BackgroundBlood 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 Findings103 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.ConclusionIn 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.
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. SEL-DI-TOF/MS features improved the diagnostic accuracy of CA 19-9 (AUCs0.883 for CA 19-9; AUCs0.935 for CA 19-9 and SELDI-TOF/MS features combined).
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 β-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 β-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 β-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.
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.
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