzare i ceppi di Clostridium difficile isolati da pazienti IBD (sensibilità agli antibiotici, tipologie di tossine prodotte, capacità di adesione all’epitelio intestinale), identificare i fattori di rischio per la CDI nei pazienti IBD (caratteristiche del soggetto, della malattia, della terapia concomitante) e valutare l'impatto della CDI sul decorso della IBD, sia nei portatori sintomatici che in quelli asintomatici. Da gennaio 2010 sono stati raccolti ed analizzati campioni da pazienti IBD ambulatoriali o degenti presso l’unità operativa complessa di gastroenterologia dell’Azienda Ospedaliera di Padova (sia in fase acuta di malattia che in remissione), da pazienti ricoverati presso la medesima unità operativa non affetti da IBD con sintomi e terapia medica suggestivi di CDAD e da un gruppo di controllo di soggetti sani appaiati per età e sesso. Dalla prima valutazione (e della raccolta del primo campione) i pazienti con IBD sono stati valutati almeno ogni sei mesi o in caso di recidiva o di ricovero ospedaliero per due anni. Su ogni campione è stata eseguita una coltura anaerobica seguita da PCR specifica per identificare eventuali colonie di C. difficile. Ogni ceppo è stato poi caratterizzato in base a: - tossine prodotte - sensibilità agli antibiotici - adesione alle cellule Caco-2 - presenza o assenza del gene tcdC nel DNA batterico Dati clinici sono stati raccolti dai pazienti con IBD per identificare eventuali fattori di rischio per la CDI. I pazienti con IBD sembrano presentare una maggiore frequenza di colonizzazione da parte del Clostridium difficile rispetto al gruppo di controllo dei soggetti sani: nei controlli la CDI è stata rilevata in 0/55 soggetti. Nei pazienti ricoverati con IBD è stata trovata in 5/55 soggetti (9%). Nei pazienti ambulatoriali è stata rilevata in 9/195 soggetti (4,6%). Il profilo di produzione delle tossine sembra essere differente nei pazienti IBD e nei pazienti non-IBD con diarrea da antibiotici, confermando l'ipotesi di ceppi acquisiti in comunità e non in ambiente ospedaliero. L’antibiogramma eseguito su ceppi isolati da pazienti con CDAD e pazienti con IBD attive o in remissione ha mostrato che tutti i ceppi sono sensibili a metronidazolo e vancomicina e marcatamente resistenti alla ciprofloxacina. Ceppi di Clostridium difficile isolati da pazienti con IBD attive, in fase di remissione e da pazienti con CDAD hanno dimostrato una diversa, seppur piccola, capacità di aderire a monostrati di cellule epiteliali intestinali umane (CACO-2), indicando che i ceppi associati ai pazienti con IBD attive hanno maggiore abilità a colonizzare di quelli in remissione. Il gene tcdC è stato identificato nell’8% dei ceppi tossigenici isolati da pazienti IBD (attivi ed in remissione) e nel 25% di quelli isolati da pazienti con CDAD, ma il genoma presentava delezioni di varia entità, indicando una potenziale aumentata virulenza dei ceppi identificati. L'analisi statistica non ha individuato fattori di rischio associati con la CDI. Nella parte prospettica dello studio la CDI non è stata identificata come fattore di rischio per la recidiva clinica o endoscopica o per la necessità di trattamento chirurgico, dimostrando invece, inaspettatamente, di avere un ruolo protettivo nei confronti della riacutizzazione della malattia.

Il processo infiammatorio è un meccanismo di difesa aspecifico innato, che costituisce una risposta protettiva dell’organismo a vari tipi di insulto (infezione, danno tissutale, trauma, stress, malattie autoimmune,). Esso comporta il rilascio in circolo di mediatori pro-infiammatori (es. citochine) and anti-infiammatori (es lipossine ed alcune citochine). Le citochine pro-infiammatorie inducono effetti infiammatori (es. anoressia e febbre) e stimolano la risposta di fase acuta (APR). Invece, le lipossine e le citochine anti-infiammatorie tendono ad attenuare l’infiammazione. Gli scopi di questa ricerca erano due: distinguere i soggetti in base al grado di severità della APR dopo il parto, e dopo stimolazione intramammaria con lipopolisaccaride (LPS) in bovine da latte sottoposte a diversi stress metabolici (NaCl, BHB, EuG e IpoG). I soggetti EuG e BHB hanno mostrato una APR più severa rispetto a IpoG e NaCl. Un ulteriore scopo è stato proposto un indice composto da alcune proteine di fase acuta al fine di stimare i processi infiammatori e le conseguenze epatiche (PICE). Le bovine con PICE più basso prima del parto, avevano più alti livelli plasmatici di citochine pro-infiammatorie e lipossine prima del parto (e mostravano una APR più severa dopo il parto), anche in assenza di sintomi clinici.
Inflammation is the innate, non-specific response of the host to disturbances in his homeostasis caused by infection, tissue injury, stress, trauma, neoplastic growth, immunological disorders. It involves pro- (e.g. cytokines) and anti-inflammatory mediators (e.g. lipoxins, some cytokines). The pro-inflammatory cytokines induce inflammatory effects (e.g. anorexia, fever) and play key roles in the stimulation of acute phase response (APR). The lipoxins and anti-inflammatory cytokines tend to mitigate the inflammation. Two were the aims of this research: to investigate in dairy cows the severity of APR at calving time as well as after intramammary lypopolysaccharide (LPS) administration in cows challenged with hyperinsulinemic hypoglycemic (HypoG, n=4), hyperinsulinemic euglycemic (EuG, n=5), hyperketonemic (BHB, n=4) and control (NaCl, n=6) clamps. Plasma samples were assayed for a wide metabolic and inflammatory profile. With respect to HypoG and NaCl animals, more severe APR was observed in EuG and BHB. A further aim was the proposal of an Index, composed by several acute phase proteins, to estimate Inflammatory Processes and Hepatic Consequences (IPHC). The dairy cows with lower IPHC after calving, had higher plasma levels of pro-inflammatory cytokines and of lipoxins before calving (and showed a stronger APR after calving); this was seen also in absence of clinical symptoms.

Il processo infiammatorio è un meccanismo di difesa aspecifico innato, che costituisce una risposta protettiva dell’organismo a vari tipi di insulto (infezione, danno tissutale, trauma, stress, malattie autoimmune,). Esso comporta il rilascio in circolo di mediatori pro-infiammatori (es. citochine) and anti-infiammatori (es lipossine ed alcune citochine). Le citochine pro-infiammatorie inducono effetti infiammatori (es. anoressia e febbre) e stimolano la risposta di fase acuta (APR). Invece, le lipossine e le citochine anti-infiammatorie tendono ad attenuare l’infiammazione. Gli scopi di questa ricerca erano due: distinguere i soggetti in base al grado di severità della APR dopo il parto, e dopo stimolazione intramammaria con lipopolisaccaride (LPS) in bovine da latte sottoposte a diversi stress metabolici (NaCl, BHB, EuG e IpoG). I soggetti EuG e BHB hanno mostrato una APR più severa rispetto a IpoG e NaCl. Un ulteriore scopo è stato proposto un indice composto da alcune proteine di fase acuta al fine di stimare i processi infiammatori e le conseguenze epatiche (PICE). Le bovine con PICE più basso prima del parto, avevano più alti livelli plasmatici di citochine pro-infiammatorie e lipossine prima del parto (e mostravano una APR più severa dopo il parto), anche in assenza di sintomi clinici.
Inflammation is the innate, non-specific response of the host to disturbances in his homeostasis caused by infection, tissue injury, stress, trauma, neoplastic growth, immunological disorders. It involves pro- (e.g. cytokines) and anti-inflammatory mediators (e.g. lipoxins, some cytokines). The pro-inflammatory cytokines induce inflammatory effects (e.g. anorexia, fever) and play key roles in the stimulation of acute phase response (APR). The lipoxins and anti-inflammatory cytokines tend to mitigate the inflammation. Two were the aims of this research: to investigate in dairy cows the severity of APR at calving time as well as after intramammary lypopolysaccharide (LPS) administration in cows challenged with hyperinsulinemic hypoglycemic (HypoG, n=4), hyperinsulinemic euglycemic (EuG, n=5), hyperketonemic (BHB, n=4) and control (NaCl, n=6) clamps. Plasma samples were assayed for a wide metabolic and inflammatory profile. With respect to HypoG and NaCl animals, more severe APR was observed in EuG and BHB. A further aim was the proposal of an Index, composed by several acute phase proteins, to estimate Inflammatory Processes and Hepatic Consequences (IPHC). The dairy cows with lower IPHC after calving, had higher plasma levels of pro-inflammatory cytokines and of lipoxins before calving (and showed a stronger APR after calving); this was seen also in absence of clinical symptoms.

Il nostro gruppo di ricerca lavora da diversi anni nel campo degli inibitori dell'elastasi dei neutrofili umani (HNE), e in questo periodo è stata progettata e sintetizzata un'ampia varietà di strutture bi-eterocicliche azotate, come indazoli, indoli, cinnoline e 7-azaindoli; inoltre, abbiamo anche studiato strutture mono-eterocicliche azotate, come isossazoloni e tiazoloni, ottenendo interessanti risultati. Il primo progetto di questa tesi di dottorato è stata divisa in due sezioni: nella prima parte sono stati progettati nuovi HNE inibitori come ulteriore modifica di indazoli e 7-azaindoli, sintetizzati in precedenza, con l'obiettivo di studiare l'impatto che, sia lo spostamento dell'azoto nell'anello piridinico, sia il numero totale degli azoti nelle molecole, possono avere sull'attività biologica. Quindi abbiamo esplorato inibitori 4-, 5- e 6-azaindolici, come risultato dello spostamento dell'azoto della piridina nel 7-azaindolo, inibitori a struttura 5H-pirrolo[2,3-b]pirazinica, inserendo un atomo di azoto in posizione 4 del nucleo 7-azaindolico, e 5- e 7-azaindazoli (1H-pirazolo[3,4-b]piridina e 1H-pirazolo[4,3-c]piridina) come aza-analoghi dei potenti indazoli. Nella seconda parte di questo primo progetto abbiamo studiato tre nuovi scaffold: 1,5,6,7-tetraidro-4H-indazol-4-one, 5,6-diidrociclopenta[c]pirazolo-4(1H)-one e 5,6,7,8-tetraidrocyclohepta[c]pirazol-4(1H)-one come inibitori di HNE. Inoltre, il nostro gruppo di ricerca ha anche sintetizzato per lungo tempo agonisti dei recettori dei peptidi formilati (FPRs) con scaffold piridazinonico, ottenendo risultati interessanti per attività e selettività; d'altra parte, molti derivati del piridazinone sono noti per esibire un effetto anti-infiammatorio, come ampiamente documentato in letteratura, interagendo con diversi target. Quindi, tenendo conto della nostra esperienza sugli agonisti FPR e delle preziose informazioni riportate in letteratura, nel secondo progetto di questa tesi di dottorato abbiamo selezionato una piccola libreria di agonisti FPR che sono stati ulteriormente studiati per capire se possono presentare un'attività anti-infiammatoria alternativa, che non è correlata alla loro attività FPR. Our research group has been working for several years in the field of human neutrophil elastase (HNE) inhibitors, and over this period a wide variety of nitrogen bi-heterocycle scaffolds have been designed and synthesized, such as indazoles, indoles, cinnolines and 7-azaindoles; moreover, we also investigated nitrogen mono-heterocyclic scaffolds such as isoxazolones and thiazolones obtaining interesting results. The first project of this PhD thesis has been divided in two sections: in the first part new HNE inhibitors have been designed as further modification of both indazoles and 7-azaindoles, synthetized previously, with the goal to investigate the impact that either the shift of the nitrogen in the pyridine ring as well as the total number of nitrogens in the molecules can have on the biological activity. Hence, we explored 4-, 5- and 6-azaindoles as the result of the 7-azaindole pyridine nitrogen shift, 5H-pyrrolo[2,3-b]pyrazines by inserting a nitrogen atom at position 4 of the 7-azaindole nucleus, and 5- and 7-azaindazoles (1H-pyrazolo[3,4-b]pyridine and 1H-pyrazolo[4,3-c]pyridine) as aza-analogues of the potent indazoles. As second part of this first project, we have investigated three new scaffolds: 1,5,6,7-tetrahydro-4H-indazol-4-one, 5,6-dihydrocyclopenta[c]pyrazol-4(1H)-one and 5,6,7,8-tetrahydrocyclohepta[c]pyrazol-4(1H)-one as HNE inhibitors. Furthermore, our research group has also been synthesizing for a long time formyl peptide receptors (FPRs) agonist with pyridazinone scaffold, obtaining interesting results for activity and selectivity; on the other hand, many pyridazinone derivatives are known to exhibit an anti-inflammatory effect, as widely documented in the literature, by interacting with different targets. Thus, taking into account our experience on FPR agonists and the valuable information reported in the literature, in the second project of this PhD thesis we have selected a small library of FPR agonists that were further investigated to understand wether they may present an alternative anti-inflammatory activity, which is not correlated to their FPR activity.

Background: The inflammatory bowel diseases (IBD) are chronic relapsing inflammatory disorders of the gastrointestinal tract, comprising Crohn’s disease (CD), ulcerative colitis (UC), and IBD-unclassified (IBD-U). The microbiome, the barrier function, and the immune system play an integrated role in the development of IBD, and all three components are likely critical for perpetuating the disease process. Aims and project: In this study we have preliminary investigated some specific aspects of the IBD pathogenesis including the intestinal dysbiosis, the alteration of the intestinal barrier and the effect of a specific dietetic treatment (the so-called exclusive enteral nutrition, EEN). Methods: IBD pediatric patients were enrolled in the period 2013-2015. The study included 3 type of analysis: 1. Microbiota study: analysis of fecal microbiota in IBD in general (through the amplification of the V3-V4 regions of the 16S rRNA gene); 2. EEN study: evaluation of the clinical, laboratory and microbiological changes induced by EEN; 3. IPT study: evaluation of the intestinal barrier damage in IBD patients by the intestinal permeability test (IPT). Results: preliminary results of the microbiota study showed a different prevalence in some specific phyla in CD patients compared to UC. Such changes were not correlated to the level of inflammation. The results of the EEN study support the efficacy of EEN in the treatment of CD and preliminary results show a shift in microbiota composition after a course of EEN. IPT was found to be a sensitive test to detect disease activity and to evaluate response to treatments. Conclusion: Our study has generated novel and intriguing data especially with regards to microbiota changes secondary to EEN and to the modification of intestinal permeability following specific treatments.