Cooperative induction of a tolerogenic dendritic cell phenotype by cytokines secreted by pancreatic carcinoma cells

Ag presentation by dendritic cells (DC) is essential to effective antitumor T cell responses in cancer patients. Depending on their origin, maturation state, and the ambient cytokine milieu, DC can differentiate into distinct subpopulations, which preferentially either induce Th1 cell activation (CD11c+,CD123- myeloid DC (MDC)) or immunosuppressive T cell development (CD11c-,CD123+ plasmacytoid DC (PDC)). The present study was undertaken to characterize the effects of pancreatic carcinoma cell-derived cytokines on immature monocyte-derived DC (iMo-DC) in vitro and in vivo. Medium conditioned by human pancreatic carcinoma cells inhibited iMo-DC proliferation, expression of costimulatory molecules (CD80 and CD40) and of HLA-DR, and functional activity as assessed by MLR and IL-12p70 production. iMo-DC generated from pancreatic carcinoma patients in advanced stages of the disease similarly showed decreased levels of HLA-DR expression and reduced ability to stimulate MLR in response to CD40L and IFN-gamma. Moreover, in tumor-patient peripheral blood, the ratio of MDC to PDC cells was lower than in healthy controls due to reduced numbers of MDC CD11c+ cells. Importantly, rather than a single cytokine, a combination of tumor-derived cytokines was responsible for these effects; these were primarily TGF-beta, IL-10, and IL-6, but not vascular endothelial growth factor. In summary, we have identified an array of pancreatic carcinoma-derived cytokines that cooperatively affect iMo-DC activation in a manner consistent with ineffective antitumor immune responses

Sequential molecular monitoring of chimerism in chronic myeloid leukemia patients receiving donor lymphocyte transfusion for relapse after bone marrow transplantation

Recent observations of chimerism in patients relapsed following an allotransplant suggest the persistence of immunotolerance, thus offering a biologic rationale for the use of donor lymphocyte transfusion (DLT), In this study, we have analyzed by PCR amplification of several VNTR regions, sequential bone marrow and peripheral blood DNA samples in four patients who received DLT for CR IL relapse after bone marrow transplantation. Prior to DLT, all patients showed mixed chimerism in peripheral blood cells while two had mixed chimerism and two no chimerism in the PM, None of these four patients showed evidence of chimerism at the cytogenetic level (all had 100% +ve metaphases), After DLT, a complete hematologic and molecular remission (ie disappearance of the BCR/ABL fusion transcript) was obtained in the two patients who had bone marrow mixed chimerism prior to DLT, The two patients without evidence of marrow chimerism prior to DLT converted to a pattern of mixed chimerism after DLT, but both developed a severe bone marrow aplasia occurring at day 56 and 36, respectively, With regard to the sequential analysis of bone marrow chimerism after DLT we observed that: (1) the disappearance of BCR/ABL +ve cells paralleled the conversion to a pattern of full donor chimerism; and (2) the time interval to achieve CR was inversely correlated with the percentage of donor DNA in bone marrow, In conclusion, we have shown here that the assessment of bone marrow pre-DLT chimerism by PCR analysis might predict the response in patients with favorable characteristics, and also might identify patients at high risk of developing severe myelosuppression

Cytokine expression profile in human pancreatic carcinoma cells and in surgical specimens: implications for survival

Cytokine shedding by tumor cells into the local microenvironment modulates host immune response, tumor growth, and metastasis. The study aimed to verify the hypothesis that the immunological microenvironment of pancreatic carcinoma exists in a prevalently immunosuppressive state, influencing survival. We analyzed expression profiles of pro-inflammatory (IL-1beta, IL-2, IL-6, IL-8, IL-12 p40, IL-18 and IFN-gamma) and anti-inflammatory (IL-10, IL-11, IL-13 and TGF-beta isoforms) cytokines. The study was performed both in vitro, in five pancreatic carcinoma cell lines (real time RT-PCR), and in specimens from 65 patients, comparing tumoral versus non-tumoral pancreatic tissues (real time RT-PCR and immunohistochemistry). Furthermore, cytokines were measured in supernatants and sera (from patients and controls) by ELISA. All cell lines expressed IL-8, IL-18, TGF-beta1, TGF-beta2 and TGF-beta3, but not IFN-gamma and IL-2 transcripts. Expression of IL-1beta, IL-6, IL-10, IL-11, IL-13 and IL-12 mRNA was variable. All the above cytokines were detected as soluble proteins in supernatants, except IL-13. Tumor tissues overexpressed IL-1beta, IL-6, IL-8, IL-10, IL-11, IL-12 p40, IL-18, IFN-gamma, TGF-beta1, TGF-beta2 and TGF-beta3 at the mRNA level and IL-1beta, IL-18, TGF-beta2 and TGF-beta3 also at the protein level. Conversely, non-tumor tissues had stronger RNA and protein expression of IL-13. Survival was significantly longer in patients with high IL-1beta and IL-11 and moderate IL-12 expression. Serum IL-8, IL-10, IL-12, IL-18, TGF-beta1 and TGF-beta2 were higher in patients than in controls, as opposed to IL-1beta and IL-13. Patients with low circulating levels of IL-6, IL-18 and TGF-beta2 survived longer. Pancreatic cancer is characterized by peculiar cytokine expression patterns, associated with different survival probabilities

Antagonistic interactions between gemcitabine and 5-fluorouracil in the human pancreatic carcinoma cell line Capan-2

Although the recently-developed Gemcitabine (GEM) has renewed interest in clinical research in pancreatic carcinoma, it offers modest improvement of tumor-related symptoms and marginal survival advantage, even when combined with other currently-available chemotherapeutic agents such as 5-Fluorouracil (5-FU). We hypothesized that this disappointing result could be due to an interaction between the two drugs affecting cytotoxic activity. We measured in-vitro growth inhibition, cell cycle distribution, gene and protein expression of apoptosis regulators bcl-2, bcl-x and survivin, NFkappaB and telomerase activities of human pancreatic carcinoma cell line Capan-2 following exposure to GEM and 5-FU singly or combined, by MTT assay and median effect analysis, flow cytometry, real-time RT-PCR, Western blotting, electrophoretic mobility shift assay (EMSA) and telomeric repeat amplification protocol (TRAP) assay, respectively. We found cell growth to be inhibited by both drugs, decreasing the percentage of cells in S and G2/M phases and inducing apoptosis, dependent on the levels of bcl-2, bcl-xL and survivin expression in the case of 5-FU, but not for GEM. Moreover, while telomerase activity was reduced equally by both drugs, 5-FU but not GEM effectively downregulated NFkappaB binding activity. Intriguingly, a substantial antagonistic effect was noticed when GEM was combined with 5-FU in the concentration range tested, with the exception of the TRAP assay. These indications of an antagonistic interaction between GEM and 5-FU in some pancreatic cancer context urge further investigation of both genetic and non-genetic differences to identify the variables most relevant for optimal selection and dosing of treatment for the individual patient

Improved rapid detection of the PML/RARalpha fusion gene in acute promyelocytic leukemia

Acute promyelocytic leukemia (APL) is a medical emergency which requires rapid diagnosis and tailored treatment. Detection of the PML/RARalpha fusion gene in APL blasts is critical to start promptly the specific therapy with all-trans retinoic acid (ATRA). APL lacking this genetic lesion have been reported as being ATRA resistant. Reverse-transcription polymerase chain reaction (RT-PCR) has been extensively used to detect the PML/RARalpha cDNA. The reported PML/RARalpha amplification techniques are laborious and time consuming, and include conventional RNA extraction, cDNA synthesis and a two-round (nested) PCR. We hereby describe a few variations of the commonly adopted RNA extraction and PML/RARalpha RT-PCR protocols which allow a molecular diagnosis of APL to be carried out in less than 5 h. Processing of small volumes of leukemic cell lysate (0.5 ml) in a microfuge allows extraction of good quality RNA in 1 h. After reverse transcription to obtain cDNA, a 'hot start' PCR procedure was adopted which enabled us to amplify clearly visible and specific products after a single (not nested) amplification round. The PML/RARalpha fusion gene was detected in the blasts of six consecutive APL at diagnosis, and an APL-tailored protocol including ATRA was started in each case within 6 h of admission. On repeated experiments, the assay proved highly specific and sensitive for the rapid detection of all PML/RARalpha transcript types. Our data should encourage the use of this rapid procedure for the diagnosis of both typical APL and, particularly, less typical cases awaiting urgent therapeutic intervention