Treatment Trends for Eosinophilic Esophagitis and the Other Eosinophilic Gastrointestinal Diseases: Systematic Review of Clinical Trials.

BACKGROUND: Eosinophilic gastrointestinal diseases (EGIDs) are chronic inflammatory disorders of the gut, including eosinophilic esophagitis (EoE), gastritis (EoG), duodenitis (EoD), gastroenteritis (EoGE), and colitis (EoC). Available treatments may be ineffective in some patients, and several clinical trials are investigating alternative treatments. AIM: We performed a systematic review of clinical trials to illustrate EGIDs treatment research trends. METHODS: We searched clinicaltrials.gov to identify studies investigating EGIDs treatment. For each trial we analysed relevant data, including therapeutic intervention, method of administration, study outcomes, and temporal trends. RESULTS: For EoE, 66 studies were eligible: 26 testing topical corticosteroids (39.4%), 17 (25.8%) monoclonal antibodies, eight (12.1%) dietary measures, five (7.6%) immunomodulators, one (1.5%) esophageal dilation, and nine (13.6%) other medical treatment strategies. With regard to EoG, EoD, and EoGE, 10 studies were testing monoclonal antibodies (71.5%), one immunomodulators (7.1%), one dietary measures (7.1%), and two other treatments (14.3%). There were no trials for EoC. Ongoing studies on corticosteroids are focused on novel delivery systems, including viscous suspensions, orally disintegrating tablets, or capsules. Increased research on monoclonal antibodies was seen from 2018, with interleukin (IL)-4 receptor-α, IL-5 receptor-α, IL-5, IL-13, IL-15, and Siglec-8 as the targets. CONCLUSION: Clinical trials on EGIDs are predominantly investigating corticosteroids or monoclonal antibodies. EGIDs therapeutic landscape will be trasnformed imminently.

Salivary microbiota composition may discriminate between patients with eosinophilic oesophagitis (EoE) and non-EoE subjects.

BACKGROUND: Data on the role of the microbiome in adult patients with eosinophilic oesophagitis (EoE) are limited. AIMS: To prospectively collect and characterise the salivary, oesophageal and gastric microbiome in patients with EoE, further correlating the findings with disease activity. METHODS: Adult patients with symptoms of oesophageal dysfunction undergoing upper endoscopy were consecutively enrolled. Patients were classified as EoE patients, in case of more than 15 eosinophils per high-power field, or non-EoE controls, in case of lack of eosinophilic infiltration. Before and during endoscopy, saliva, oesophageal and gastric fundus biopsies were collected. Microbiota assessment was performed by 16 s rRNA analysis. A Sparse Partial Least Squares Discriminant Analysis (sPLS-DA) was implemented to identify biomarkers. RESULTS: Saliva samples were collected from 29 EoE patients and 20 non-EoE controls;, biopsies from 25 EoE and 5 non-EoE controls. In saliva samples, 23 Amplicon Sequence Variants (ASVs) were positively associated with EoE and 27 ASVs with controls, making it possible to discriminate between EoE and non-EoE patients with a classification error (CE) of 24%. In a validation cohort, the accuracy, sensitivity, specificity, positive predictive value and negative predictive value of this model were 78.6%, 80%, 75%, 80% and 60%, respectively. Moreover, the analysis of oesophageal microbiota samples observed a clear microbial pattern able to discriminate between active and inactive EoE (CE = 8%). CONCLUSION: Our preliminary data suggest that salivary metabarcoding analysis in combination with machine learning approaches could become a valid, cheap, non-invasive test to segregate between EoE and non-EoE patients.

Low Glucose Concentrations Induce a Similar Inflammatory Response in Monocytes from Type 2 Diabetic Patients and Healthy Subjects.

This study aims to assess the proinflammatory interleukin 1ß (IL-1ß) and anti-inflammatory IL-10 production by monocytes from 38 patients with type 2 diabetes and 31 controls in different glucose concentrations. Monocytes were incubated in low (2.5 mmol/L)-, normal (5.0 mmol/L)-, and high (20 mmol/L)-glucose conditions in the presence and absence of lipopolysaccharide (LPS). Monocytes from both patients and controls only produced a significant increase in IL-1ß in low-glucose conditions (p < 0.01), and this phenomenon was amplified in the presence of LPS, while it was not seen in normal- or high-glucose conditions, not even in the presence of LPS stimulation. There was no increase in IL-10 production by monocytes from either diabetic patients or controls using whatever glucose concentrations, except when treated with LPS in normal-glucose conditions. These findings seem to suggest that low-glucose conditions induce an inflammatory response in monocytes in all individuals, as an intrinsic capacity of this cell line. On the other hand, monocytes only retain their anti-inflammatory ability in response to known inflammatory stimuli such as LPS, under normal-glucose concentrations. In conclusion, human monocytes express an inflammatory pattern in low-glucose conditions in vitro. This response could contribute to explaining the higher cardiovascular risk induced by hypoglycemia in diabetic patients.

TNFA Haplotype Genetic Testing Improves HLA in Estimating the Risk of Celiac Disease in Children.

BACKGROUND: TNF-α and IFN-γ play a role in the development of mucosal damage in celiac disease (CD). Polymorphisms of TNFA and IFNG genes, as well as of the TNFRSF1A gene, encoding the TNF-α receptor 1, might underlie different inter-individual disease susceptibility over a common HLA risk background. The aims of this study were to ascertain whether five SNPs in the TNFA promoter (-1031T>C,-857C>T,-376G>A,-308G>A,-238G>A), sequence variants of the TNFRSF1A gene and IFNG +874A>T polymorphism are associated with CD in a HLA independent manner. METHODS: 511 children (244 CD, 267 controls) were genotyped for HLA, TNFA and INFG (Real Time PCR). TNFRSF1A variants were studied (DHPLC and sequence). RESULTS: Only the rare TNFA-1031C (OR=0.65, 95% CI:0.44-0.95), -857T (OR=0.42, 95% CI:0.27-0.65), -376A (OR=2.25, 95% CI:1.12-4.51) and -308A (OR=4.76, 95% CI:3.12-7.26) alleles were significantly associated with CD. One TNFRSF1A variant was identified (c.625+10A>G, rs1800693), but not associated with CD. The CD-correlated TNFA SNPs resulted in six haplotypes. Two haplotypes were control-associated (CCGG and TTGG) and three were CD-associated (CCAG, TCGA and CCGA). The seventeen inferred haplotype combinations were grouped (A to E) based on their frequencies among CD. Binary logistic regression analysis documented a strong association between CD and HLA (OR for intermediate risk haplotypes=178; 95% CI:24-1317; OR for high risk haplotypes=2752; 95% CI:287-26387), but also an HLA-independent correlation between CD and TNFA haplotype combination groups. The CD risk for patients carrying an intermediate risk HLA haplotype could be sub-stratified by TNFA haplotype combinations. CONCLUSION: TNFA promoter haplotypes associate with CD independently from HLA. We suggest that their evaluation might enhance the accuracy in estimating the CD genetic risk.

The novel S59P mutation in the TNFRSF1A gene identified in an adult onset TNF receptor associated periodic syndrome (TRAPS) constitutively activates NF-κB pathway.

INTRODUCTION: Mutations in the TNFRSF1A gene, encoding tumor necrosis factor receptor 1 (TNF-R1), are associated with the autosomal dominant autoinflammatory disorder, called TNF receptor associated periodic syndrome (TRAPS). TRAPS is clinically characterized by recurrent episodes of long-lasting fever and systemic inflammation. A novel mutation (c.262 T > C; S59P) in the TNFRSF1A gene at residue 88 of the mature protein was recently identified in our laboratory in an adult TRAPS patient. The aim of this study was to functionally characterize this novel TNFRSF1A mutation evaluating its effects on the TNF-R1-associated signaling pathways, firstly NF-κB, under particular conditions and comparing the results with suitable control mutations. METHODS: HEK-293 cell line was transfected with pCMV6-AC construct expressing wild-type (WT) or c.262 T > C (S59P), c.362G > A (R92Q), c.236C > T (T50M) TNFRSF1A mutants. Peripheral blood mononuclear cells (PBMCs) were instead isolated from two TRAPS patients carrying S59P and R92Q mutations and from five healthy subjects. Both transfected HEK-293 and PBMCs were stimulated with tumor necrosis factor (TNF) or interleukin 1ß (IL-1ß) to evaluate the expression of TNF-R1, the activation of TNF-R1-associated downstream pathways and the pro-inflammatory cytokines by means of immunofluorescent assay, array-based technique, immunoblotting and immunometric assay, respectively. RESULTS: TNF induced cytoplasmic accumulation of TNF-R1 in all mutant cells. Furthermore, all mutants presented a particular set of active TNF-R1 downstream pathways. S59P constitutively activated IL-1ß, MAPK and SRC/JAK/STAT3 pathways and inhibited apoptosis. Also, NF-κB pathway involvement was demonstrated in vitro by the enhancement of p-IκB-α and p65 nuclear subunit of NF-κB expression in all mutants in the presence of TNF or IL-1ß stimulation. These in vitro results correlated with patients' data from PBMCs. Concerning the pro-inflammatory cytokines secretion, mainly IL-1ß induced a significant and persistent enhancement of IL-6 and IL-8 in PBMCs carrying the S59P mutation. CONCLUSIONS: The novel S59P mutation leads to defective cellular trafficking and to constitutive activation of TNF-R1. This mutation also determines constitutive activation of the IL-1R pathway, inhibition of apoptosis and enhanced and persistent NF-κB activation and cytokine secretion in response to IL-1ß stimulation.

Inflammation and pancreatic cancer: molecular and functional interactions between S100A8, S100A9, NT-S100A8 and TGFß1.

BACKGROUND: In order to gain further insight on the crosstalk between pancreatic cancer (PDAC) and stromal cells, we investigated interactions occurring between TGFß1 and the inflammatory proteins S100A8, S100A9 and NT-S100A8, a PDAC-associated S100A8 derived peptide, in cell signaling, intracellular calcium (Cai2+) and epithelial to mesenchymal transition (EMT). NF-κB, Akt and mTOR pathways, Cai2+ and EMT were studied in well (Capan1 and BxPC3) and poorly differentiated (Panc1 and MiaPaCa2) cell lines. RESULTS: NT-S100A8, one of the low molecular weight N-terminal peptides from S100A8 to be released by PDAC-derived proteases, shared many effects on NF-κB, Akt and mTOR signaling with S100A8, but mainly with TGFß1. The chief effects of S100A8, S100A9 and NT-S100A8 were to inhibit NF-κB and stimulate mTOR; the molecules inhibited Akt in Smad4-expressing, while stimulated Akt in Smad4 negative cells. By restoring Smad4 expression in BxPC3 and silencing it in MiaPaCa2, S100A8 and NT-S100A8 were shown to inhibit NF-κB and Akt in the presence of an intact TGFß1 canonical signaling pathway. TGFß1 counteracted S100A8, S100A9 and NT-S100A8 effects in Smad4 expressing, not in Smad4 negative cells, while it synergized with NT-S100A8 in altering Cai2+ and stimulating PDAC cell growth. The effects of TGFß1 on both EMT (increased Twist and decreased N-Cadherin expression) and Cai2+ were antagonized by S100A9, which formed heterodimers with TGFß1 (MALDI-TOF/MS and co-immuno-precipitation). CONCLUSIONS: The effects of S100A8 and S100A9 on PDAC cell signaling appear to be cell-type and context dependent. NT-S100A8 mimics the effects of TGFß1 on cell signaling, and the formation of complexes between TGFß1 with S100A9 appears to be the molecular mechanism underlying the reciprocal antagonism of these molecules on cell signaling, Cai2+ and EMT.

Chemiluminescence and ELISA-based serum assays for diagnosing and monitoring celiac disease in children: a comparative study.

BACKGROUND: Anti-transglutaminase (tTG) or anti-deamidated gliadin peptides (DGP) serum determination is the first step in diagnosing celiac disease (CD). Our aims were to: compare the performance of novel chemiluminescent tool in the detection of tTG and DGP (Q-Flash®, Inova) with that of the established ELISA (Q-Lite®, Inova) methods; identify the more reliable index for making a sound diagnosis and monitoring therapy. METHODS: Using Q-Flash® and Q-Lite®, IgA and IgG class tTG and DGP were measured in the sera of 155 CD pediatric patients and 166 healthy age-matched controls. Forty-two of the patients had a follow-up one year after starting gluten free diet (GFD). RESULTS: Q-Flash® IgA tTG, the more accurate (intra-assay CV for low, intermediate and high values: 2.2%, 1.6%, and 1.1%; inter-assay CV: 2.8%, 4%, and 3%), sensitive (96.1%) and specific (97%) test for diagnosing CD, was the only variable to be significantly correlated with CD at binary logistic regression analysis (r=0.263, p<0.0001, Exp(B)=1.0506, 95% CI=1.0286-1.0731). Q-Flash® IgA tTG or DGP screen were more accurate than Q-Lite® IgA tTG in monitoring CD patients on GFD (p=0.003). CONCLUSION: Q- Flash® IgA tTG measurement is an extremely precise, sensitive and specific index for not only diagnosing CD, but also monitoring therapy.

Pancreatic tumors and immature immunosuppressive myeloid cells in blood and spleen: role of inhibitory co-stimulatory molecules PDL1 and CTLA4. An in vivo and in vitro study.

BACKGROUND: Blood and spleen expansion of immature myeloid cells (IMCs) might compromise the immune response to cancer. We studied in vivo circulating and splenic T lymphocyte and IMC subsets in patients with benign and malignant pancreatic diseases. We ascertained in vitro whether pancreatic adenocarcinoma (PDAC)-associated IMC subsets are induced by tumor-derived soluble factors and whether they are immunosuppressive focusing on the inhibitory co-stimulatory molecules PDL1 and CTLA4. METHODOLOGY AND PRINCIPAL FINDINGS: 103 pancreatic and/or splenic surgical patients were enrolled including 52 PDAC, 10 borderline and 10 neuroendocrine tumors (NETs). Lymphocytes and IMCs were analysed by flow cytometry in blood, in spleen and in three PDAC cell conditioned (CM) or non conditioned PBMC. PDL1 and CTLA4 were studied in 30 splenic samples, in control and conditioned PBMC. IMCs were FACS sorted and co-coltured with allogenic T lymphocytes. In PDAC a reduction was found in circulating CD8(+) lymphocytes (p = 0.004) and dendritic cells (p = 0.01), which were reduced in vitro by one PDAC CM (Capan1; p = 0.03). Blood myeloid derived suppressive cells (MDSCs) CD33(+)CD14(-)HLA-DR(-) were increased in PDAC (p = 0.022) and were induced in vitro by BxPC3 CM. Splenic dendritic cells had a higher PDL1 expression (p = 0.007), while CD33(+)CD14(+)HLA-DR(-) IMCs had a lower CTLA4 expression (p = 0.029) in PDAC patients. In vitro S100A8/A9 complex, one of the possible inflammatory mediators of immune suppression in PDAC, induced PDL1 (p = 0.018) and reduced CTLA4 expression (p = 0.028) among IMCs. IMCs not expressing CTLA4 were demonstrated to be immune suppressive. CONCLUSION: In PDAC circulating dendritic and cytotoxic T cells are reduced, while MDSCs are increased and this might favour tumoral growth and progression. The reduced CTLA4 expression found among splenic IMCs of PDAC patients was demonstrated to characterize an immune suppressive phenotype and to be consequent to the direct exposure of myeloid cells to pancreatic cancer derived products, S100A8/A9 complex in particular.

Usefulness of MALDI-TOF/MS identification of low-MW fragments in sera for the differential diagnosis of pancreatic cancer.

OBJECTIVES: To identify new biomarkers of pancreatic cancer (PaCa), we performed MALDI-TOF/MS analysis of sera from 22 controls, 51 PaCa, 37 chronic pancreatitis, 24 type II diabetes mellitus (DM), 29 gastric cancer (GC), and 24 chronic gastritis (CG). METHODS: Sera were purified by Sep-Pak C18 before MALDI-TOF/MS Anchorchip analysis. RESULTS: Features present in at least 5% of all spectra were selected (n = 160, m/z range, 1200-5000). At univariate analysis, 2 features (m/z 2049 and 2305) correlated with PaCa, 3 (m/z 1449, 1605, and 2006) with DM. No feature characterized gastric cancer or chronic gastritis. Ten-fold cross-validation binary recursive partitioning trees were obtained for patients' classification. The tree (CA 19-9, age, m/z 2006, 2599, 2753, and 4997), built considering only patients with diabetes, allowed a distinction between DM [area under the receiver operating characteristic curve (AUC), 0.997], chronic pancreatitis (AUC, 0.968), and PaCa (AUC, 0.980), with an overall correct classification rate of 89%. The tree including CA 19-9, 1550, and 2937 m/z features, achieved an AUC of 0.970 in distinguishing localized from advanced PaCa. MALDI-TOF-TOF analysis revealed the 1550 feature as a fragment of Apo-A1, which was determined as whole protein and demonstrated to be closely correlated with PaCa. CONCLUSIONS: The findings made demonstrate a role for serum peptides identified using MALDI-TOF/MS for addressing PaCa diagnosis.

Pancreatic cancer alters human CD4+ T lymphocyte function: a piece in the immune evasion puzzle.

OBJECTIVES: To verify whether the dysregulation of CD4 T cells concurs in worsening the outcome of pancreatic cancer, we compared the effects of pancreatic cancer and other gastrointestinal cancer cell-conditioned media on the (1) proliferation, migration, and differentiation of CD4 T cells and (2) expansion of CD4 memory (CD45RO), naive (CD45RA), activated (CD69), and regulatory (CD25) subsets. METHODS: After culture of CD4 T cells in control, pancreatic (BxPC3, Capan1, MiaPaCa2), or gastrointestinal cancer (AGS, HepG2, HT29) cell-conditioned media, we evaluated proliferation, migration, interferon γ (IFNγ) production, and CD45RA, CD45RO, CD69, and CD25 membrane expression in control and conditioned CD4 T cells. RESULTS: Only pancreatic cancer-conditioned media (1) inhibited CD4 T-cell proliferation (P < 0.001) and migration under human stromal cell-derived factor-α chemotaxis (P < 0.001) and (2) induced CD4 T-cell IFNγ production (P < 0.05) and the expansion of the CD69-positive subset (P < 0.001) with respect to the control, with no changes being found in the CD45RA, CD45RO, and CD25 subsets. CONCLUSIONS: The in vitro findings achieved in the present study demonstrate that pancreatic cancer cells inhibit CD4 T-cell proliferation and migration, induce IFNγ production, and favor a CD69 subset expansion, suggesting that CD4 T cells play an important role in pancreatic cancer immune evasion.

New screening tests enrich anti-transglutaminase results and support a highly sensitive two-test based strategy for celiac disease diagnosis.

BACKGROUND: The identification of specific serological algorithms allowing the diagnosis of celiac disease (CD) is a new challenge for both the clinic and the laboratory. We compared the diagnostic accuracy of three new tests proposed for CD screening with that of the well established IgA tTG, and ascertained whether any combination of these tools might enhance accuracy in diagnosing CD. METHODS: In sera from 329 CD and 374 control children, the following were assayed: IgA tTG; IgA/IgG, which identify tTG-gliadin complexes (Aeskulisa Celi Check and CeliCheck IgGA); IgA/IgG, which identify deamidated gliadin peptides and tTG (QUANTA Lite(TM) h-tTG/DGP Screen). RESULTS: When specificity was set at 100%, the most sensitive index of CD was IgA tTG (75.7%, cut-off=100U), followed by QUANTA Lite(TM) h-tTG/DGP Screen (65.3%, cut-off 145U), Aeskulisa Celi Check (62.6%, cut-off 909U/mL) and CeliCheck IgGA (59.6%, cut-off 977U/mL). Three algorithms were obtained by combining IgA tTG with each of the new tests. The algorithm obtained by measuring IgA tTG and QUANTA Lite(TM) h-tTG/DGP Screen allowed the correct identification of CD in 78.7% of cases (negative predictive value=97.3%). CONCLUSIONS: The two-test based strategy could be used for the cost effective diagnosis of CD.

Altered intracellular calcium fluxes in pancreatic cancer induced diabetes mellitus: Relevance of the S100A8 N-terminal peptide (NT-S100A8).

After isolating NT-S100A8 from pancreatic cancer (PC) tissue of diabetic patients, we verified whether this peptide alters PC cell growth and invasion and/or insulin release and [Ca(2+)](i) oscillations of insulin secreting cells and/or insulin signaling. BxPC3, Capan1, MiaPaCa2, Panc1 (PC cell lines) cell growth, and invasion were assessed in the absence or presence of 50, 200, and 500 nM NT-S100A8. In NT-S100A8 stimulated ß-TC6 (insulinoma cell line) culture medium, insulin and [Ca(2+)] were measured at 2, 3, 5, 10, 15, 30, and 60 min, and [Ca(2+)](i) oscillations were monitored (epifluorescence) for 3 min. Five hundred nanomolars NT-S100A8 stimulated BxPC3 cell growth only and dose dependently reduced MiaPaCa2 and Panc1 invasion. Five hundred nanomolars NT-S100A8 induced a rapid insulin release and enhanced ß-TC6 [Ca(2+)](i) oscillations after both one (F = 6.05, P < 0.01) and 2 min (F = 7.42, P < 0.01). In the presence of NT-S100A8, [Ca(2+)] in ß-TC6 culture medium significantly decreased with respect to control cells (F = 6.3, P < 0.01). NT-S100A8 did not counteract insulin induced phosphorylation of the insulin receptor, Akt and IκB-α, but it independently activated Akt and NF-κB signaling in PC cells. In conclusion, NT-S100A8 exerts a mild effect on PC cell growth, while it reduces PC cell invasion, possibly by Akt and NF-κB signaling, NT-S100A8 enhances [Ca(2+)](i) oscillations and insulin release, probably by inducing Ca(2+) influx from the extracellular space, but it does not interfere with insulin signaling.

Analogs of vitamin E epitomized by alpha-tocopheryl succinate for pancreatic cancer treatment: in vitro results induce caution for in vivo applications.

OBJECTIVES: alpha-Tocopheryl succinate (alpha-TOS) is thought to be toxic only for cancer cells. We ascertained in vitro alpha-TOS effects on pancreatic cancer (PC) and normal cell growth and verified whether the combination of nontoxic alpha-TOS and 5-fluorouracil (5-FU) doses causes cancer cell death and whether alpha-TOS effects are mediated by the proapoptotic proteins Bax/Bak and/or SMAD4/DPC4 status. METHODS: Five PC cell lines, myoblasts, normal monocytes, wild-type (WT) and Bax/Bak double knockout mouse embryonic fibroblast (MEF) cells, and permanently SMAD4/DPC4-transfected PSN1 cells were cultured in 1% and 10% fetal calf serums (FCSs), without or with alpha-TOS (5-500 micromol/L). Nontoxic 5-FU (0.0001 mmol/L) and alpha-TOS alone or in combination were also evaluated. RESULTS: Only PSN1 PC cell line, which had SMAD4/DPC4 homozygous deletion, was sensitive to nontoxic alpha-TOS doses (5 micromol/L in 1% FCS and 50 micromol/L in 10% FCS). A 20-micromol/L alpha-TOS inhibited MEF-WT, not MEF-double knockout growth. Only PSN1 cells were sensitive to nontoxic 5-FU and alpha-TOS combination. SMAD4/DPC4 transfection restored PSN1 resistance to the effects of combined 5-FU and alpha-TOS effects. CONCLUSIONS: Only a minority of PC cells are sensitive to the antiproliferative effects of alpha-TOS, any sensitivity appearing to be correlated with SMAD4/DPC4 homozygous deletion and Bax/Bak expression.

Pancreatic cancer biomarkers discovery by surface-enhanced laser desorption and ionization time-of-flight mass spectrometry.

BACKGROUND: Surface-enhanced laser desorption and ionization time-of-flight mass spectrometry (SELDI-TOF/MS), a laboratory-friendly technique, is used to identify biomarkers for cancer. The aim of the present study was to explore the application of SELDI proteomic patterns in serum for distinguishing between cases of pancreatic cancer, chronic pancreatitis, type 2 diabetes mellitus and healthy controls. METHODS: Sera from 12 healthy controls, 24 patients with type 2 diabetes mellitus, 126 with pancreatic cancer, including 84 with diabetes, and 61 with chronic pancreatitis, 32 of which were diabetics, were analyzed using SELDI-TOF/MS. Spectra (IMAC-30) were clustered and classified using Biomarker Wizard and Biomarker Pattern software. RESULTS: Two decision tree classification algorithms, one with and one without CA 19-9, were constructed. In the absence of CA 19-9, the splitting protein peaks were: m/z 1526, 1211, and 3519; when CA 19-9 was used in the analysis, it replaced the m/z 3519 splitter. The two algorithms performed equally for classifying patients. A classification tree that considered diabetic patients only was constructed; the main splitters were: 1211, CA 19-9, 7903, 3359, 1802. With this algorithm, 100% of patients with type 2 diabetes mellitus, 97% with chronic pancreatitis and 77% of patients with pancreatic cancer were correctly classified. SELDI-TOF/MS features improved the diagnostic accuracy of CA 19-9 (AUC = 0.883 for CA 19-9; AUC = 0.935 for CA 19-9 and SELDI-TOF/MS features combined). CONCLUSIONS: SELDI-TOF/MS allows identification of new peptides which, in addition to CA 19-9, enable the correct classification of the vast majority of patients with pancreatic cancer, which can be distinguished from patients with chronic pancreatitis or type 2 diabetes mellitus.

GastroPanel: evaluation of the usefulness in the diagnosis of gastro-duodenal mucosal alterations in children.

BACKGROUND: The combined evaluation of serum pepsinogens A (PGA) and C (PGC), gastrin-17 (G17) and anti-H. pylori antibodies (anti-H. pylori)(GastroPanel) has recently been proposed as a useful aid for investigating H. pylori-associated gastric mucosal inflammation. Our aim was to evaluate whether GastroPanel can correctly classify children who need or not endoscopy (EGD). METHODS: GastroPanel was performed in 554 consecutive children subjected to EGD. RESULTS: PGC and anti-H. pylori were sensitive (82.5% and 73.1%) and specific (58.1% and 84.0%) indices of H. pylori infection. Antral H. pylori colonization density, inflammation and activity grades were correlated with PGC. PGC and G17 were significantly higher in children with celiac disease (14.9+/-0.88 microg/L and 5.6+/-0.79 pmol/L) than in controls (8.5+/-0.38 microg/L and 2.4+/-0.24 pmol/L). The best cut-offs to distinguish H. pylori infected children from controls were 7.45 microg/L for PGC, 4.2 pmol/L for G17, 18 U for anti-H. pylori and 25 microg/L for PGA. With these cut-offs, GastroPanel had a NPV of 89.6% and a PPV of 66.8%. CONCLUSIONS: A negative GastroPanel result in children with upper abdominal non alarm symptoms, should allow the paediatrician to reasonably rule out the presence of major gastro-duodenal diseases and therefore avoid EGD.

Antibodies against synthetic deamidated gliadin peptides for celiac disease diagnosis and follow-up in children.

BACKGROUND: AGA IgA II and AGA IgG II have recently been suggested as reliable tools for celiac disease (CD) diagnosis. We compared their utility for diagnosis and monitoring CD in children with that of tTG IgA, an established CD marker. METHODS: We studied a cohort of 161 CD and 129 control children in whom CD was histologically confirmed or ruled out. We followed 37 children with CD on a gluten-free diet for 12-84 months. In fasting sera, we measured AGA IgA II, AGA IgG II, and tTG IgA using ELISAs. RESULTS: The best sensitivity (92.5%), specificity (97.6%), positive predictive value (98%), and negative predictive value (91.2%) were obtained using tTG IgA. AGA IgG II correctly identified 3 of 3 children with CD with total IgA deficiency who had negative AGA IgA II and tTG IgA results. In children <2 years old without total IgA deficiency, AGA IgG II and tTG IgA performed equally well (sensitivity 96.4% and specificity 100%). AGA IgA II, AGA IgG II, and tTG IgA concentrations diminished significantly (P < 0.0001) after 1 year of a gluten-free diet, reaching values below the cutoff in 87%, 70%, and 51% of cases, respectively. CONCLUSIONS: The best available index for diagnosing CD in children was tTG IgA. In infants <2 years old, AGA IgG II performed as well as tTG IgA in cases without total IgA deficiency and allowed detection of CD when total IgA was <0.06 g/L. Gluten-free diet monitoring can be achieved using any of the studied serum markers.

IL-4 -588C>T polymorphism and IL-4 receptor alpha [Ex5+14A>G; Ex11+828A>G] haplotype concur in selecting H. pylori cagA subtype infections.

BACKGROUND: The Th2 cytokine IL-4 might limit H. pylori associated gastric inflammation and favour H. pylori clearance. The aim of the study was to verify whether IL-4 -588C>T SNP, or two SNPs of the gene coding the alpha chain of IL-4 receptor (IL-4RA Ex5+14A>G, IL-4RA Ex11+828A>G) considered singly or as haplotypes, are correlated with H. pylori virulence genes or H. pylori associated diseases. METHODS: We studied 144 patients with non-cardia gastric cancer (NCGC)(41/50 with present or past H. pylori infection), 75 with duodenal ulcer (DU)(66 H. pylori infected) and 171 with gastritis (CG)(107 H. pylori infected). cagA gene was present in 24/28 NCGC, 45/59 DU and 56/107 CG. RESULTS: All SNPs were in Hardy-Weinberg equilibrium. IL-4RA haplotypes frequencies were estimated using Arlequin software. Neither the SNPs nor the IL-4RA haplotype correlated with disease diagnosis, H. pylori infection, degree of mucosal inflammation or intestinal metaplasia. IL-4 -588T allele (OR=3.69, 95% CI:1.34-10.16) and IL-4RA GA haplotype (p<0.05) enhanced the risk for cagA positive infections. IL-4RA GA haplotype correlated with IL-4 protein levels in H. pylori infected gastric mucosa. CONCLUSIONS: IL-4 and IL-4RA gene polymorphisms concur in selecting the H. pylori infecting strain, probably influencing the IL-4 signalling pathway.

Suicide gene therapy with the yeast fusion gene cytosine deaminase/uracil phosphoribosyltransferase is not enough for pancreatic cancer.

OBJECTIVES: Suicide gene therapy with FCY1 gene, encoding cytosine deaminase (CD), together with FUR1, encoding uracil phosphoribosyltransferase (UPRT), has been proposed for pancreatic cancer therapy in vivo. We ascertained whether gene therapy with FCY1-FUR1 is effective in killing pancreatic cancer cells after 5-fluorocytosine (5-FC) treatment. METHODS: AsPC1, BxPC3, Capan1, MIA PaCa2, and Panc1 cell lines were transfected using 2 plasmid vectors expressing CD only (pRSV-CD) or the chimera CD-UPRT (pRSV-CD-UPRT). Control and pRSV-CD- or pRSV-CD-UPRT-transfected cell lines were treated with 0, 0.1, 0.5, 1, 5, and 10 mM of 5-FC for 1, 3, 6, 8, 10, and 13 days. RESULTS: FCY1 alone did not confer sensitivity to 5-FC. The CD-UPRT-transfected BxPC3 and Panc1 were sensitive to very low 5-FC doses (0.1 mM). 5-Fluorocytosine-sensitive transfected cell lines rapidly converted 5-FC into 5-fluorouracil, whereas the 5-FC resistant cell lines had an impaired 5-FC conversion. CONCLUSIONS: Suicide gene therapy with the FCY1 gene alone was ineffective in the treatment of pancreatic cancer in vitro. The pRSV-CD-UPRT construct conferred 5-FC sensitivity to some pancreatic cancer cell lines. Therefore, the application in vivo of suicide gene therapy with FCY1 alone or in combination with the FUR1 gene is probably destined to fail.

Clarithromycin resistance, tumor necrosis factor alpha gene polymorphism and mucosal inflammation affect H. pylori eradication success.

Several bacterial and host-related factors concur in causing Helicobacter pylori eradication failure. We ascertained the role of bacterial virulence genes (cagA, vacA), clarithromycin resistance [Cla(R), 23S ribosomal RNA (rRNA) mutations], host polymorphism of CYP2C19 (polyphosphoinositide, PPI, metabolism) and of the cytokines IL-1B-31C>T, IL-1RN VNTR, IFN-gamma+874A>T, TNF-alpha-1031T>C, TNF-alpha-857C>T, TNF-alpha-376G>A, TNF-alpha-308G>A, TNF-alpha-238G>A, IL-10-1082A>G, IL-10-819C>T, IL-10-592C>A, IL-12A+6686G>A, IL-12B+15485A>C. Two groups of H. pylori-infected and H. pylori-treated patients were retrospectively identified: 45 not eradicated and 57 eradicated. Treatment failure was significantly correlated with Cla(R) (all resistant strains in non-eradicated patients); with TNF-alpha-238, IL10-819, IL10-592, IL-12B+15485 single nucleotide polymorphism (SNP); with IL10 ATA/ATA haplotype; and with antral inflammatory grade. On considering Cla(S)-infected patients only, logistic regression analysis (eradication = dependent; TNF-alpha-238, IL12B + 15485 genotypes, IL10 ATA/ATA as present or absent, antral gastritis grade = covariates) confirmed as significantly correlated with eradication antral gastritis grade only (Exp(B) = 6.48; 95% CI, 1.2-35.01). In conclusion, the bacterial determinant causing triple therapy failure is clarithromycin resistant, being virulence genes not involved. The host related factors that favor eradication are those linked to inflammation: a higher inflammatory infiltrate in the mucosa, possibly favored by genotypes able to down regulate the anti-inflammatory cytokine response, enhance the chance of eradication success.

DNA repair pathways and mitochondrial DNA mutations in gastrointestinal carcinogenesis.

This work focuses on the main DNA repair pathways, highlighting their role in gastrointestinal carcinogenesis and the role of mitochondrial DNA (mtDNA), mutations being described in several tumor types, including those of the gastrointestinal tract. The mismatch repair (MMR) system is inherently altered in patients with hereditary non-polyposis colorectal cancer, and plays a role in carcinogenesis in a subset of sporadic colorectal, gastric and esophageal cancers. Alterations in homologous recombination (HR) and non-homologous end-joining (NHEJ) also contribute to the development of pancreatic cancer. Gene polymorphisms of some X-ray cross-complementing (XRCCs), cofactor proteins involved in the base excision repair pathway, have been investigated in relation to gastric, colorectal and pancreatic cancer. Yet only one polymorphism, XRCC1 Arg194Trp, appears to be involved in smoking-related cancers and in early onset pancreatic cancer. Although evidence in the literature indicates that mtDNA somatic mutations play a role in gastric and colorectal carcinogenesis, no sound conclusions have yet been drawn regarding this issue in pancreatic cancer, although an mtDNA variant at 16519 is believed to worsen the outcome of pancreatic cancer patients, possibly because it is involved in altering cellular metabolism.