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L Piemonti

Publications and source records attributed to L Piemonti.

At least 19 recordsLinked to original sources
PubMed2006

Increased intestinal permeability precedes clinical onset of type 1 diabetes.

AIMS/HYPOTHESIS: Recent observations have shown subclinical intestinal abnormalities in human type 1 diabetes. Whether these are related to the pathogenetic process or secondary to the diabetes remains to be clarified. The aim of this study was to investigate this issue by examining intestinal permeability to sugars in subjects at different stages of type 1 diabetes: preclinical, new-onset and long-term established disease. METHODS: Eighty-one subjects with islet autoimmunity (18 preclinical, 28 new-onset and 35 long-term type 1 diabetes) and 40 healthy control subjects were investigated by a lactulose-mannitol test, consisting of oral administration of the two sugars and measurement of their urinary excretion. RESULTS: All groups of subjects with islet autoimmunity showed an increase in intestinal permeability (p < or = 0.009 vs controls) to the disaccharide lactulose, indicative of a damaged barrier, but a similar permeability to the monosaccharide mannitol (NS vs controls), indicative of an integral surface mucosa; consequently there was an increase in the lactulose:mannitol excretion ratio (p < or = 0.025 vs controls). CONCLUSIONS/INTERPRETATION: These findings indicate the presence of a subclinical enteropathy associated with type 1 diabetes that is already detectable before clinical onset of the disease, and suggest that the small intestine is an organ participating in the pathogenetic process of type 1 diabetes.

Adolescent↗
PubMed2006

Culture medium modulates proinflammatory conditions of human pancreatic islets before transplantation.

A portion of transplanted islets is lost during engraftment as a result of stressful events, involving hypoxia and production of proinflammatory molecules by islets. Two of these molecules (monocyte chemoattractant protein-1, CCL2/MCP-1 and tissue factor, TF) are directly correlated with reduced graft function. We evaluated which factors reduce islet proinflammatory conditions. In particular the effects of different culture media supplemented with proteins or antioxidant agents on CCL2/MCP-1 and TF human islet release were evaluated. We observed that human islets after culture in final wash culture medium (FW) significantly decreased CCL2/MCP-1 release and TF production compared with CMRL and M199. These effects were independent from the type of protein added to the media (human serum, human albumin, fetal calf serum). Glutathione in FW further decreased CCL2/MCP-1 in a dose-dependent manner. Culture conditions can modulate the proinflammatory state of islets, and could be used in clinical islet transplantation to reduce inflammation during engraftment.

Amino Acids↗
PubMed2006

Raltitrexed-eloxatin salvage chemotherapy in gemcitabine-resistant metastatic pancreatic cancer.

Limited information on salvage treatment in patients affected by pancreatic cancer is available. At failure, about half of the patients present good performance status (PS) and are candidate for further treatment. Patients >18 years, PS >or=50, with metastatic pancreatic adenocarcinoma previously treated with gemcitabine-containing chemotherapy, and progression-free survival (PFS) <12 months received a combination of raltitrexed (3 mg m(-2)) and oxaliplatin (130 mg m(-2)) every 3 weeks until progression, toxicity, or a maximum of six cycles. A total of 41 patients received 137 cycles of chemotherapy. Dose intensity for both drugs was 92% of the intended dose. Main grade >2 toxicity was: neutropenia in five patients (12%), thrombocytopenia, liver and vomiting in three (7%), fatigue in two (5%). In total, 10 patients (24%) yielded a partial response, 11 a stable disease. Progression-free survival at 6 months was 14.6%. Median survival was 5.2 months. Survival was significantly longer in patients with previous PFS >6 months and in patients without pancreatic localisation. A clinically relevant improvement of quality of life was observed in numerous domains. Raltitrexed-oxaliplatin regimen may constitute a treatment opportunity in gemcitabine-resistant metastatic pancreatic cancer. Previous PFS interval may allow the identification of patients who are more likely to benefit from salvage treatment.

Adult↗
PubMed2006

Rapamycin induces a caspase-independent cell death in human monocytes.

The immunosuppressive activity of rapamycin (RAPA) and its efficacy as an anti-rejection agent in organ transplantation have been ascribed principally to its anti-proliferative effects on T cells, while the activity on monocytes is partially unknown. In vitro, RAPA reduced monocyte survival by inducing a caspase-independent cell death. RAPA-induced monocyte cell death (RAPA-CD) was impeded by activation of granulocyte macrophage-colony stimulating factor family receptors or toll-like receptor 4, and by exposure to inflammatory cytokines. In vivo, in patients who received RAPA monotherapy as part of pre-conditioning for islet transplantation, RAPA affected survival of myeloid lineage cells. In the peripheral blood, CD33(+) and CD14(+) cells decreased, whereas lymphocytes appeared unaffected. In the bone marrow, myeloid precursors such as CD15(+) and CD15(+)/CD16(+) were selectively and significantly decreased, but no major cytotoxic effects were observed. The RAPA-CD suggests a dependence of monocytes on mammalian target of RAPA pathways for nutrient usage, and this feature implies that RAPA could be selectively useful as a treatment to reduce monocytes or myeloid cells in conditions where these cells negatively affect patient, suggesting a potential anti-inflammatory action of this drug.

Antigens, CD↗
PubMed2004

In vitro modulation of monocyte chemoattractant protein-1 release in human pancreatic islets.

Islet transplantation is a new approach to treat type 1 diabetic patients. Despite its great potential and progressively increasing success rate, islet engraftment still represents an unsolved problem. Only part of the transplanted beta-cell mass survives after infusion due to hypoxia and inflammatory reactions, principally mediated by macrophages. We have demonstrated that human islets release monocyte chemoattractant protein-1 (MCP-1), one of the most powerful macrophage chemokines, which may impair the fate of a transplant. In this study we have attempted to modulate in vitro MCP-1 release by human islets. Human islets isolated using the automated method were cultured in CMRL or M199 standard culture media alone or supplemented with (1) two intracellular kinase inhibitors (10 micromol/L RO8220, a protein kinase C inhibitor, and rcAMP 20 micromol/L, a protein kinase A inhibitor) or (2) two antioxidant and cell-protective agents (vitamin E, vitamin B); or (3) immunosuppressive drugs (0.001 to 10 ng/mL cyclosporine, 0.1 to 100 ng/mL rapamycin, 0.1 to 10 ng/mL tacrolimus, 0.001 to 10 ng/mL mycophenolate acid). We observed that the only culture condition that significantly decreased MCP-1 in human islets were CMRL (31 +/- 12 in CMRL vs 539 +/- 184 pg/mL, in M199, P <.05) or cyclosporine (514 +/- 83 pg/mL in control islet vs 307 +/- 13, 231 +/- 44, 192 +/- 4, 242 +/- 113, 169 +/- 15 pg/mL in islet plus cyclosporine ranging from 0.001 to 10 ng/mL, respectively, P >.05). The capacity of in vitro factors to decrease human islet MCP-1 release suggests strategies to increase the success of islet transplantation.

Cell Culture Techniques↗
PubMed2000

Vitamin D3 affects differentiation, maturation, and function of human monocyte-derived dendritic cells.

We studied the effects of 1alpha,25-dihydroxyvitamin D3 (1alpha, 25-(OH)2D3) on differentiation, maturation, and functions of dendritic cells (DC) differentiated from human monocytes in vitro in the presence of GM-CSF and IL-4 for 7 days. Recovery and morphology were not affected by 1alpha,25-(OH)2D3 up to 100 nM. DC differentiated in the presence of 10 nM 1alpha,25-(OH)2D3 (D3-DC) showed a marked decrease in the expression of CD1a, while CD14 remained elevated. Mannose receptor and CD32 were significantly increased, and this correlated with an enhancement of endocytic activity. Costimulatory molecules such as CD40 and CD86 were slightly decreased or nonsignificantly affected (CD80 and MHC II). However, after induction of DC maturation with LPS or incubation with CD40 ligand-transfected cells, D3-DC showed marginal increases in MHC I, MHC II, CD80, CD86, CD40, and CD83. The accessory cell function of D3-DC in classical MLR was also inhibited. Moreover, allogeneic T cells stimulated with D3-DC were poor responders in a second MLR to untreated DC from the same or an unrelated donor, thus indicating the onset of a nonspecific hyporesponsivity. In conclusion, our data suggest that 1alpha,25-(OH)2D3 may modulate the immune system, acting at the very first step of the immune response through the inhibition of DC differentiation and maturation into potent APC.

Adjuvants, Immunologic↗
PubMed2000

Generation and functional characterisation of dendritic cells from patients with pancreatic carcinoma with special regard to clinical applicability.

BACKGROUND: We studied dendritic cell (DC) function in patients affected by pancreatic carcinoma, and the possibility of obtaining DC adequate for immunological treatment modalities. METHODS: Leucocytes were isolated from buffy coats obtained by autotransfusion of six patients undergoing pancreatico-duodenectomy. The leucocytes were cryopreserved and, after thawing, were purified by density gradient and/or plastic adhesion. They were then cultured in vitro in cytokine-enriched medium (granulocyte/macrophage-colony-stimulating factor + interleukin-4) with different sources of serum: 10% fetal calf serum (FCS), 2% autologous human serum or 2% pooled human AB serum. RESULTS: The DC obtained were identical to those from healthy donors in terms of phenotype, antigen uptake capacity, capacity for antigen presentation and their capacity to mature after exposure to stimuli like CD40L. DC differentiated in human serum demonstrated more mature behaviour than did DC cultured in FCS but, after exposure to CD40L, this difference disappeared. In one patient soluble factors in serum were able to inhibit the capacity of DC to stimulate T cells. CONCLUSION: It's possible to obtain DC from autotransfusion of patients with pancreatic carcinoma: these cells do not show evident quantitative or qualitative alterations, are able to present soluble antigen even when cultured in the presence of human serum and may be used in immunological tumour treatments.

Adenocarcinoma↗
PubMed1999

Effects of cryopreservation on in vitro and in vivo long-term function of human islets.

BACKGROUND: The possibility of performing transplantation several days after explant seems to be a peculiarity of islet grafts, and the opportunity to cryopreserve human islets may permit an indefinite period for modulating the recipient immune system. The aim of the present study was the evaluation of in vitro and in vivo functional properties of cryopreserved human islets. METHODS: We used six consecutive human islet preparations not suitable for an immediate transplantation in diabetic patients because the limited islet mass separated. The in vitro function of cryo and fresh islets was studied by determination of insulin and glucagon secretion in response to such classical stimuli as glucose (16.7 mM), glucose (16.7 mM) + 3-isobutyl-1-methylxanthine (0.1 mM), arginine (10 mM), and tolbutamide (100 microM). In vivo islet function was assessed through intravenous glucose tolerance tests performed at 15, 30, 60, and 90 days after transplantation of 1000 hand-picked fresh or cryopreserved islets in nude mice. RESULTS: Basal secretion of true insulin was significantly higher in cryopreserved islets than in fresh ones. The response of cryopreserved islets to arginine and glucose + isobutyl-1-methylxanthine seemed partially impaired. Proinsulin-like molecule secretion seemed higher in cryopreserved than in fresh islets in response to all secretagogues used, and the difference was statistically significant for arginine. The capacity of human cryopreserved islets to maintain a correct metabolic control in diabetic nude mice was progressively lost in 3 months. CONCLUSIONS: These findings showed that cryopreservation affects the function of isolated human islets, maintaining in vivo function for a limited period of time.

1-Methyl-3-isobutylxanthine↗
PubMed1999

Glucocorticoids affect human dendritic cell differentiation and maturation.

Because dendritic cells (DC) play a major role in the initiation of T cell-mediated immunity, we studied the effects of glucocorticoids, well-known inhibitors of the immune and inflammatory response, on the differentiation and maturation of human DC. DC were differentiated from human monocytes by culture with GM-CSF and IL-4 for 7 days with and without dexamethasone (Dex). Cells treated with Dex (10-8 M) (Dex-DC) developed a characteristic dendritic morphology; however, membrane phenotype analysis demonstrated that they were not fully differentiated. Dex-DC expressed low levels of CD1a and, unlike untreated cells, high levels of CD14 and CD16. Molecules involved in Ag presentation (CD40, CD86, CD54) were also impaired. In contrast, molecules involved in Ag uptake (mannose receptor, CD32) and cell adhesion (CD11/CD18, CD54) were up-regulated. After exposure to TNF-alpha or CD40 ligand, Dex-DC expressed lower levels of CD83 and CD86 than untreated cells. Dex-DC showed a higher endocytic activity, a lower APC function, and a lower capacity to secrete cytokines than untreated cells. Overall, these results indicate that DC differentiated in the presence of Dex are at a more immature stage. Moreover, Dex also partially blocked terminal maturation of already differentiated DC. In conclusion, our data suggest that glucocorticoids may act at the very first step of the immune response by modulating DC differentiation, maturation, and function.

Antigen Presentation↗
PubMed1999

Glucocorticoids increase the endocytic activity of human dendritic cells.

We have investigated the effect of glucocorticoids (GC) on antigen uptake molecule expression and on endocytic activity of human dendritic cells (DC). Human monocyte-derived DC were differentiated in vitro for 7 days with granulocyte macrophage colony stimulating factor and IL-4 in the presence or absence of dexamethasone 10(-8) M (Dex). Dex-treated DC showed an enhancement of mannose receptor (MR)-mediated endocytosis (measured as uptake of FITC-dextran) and of fluid-phase endocytosis [measured as uptake of Lucifer yellow (LY)] The effect was dose dependent and correlated with the length of exposure to Dex. The expression of receptors involved in antigen capture was investigated by FACS analysis. Dex up-regulates MR, CD16 and CD32 expression on DC. After maturation with tumor necrosis factor-alpha or CD40 ligand in Dex-treated DC, despite a reduction induced by maturation the endocytic activity of FITC-dextran and LY, the expression of MR, CD16 and CD32 remained higher than in control DC. In view of the fact that antigen capture was increased in cells cultured with Dex, we evaluated the ability to present soluble antigen that needs to be taken up and processed. Cells differentiated in the presence of Dex showed much lower efficiency in presenting tetanus toxin to specific autologous T cell lines. In conclusion our data suggest a new mechanism by which GC may influence immune responses. In fact with the increase in endocytic activity, Dex favors the scavenging of antigen from the external milieu, decreasing antigen concentration and availability, and simultaneously inhibiting the capacity to stimulate T cells.

Antigen Presentation↗
PubMed1998

Adhesion, transendothelial migration, and reverse transmigration of in vitro cultured dendritic cells.

Dendritic cells (DC) are migratory cells which exhibit complex trafficking properties in vivo, involving interaction with vascular and lymphatic endothelium and extracellular matrix (ECM). The underlying mechanisms involved in these processes are still ill defined. In the present study we have investigated the ability of DC to interact in vitro with human vascular endothelial cells (EC) and ECM. DC were differentiated from monocytes by in vitro exposure to granulocyte-macrophage colony-stimulating factor and interleukin-13 for 7 days. In adhesion assays a considerable proportion of DC bound to resting EC monolayers: (17% +/- 4%, mean +/- SE of eight experiments). Adhesion to tumor necrosis factor (TNF)-activated EC was increased to 29% +/- 5% (n = 8). Binding to resting EC was strongly inhibited by anti-CD11a and CD11b, but not by CD11c monoclonal antibodies (MoAbs); on TNF-activated EC, anti-VLA-4 in concert with anti-CD18 inhibited adhesion by more than 70%. Binding to a natural ECM, derived from cultured EC, or to purified fibronectin was high: 52% +/- 6% (n = 8) involved VLA-4 and VLA-5 integrins. In a transmigration assay, 10% +/- 2% (n = 6) of input cells were able to cross the EC monolayer. Unlike adhesion, transendothelial migration was significantly reduced by anti-CD31 MoAb. The amount of DC transmigrated through a monolayer of EC was increased twofold to threefold by a defined set of C-C chemokines including RANTES, MIP1alpha, MIP5, and, to a lesser extent, by MIP1beta and MCP-3. Most importantly, in view of the trafficking pattern of these cells, a significant proportion of DC (13% +/- 4% of input cells seeded) was able to migrate across the endothelial basement membrane and, subsequently, across the endothelial barrier (reverse transmigration). The adhesion molecules and chemoattractants characterized herein are likely to underlie the complex trafficking of DC in vivo.

CD11 Antigens↗
PubMed1998

IL-10 prevents the differentiation of monocytes to dendritic cells but promotes their maturation to macrophages.

Human monocytes cultured with granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-13 for 7 days differentiate into cells with the morphology and function of dendritic cells (DC). We have investigated the effect of IL-10 on this differentiation pathway. In the presence of IL-10 cells did not develop DC morphology, did not express CD1a and had lower levels of MHC class II. IL-10 promoted the differentiation of large cells with the morphology, cytochemistry and membrane phenotype of macrophages, including staining for nonspecific esterase and high levels of CD14, CD16 and CD68. The effect of IL-10 was dose dependent and was best appreciated when the cytokine was added at the initiation of the culture, as addition on day 3 was less inhibitory. When added to already differentiated DC on day 6, IL-10 caused only a modest reduction of MHC class II and CD1a expression, and no acquisition of the macrophage markers CD14, CD16 and CD68. Prolonged incubation up to 5 days with IL-10 did not induce a shift of differentiated DC to macrophages. On the other hand, the macrophages obtained by culturing for 7 days with GM-CSF+IL-13+IL-10 did not shift to DC upon removal of IL-10 for up to 3 days. Thus, the effect of IL-10 on monocyte differentiation, occurs only at the precursor level and confers an irreversible phenotype. From a functional point of view, cells cultured in the presence of IL-10 were poor stimulators of allogeneic cord blood T cells in mixed lymphocyte reaction (MLR) and presented tetanus toxin (TT) to specific T cell lines with much less efficiency than control DC. In contrast, IL-10-cultured DC showed 7 times greater endocytosis of FITC-dextran. This increased endocytosis was mostly mediated via the mannose receptor, as demonstrated by blocking with unlabeled mannose. In conclusion, IL-10 inhibits DC differentiation from monocytes and, in a substantial proportion of the cells, promotes the differentiation to mature macrophages. Intriguingly, IL-10 inhibits antigen presentation while it stimulates endocytic activity.

Antigen Presentation↗
PubMed1998

Interleukin-10 increases mannose receptor expression and endocytic activity in monocyte-derived dendritic cells.

Human monocyte-derived dendritic cells were differentiated in vitro for 7 days with granulocyte macrophage-colony stimulating factor and interleukin-13. These cultured dendritic cells are at an immature stage of differentiation and exhert high endocytic activity via surface mannose receptor and via fluid-phase macropinocytosis. We have investigated the modulation of endocytosis by interleukin-10 in these cells. When added during the last 24 h of the 7-day culture, interleukin-10 significantly stimulated the uptake of fluorescein-labelled dextran (39 +/- 16% increase, mean +/- SD of 6 experiments), a sugar binding to the mannose receptor. This effect was dose dependent and correlated with the length of exposure to interleukin-10, with a maximal effect (more than seven-fold increase) when the cytokine was added at the beginning of the culture (day 0). The interleukin-10-increased fluorescein-labelled-dextran endocytosis was mostly mediated via the mannose receptor, as unlabelled mannose and specific antimannose receptor monoclonal antibody inhibited most of the uptake. Moreover, interleukin-10-treated cells expressed increased levels (up to four-fold) of mannose receptor. Interleukin-10 also increased, although to a lesser extent, the fluid-phase endocytosis (macropinocytosis) of fluorescein-labelled albumin. Interleukin-10 had the opposite effect on the differentiation and functional activity of monocyte-derived dendritic cells; cells having a very low stimulatory capacity and reduced expression of MHC class II and CD1a after a 7-day exposure. Thus interleukin-10 had a strong immunosuppressive effect on the differentiation and functional activity of monocyte-derived dendritic cells and yet strongly stimulated endocytosis in these cells. We speculate that an increased endocytic activity would eventually result in a decreased availability of antigens in the external milieu, thus contributing to the immunosuppressive and tolerogenic activity of interleukin-10.

Antibodies, Monoclonal↗
PubMed1997

Receptor expression and responsiveness of human dendritic cells to a defined set of CC and CXC chemokines.

Dendritic cells (DC) are migratory cells that exhibit complex trafficking properties in vivo. The present study was designed to characterize receptor expression and responsiveness to chemoattractants of human DC obtained from PBMC by culture with granulocyte/macrophage-CSF and IL-13. DC expressed appreciable levels of the CCR1, CCR2, and CCR5 receptors for the CC chemokines and the chemokine receptors CXCR1, CXCR2, and CXCR4. DC increased intracellular free calcium and migrated in response to the CC chemokines MCP-3, MCP-4, RANTES, MIP-1alpha, MIP-1beta, and MIP-5/HCC2 and the CXC chemokine SDF-1. In contrast, the CC chemokines MCP-1 and eotaxin had little or no activity in the concentration range tested (up to 1 microg/ml). IL-8 and Gro-beta (CXC) and lymphotactin (C chemokines) were also inactive. DC did not respond to 5-HETE, whereas platelet-activating factor was an active agonist. Selected chemokines active on DC in terms of migration and calcium fluxes were examined for their capacity to modulate endocytosis and Ag presentation. Under conditions in which TNF-alpha was active, MCP-1, MCP-3, MIP-1alpha, and RANTES did not affect these two responses. Thus, among hemopoietic elements, DC respond to a unique set of CC and CXC chemokines, and their responsiveness is restricted to migration with no effect on Ag capture and presentation. Chemokines may play a role in the trafficking of DC under resting or stimulated conditions. Chemokine receptors expressed in DC are likely to underlie HIV infection of this cell type.

Base Sequence↗
PubMed1997

Islet transplantation in IDDM patients.

This single-centre study investigated parameters that positively correlated with the success rate after islet allotransplantation in insulin-dependent diabetic (IDDM) patients. Twenty-one intrahepatic, fresh islet transplantations were performed in 20 IDDM patients (one patient had two transplants), after or simultaneous with kidney transplantation. The correlation between number and purity of transplanted islets and final outcome was investigated. One patient died of a cardiac arrest several hours after islet transplantation; this patient was not included in the follow-up analysis. Three patients (15%) experienced acute, irreversible, early failure of islet function, which was considered as a 'presumed rejection'. Nine patients (45%) achieved either complete insulin-independence (seven cases) or a reduction (> 50%) of exogenous insulin requirement (two cases), with sustained serum C-peptide secretion (0.89 +/- 0.04 nmol/l; duration: 21 +/- 7 months, range 2-58 months). Liver biopsy, performed 3 years after transplantation in one successful case, showed normal islets within the hepatic parenchyma. Eight cases (40%) did not show any metabolic effect of islet transplantation, with low serum C-peptide levels ('presumed function exhaustion'). Metabolic investigations performed in successful cases showed an early phase of insulin release after arginine, mild and reversible postprandial hyperglycaemia and normal HbA1c levels. Success of islet transplantation positively correlates with the number (p < 0.05) of the transplanted islets. Islet transplantation is a safe procedure, with 45% success rate, in terms of insulin-independence or relevant reductions of exogenous insulin requirement, although success can be transient.

Adult↗
PubMed1995

Migration of dendritic cells in response to formyl peptides, C5a, and a distinct set of chemokines.

Trafficking to tissues and then to lymph nodes is a crucial aspect of the immunobiology of dendritic cells. The present study was designed to identify molecules able to direct the migration of human blood-derived dendritic cells. fMLP (representative of formyl peptides of bacterial origin), C5a, and the C-C chemokines monocyte chemotactic protein (MCP)-3, MIP-1 alpha/LD78, and RANTES elicited chemotactic migration and a rise of intracellular free calcium in dendritic cells. In contrast, the C-X-C chemokines IL-8 and IP-10 and the C-C chemokines MCP-1 and MCP-2 were inactive as chemoattractants. Thus, dendritic cells respond to classical chemotactic signals and to a set of chemokines distinct from that active on monocytes and neutrophils. Chemoattractants are likely to contribute to localization and trafficking of dendritic cells and provide tools to recruit these cells in the design of immunization strategies.

Calcium↗