Defensins are small cationic peptides with antibacterial activity expressed in Paneth cells (α-defensins) or generally in intestinal epithelial cells (β-defensins) that have a profound effect on gut ...microbiota. Chronic pouchitis, which occurs in 5% of patients after restorative proctocolectomy and can cause pouch failure, is associated to a significant increase of Clostridiaceae spp. The aim of this study was to gain further insight in the pathogenesis of pouch dysbiosis by exploring defensin expression. Thirty-two consecutive patients coming for follow-up endoscopy were recruited. On pouch biopsies, we cultured bacteria adherent to the mucosa and determined α- and β-defensins and toll-like receptor-4 and −2 mRNA by quantitative real-time RT-PCR. Serum and mucosal levels of IL-1β, IL-6 and TNF-α were measured with immunometric assays. Faecal lactoferrin was analysed by quantitative ELISA. After a median follow-up of 23 (IQR 20–24) months, the patients were contacted for a reassessment of current and past disease activity. During the follow-up, chronic/relapsing pouchitis was diagnosed in six patients. The mucosal level of α-5 and α-6 defensins correlated with chronic/relapsing pouchitis onset (
τ
= 0.30,
p
= 0.034 and
τ
= 0.28,
p
= 0.053, respectively). High levels of α-5 defensin resulted to be predictive of chronic/relapsing pouchitis AUC = 74% (95% CI = 53–89%),
p
= 0.052. Patients with high levels of α-5 and α-6 defensins had earlier pouchitis relapses (
p
= 0.009 and
p
= 0.034, respectively). High levels of α-5 defensin were associated to a significant risk of chronic/relapsing pouchitis OR = 10.6 (95% CI = 1.2–97.6),
p
= 0.027. At multivariate analysis, the mucosal levels of α-5 defensin and the number of CFU of mucosa-associated Clostridiaceae spp resulted to be independent predictors of chronic/relapsing pouchitis
β
= 0.46 (0.18),
p
= 0.024 and
β
= 0.44 (0.18),
p
= 0.027, respectively. In conclusion, chronic/relapsing pouchitis is associated to increased expression of mucosal HD-5 and to increased antimicrobial activity against
Escherichia coli
. In patients with chronic/relapsing pouchitis, HD-5 and TLR-4 over-expression is likely to create a hostile environment against Enterobacteriaceae, thus favouring Clostridiaceae spp by decreasing competing bacteria families.
Background. Nonselective beta-blockers (NSBB) have been associated with increased incidence of paracentesis-induced circulatory dysfunction (PICD) and reduced survival in patients with cirrhosis and ...refractory ascites. Aim. To prospectively evaluate a hemodynamic response to NSBB in cirrhotics listed for liver transplantation with refractory ascites undergoing large volume paracentesis (LVP). Methods. Patients with cirrhosis and refractory ascites, with an indication to start NSBB in primary prophylaxis for variceal bleeding, were enrolled. During two consecutive LVP, while being, respectively, off and on NSBB, cardiac output (CO), systemic vascular resistances (SVR), peripheral vascular resistances (PVR), and plasma renin activity (PRA) were noninvasively assessed. Results. Seventeen patients were enrolled, and 10 completed the study. Before NSBB introduction, SVR (1896 to 1348 dyn x s x cm.sup.-5; p = 0.028) and PVR (47 to 30 mmHg x min x dl x ml.sup.-1; p = 0.04) significantly decreased after LVP, while CO showed an increasing trend (3.9 to 4.5 l/m; p = 0.06). After NSBB introduction, LVP was not associated with a significant increase in CO (3.4 to 3.8 l/m; p = 0.13) nor with a significant decrease in SVR (2002 versus 1798 dyn x sx cm.sup.-5; p = 01). Incidence of PICD was not increased after NSBB introduction. Conclusion. The negative inotropic effect of NSBB was counterbalanced by a smaller decrease of vascular resistances after LVP, probably due to splanchnic beta2- blockade. This pilot study showed that NSBB introduction may be void of detrimental hemodynamic effects after LVP in cirrhotics with refractory ascites.
Defensins are small cationic peptides with antibacterial activity expressed in Paneth cells ( alpha -defensins) or generally in intestinal epithelial cells ( beta -defensins) that have a profound ...effect on gut microbiota. Chronic pouchitis, which occurs in 5% of patients after restorative proctocolectomy and can cause pouch failure, is associated to a significant increase of Clostridiaceae spp. The aim of this study was to gain further insight in the pathogenesis of pouch dysbiosis by exploring defensin expression. Thirty-two consecutive patients coming for follow-up endoscopy were recruited. On pouch biopsies, we cultured bacteria adherent to the mucosa and determined alpha - and beta -defensins and toll-like receptor-4 and -2 mRNA by quantitative real-time RT-PCR. Serum and mucosal levels of IL-1 beta , IL-6 and TNF- alpha were measured with immunometric assays. Faecal lactoferrin was analysed by quantitative ELISA. After a median follow-up of 23 (IQR 20-24) months, the patients were contacted for a reassessment of current and past disease activity. During the follow-up, chronic/relapsing pouchitis was diagnosed in six patients. The mucosal level of alpha -5 and alpha -6 defensins correlated with chronic/relapsing pouchitis onset ( tau =0.30, p=0.034 and tau =0.28, p=0.053, respectively). High levels of alpha -5 defensin resulted to be predictive of chronic/relapsing pouchitis AUC=74% (95% CI=53-89%), p=0.052. Patients with high levels of alpha -5 and alpha -6 defensins had earlier pouchitis relapses (p=0.009 and p=0.034, respectively). High levels of alpha -5 defensin were associated to a significant risk of chronic/relapsing pouchitis OR=10.6 (95% CI=1.2-97.6), p=0.027. At multivariate analysis, the mucosal levels of alpha -5 defensin and the number of CFU of mucosa-associated Clostridiaceae spp resulted to be independent predictors of chronic/relapsing pouchitis beta =0.46 (0.18), p=0.024 and beta =0.44 (0.18), p=0.027, respectively. In conclusion, chronic/relapsing pouchitis is associated to increased expression of mucosal HD-5 and to increased antimicrobial activity against Escherichia coli. In patients with chronic/relapsing pouchitis, HD-5 and TLR-4 over-expression is likely to create a hostile environment against Enterobacteriaceae, thus favouring Clostridiaceae spp by decreasing competing bacteria families.
Abstract
Medullary thyroid cancer (
MTC
) is an aggressive malignancy responsible for up to 14% of all thyroid cancer‐related deaths. It is characterized by point mutations in the rearranged during ...transfection (
RET
) proto‐oncogene. The activated
RET
kinase is known to signal
via
extracellular signal regulated kinase (
ERK
) and phosphoinositide 3‐kinase (
PI
3K), leading to enhanced proliferation and resistance to apoptosis. In the present work, we have investigated the effect of two serine/threonine‐protein kinase B‐Raf (BRAF) inhibitors (
RAF
265 and
SB
590885), and a
PI
3K inhibitor (
ZSTK
474), on
RET
‐mediated signalling and proliferation in a
MTC
cell line (
TT
cells) harbouring the
RETC
634W
activating mutation. The effects of the inhibitors on
VEGFR
2,
PI
3K/Akt and mitogen‐activated protein kinases signalling pathways, cell cycle, apoptosis and calcitonin production were also investigated. Only the
RAF
265+
ZSTK
474 combination synergistically reduced the viability of treated cells. We observed a strong decrease in phosphorylated
VEGFR
2 for
RAF
265+
ZSTK
474 and a signal reduction in activated Akt for
ZSTK
474. The activated
ERK
signal also decreased after
RAF
265 and
RAF
265+
ZSTK
474 treatments. Alone and in combination with
ZSTK
474,
RAF
265 induced a sustained increase in necrosis. Only
RAF
265, alone and combined with
ZSTK
474, prompted a significant drop in calcitonin production. Combination therapy using
RAF
265 and
ZSTK
47 proved effective in
MTC
, demonstrating a cytotoxic effect. As the two inhibitors have been successfully tested individually in clinical trials on other human cancers, our preclinical data support the feasibility of their combined use in aggressive
MTC
.
Breast-fed infants have higher bilirubin levels than formula-fed infants, possibly because of variations in the composition of the breast milk. The aim of this study was to investigate whether there ...is a relationship between cytokine levels in the colostrum of nursing mothers and neonatal jaundice (NJ). Breast milk samples were collected from breast-feeding mothers of healthy full-term neonates, 32 with NJ and 29 without jaundice. The concentrations of IL-1beta, IL-6, IL-8, IL-10, and tumor necrosis factor (TNF)-alpha were measured by chemiluminescence enzyme immunometric assays. Mothers of infants with NJ had a higher concentration of IL-1beta in colostrum, compared with those feeding neonates without NJ, and similar trends were seen for IL-6, IL-8, IL-10, and for TNF-alpha. The concentrations of IL-1beta significantly correlated with IL-6, IL-8, IL-10, and TNF-alpha concentrations, but not with serum bilirubin levels of infants with NJ. In conclusion, the concentrations of IL-1beta were increased in colostrum from breast-feeding mothers whose infants had NJ. The correlation between the concentrations of cytokines involved in the function of hepatic uptake and excretory systems and in the enterohepatic circulation of bilirubin provides additional data to the delineation of the cascade of pathophysiological events that can lead to NJ.
Abstract Background In patients with ulcerative colitis (UC) the inconsistency between the rate of dysplasia and actual cancer incidence suggests the presence of an immunosurveillance mechanism. The ...aim of our study was to analyse the expression of CD80 and CD86 during the different stages of UC-associated and in non-inflammatory carcinogenesis. Patients and methods Sixty-two patients affected with UC, UC with colonic dysplasia, UC and cancer, colonic adenoma, or colonic cancer and 11 healthy subjects were enroled in our study. Tissue samples were taken from surgical specimens during colonic resection or during colonoscopy. Mucosal mRNA expression of CD80 and CD86 was quantified with real time polymerase chain reaction (RT-PCR). CD80, CD86 and p53 expressions and lamina propria mononuclear cell populations (CD3, CD20 and CD68) were analysed by immunohistochemistry. Mucosal levels of IL-1β, IL-2 and IFN-γ were measured with immunometric assays. Results Among UC patients, CD80 protein expression was higher in those with dysplasia ( p = 0.017). In non-inflammatory carcinogenesis pathway CD80 protein and mRNA expressions were lower compared to the corresponding steps in the UC pathway. CD80 expression was directly correlated with the lamina propria mononuclear cell populations (T and B lymphocytes and monocytes). CD80 protein, but not CD80 mRNA, expression was significantly and directly correlated with IL-2 expression. Conclusion CD80 resulted to be up-regulated in UC with dysplasia, while it was down-regulated in cancer. CD80 mucosal levels correlate with lamina propria T-cell and with IL-2 expression suggesting that it may elicit an active role in the immunosurveillance mechanism.
Background/Aims
Fecal lactoferrin is the direct expression of intestinal inflammation in Crohn’s disease (CD). The aim of this study was to analyze the in vivo intimate correlation between intestinal ...and systemic inflammation in CD patients in clinical remission following bowel resection. The secondary end point was to evaluate the prognostic value of lactoferrin levels and serum cytokines in terms of need of surgery for recurrence in these patients.
Patients and Methods
Fecal lactoferrin and serum cytokine (interleukin (IL)-1β, IL-6, IL-12, tumor necrosis factor (TNF)-α, and transforming growth factor (TGF)-β1) levels were assessed; hematological and biochemical investigations were carried out, and Crohn’s Disease Activity Index was evaluated in the 36 patients who had undergone bowel resection. The prognostic value of lactoferrin and cytokine levels in terms of surgical recurrence was assessed by re-calling patients after 24 months from the enrolment in the study.
Results
All patients, evaluated after a follow-up of 36 ± 5 months, were in clinical remission. Fecal lactoferrin levels were found to be significantly correlated with IL-6 (
R
= 0.431,
p
= 0.025) and C-reactive protein (CRP;
R
= 0.507,
p
= 0.007), while no correlation was observed between lactoferrin and IL-1β, IL-12, TNF-α, or TGF-β1. Reoperation for anastomotic recurrence tended to occur significantly more frequently in patients with higher IL-6 (
p
= 0.10).
Conclusions
Subclinical intestinal inflammation, expressed by fecal lactoferrin, seems to keep the systemic inflammation alive in CD patients through the IL-6-CRP cascade. IL-6 seems to be predictive of the outcome of CD patients undergoing surgery.
Chronic pouchitis, which can lead to pouch failure, occurs in approximately 5% of patients after restorative proctocolectomy for ulcerative colitis (UC). This work examined the interplay between the ...microbiota adherent to the ileal pouch mucosa and the mucosal immune system in chronic/relapsing pouchitis.
Thirty-two consecutive patients attending our surgical gastroenterological department following restorative proctocolectomy with ileal pouch-anal anastomosis (IPAA) for UC were considered eligible candidates for this study. Biopsy samples of bacteria adherent to the mucosa were collected. TLR4 and TLR2 mucosal expression was measured by Real Time RT-PCR. Serum and mucosal IL-1β, IL-6, and TNF-α levels were assessed using immunometric assays. Fecal lactoferrin concentrations were determined by quantitative ELISA. After a median follow-up of 23 months (IQR 20-24 months) each patient underwent a global assessment of their clinical condition and disease activity status.
Six patients were diagnosed with relapsing/chronic pouchitis during the follow-up period. Mucosal TLR2 and TLR4 expression was higher in the chronic/relapsing pouchitis group than in the no or only one episode of pouchitis group (P = 0.036 and P = 0.016, respectively). The number of colony forming units (CFU) of mucosa-associated Clostridiaceae spp. was higher in the former than in the latter group (P = 0.031). Clostridiaceae were associated to a significant risk of chronic/relapsing pouchitis OR: 14 (95% CI 0.887-224.021), P = 0.045.
Chronic/relapsing pouchitis is associated to higher mucosal TLR2 and TLR4 expression. Mucosal colonization by Clostridiaceae spp seems to play a role in the pathogenesis of chronic/relapsing pouchitis.
Abstract 5193
Essenthial thrombocythemia (ET) patients are at risk of developing an arterious or venous thrombotic event. Recent studies have shown that there is no clear association between platelet ...(PLT) count and thrombotic events, but that white cell count plays a role. Qualitative PLT abnormalities, activation of endothelial cells (EC) and PLT may be involved in these processes. Following activation of blood cells, plasma membrane vescicles are released, these are known as microparticles (MP) (<1. 5I′¼m of size). They may originate from PLT (PMP), EC (EMP) or red cells (RMP). MP may affect endothelial modulators such as vasorelaxant nitric oxide (NO), adrenomedullin (ADM) and endothelin-1 (ET-1), a potent vasocostrictor agent. Previous studies Trappenburg MC et al 2009 demonstrated that MP are present in elevated numbers in ET. However, the relationship between MP with treatment and MP with endothelial modulators is unknown. We investigated the relationship between MP, NO, ADM and ET-1 in ET patients on treatment with hydroxyurea (HU), anagrelide (AN) and aspirin(ASA) only. 52 patients with ET diagnosis were studied: 18 on HU, 15 on AN, and 19 on ASA. Platelet-poor-plasma aliquotes were stored at −80A′°C and subsequently processed. Samples were analysed for MP numbers (absolute total values) and functional markers (percentage values) by flow cytometry, as previously described Jy W et al JTH 2004. PMP was identified using CD62P, CD36, and CD63, EMP using CD105 and RMP using CD235a. Endothelial modulator markers NO, ADM and ET-1 were measured by ELISA. Statistical analysis included Kruskal-Wallis test, Bonferroni test, correlation matrix, principal component (PCA) and partial least square regression analysis (PLSR).
Total-MP were increased in all patients groups compared to controls (p<0. 01). Strong correlations were seen for tot-MP and %MP CD62P (R=0. 89, p<0. 05), tot-MP and %MPCD36 (R=0. 97, p<0. 05), tot-MP CD62P+ and CD36+ (R=0. 85, p<0. 05) and for tot -MP TF+ and %MP CD105+ (R=0. 66, p<0. 05). Bonferroni test, showed higher MP total number in the AN group compared to the HU and the ASA group (p <0. 05). The percentage of CD63 MP was higher than control both in the HU and in the AN group (p <0. 05), while CD36 MP% was higher in the AN group compared to the HU group (p <0. 05). NO and ADM values were higher in the HU group, while ET-1 values were lower in the AN group (p <0. 05). The principle components (PC) responsible for value variations were PC1 positive loading for WCC, PMP and EMP reflecteing PLT and EC activation, and PC4 positive loadings for PLT, PMP; negative loadings for RMP, NO. Both PC1 and PC4 were statistically significant for treatment.
This study confirms previous findings that MP are present in ET. We also showed that MP are affected by treatment and that MP are mainly PMP and express CD62P and CD36, a marker of immature PLT, which suggests that reticulated PLT partecipate in these processes. We also showed that HU increase NO and ADM levels, while AN reduces ET-1. As NO and ET-1 have antagonistic actions on endothelial cells, we suggest that HU and AN may have overlapping effects on endothelium, although using different biochemichal pathways.
Display omitted
The finding that treatment influences PC1 (PMP and EMP) supports the idea that in ET, treatment impacts directly on MP. The finding that NO and RMP have a negative loading within PC4, suggests that they have an antagonist-action to PMP and EMP in ET.
Pizzolo:Hoffmann-La Roche: Consultancy, Honoraria.
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