Comprehensive flow cytometric reference intervals of leukocyte subsets from six study centers across Europe.

A group of European FOCIS Centers of Excellence adapted panels of the Human Immunophenotyping Consortium (HIPC) for whole blood analysis. Using four core panels [T/regulatory T cell/B/natural killer (T/Treg /B/NK) and myeloid cells] the main leukocyte populations were analyzed in a clinical-diagnostic setting in a harmonized manner across different platforms. As a first step, the consortium presents here the absolute and relative frequencies of the leukocyte subpopulations in the peripheral blood of more than 300 healthy volunteers across six different European centers.

A study of 51 subtypes of peripheral blood immune cells in newly diagnosed young type 1 diabetes patients.

Type 1 diabetes (T1D) results from autoimmune destruction of insulin-producing beta cells in pancreatic islets. Various immune cell populations are involved in disease development and natural course. However, to our knowledge, so far there are no comprehensive comparative investigations of all main immune cell populations and their most important subsets at the onset of disease. Therefore, in the current study, we analyzed 51 peripheral blood immune cell populations in 22 young T1D patients and in 25 age-matched controls using a comprehensive polychromatic flow cytometry panel developed for whole blood by the COST Action no. BM0907 ENTIRE (European Network for Translational Immunology Research and Education: From Immunomonitoring to Personalized Immunotherapy) consortium. We found that in T1D patients, frequencies and absolute counts of natural killer (NK) cells, dendritic cells (DC) and T cells, as well as their respective subsets, were significantly altered compared to controls. Further, we observed that changes in several cell populations (e.g. CD14+ CD16+ non-classical monocytes, plasmablasts) were dependent on the age of the patient. In addition to age-related changes, we also found that alterations in immune cell patterns were associated with parameters such as the presence of ketoacidosis and C-peptide serum levels. Our study provides a foundation for future studies investigating different cell lineages and their role in T1D and illustrates the value of polychromatic flow cytometry for evaluating all main peripheral immune cells and their subsets in whole blood samples.

Blood immune cell biomarkers in lung cancer.

Characterization of host immune cell parameters prior to treatment is expected to identify biomarkers predictive of clinical outcome as well as to elucidate why some patients fail to respond to immunotherapy. We monitored blood immune cells from 58 patients with non-small- cell lung cancer (NSCLC) undergoing surgery of the primary tumor and from 50 age-matched healthy volunteers. Complete leukocyte blood count, the number of circulating dendritic cells (DC), HLA-DRlow monocytes and several lymphocytic subpopulations were determined by eight-color flow cytometry. Furthermore, the prognostic value of the immune cell parameters investigated was evaluated by patients' survival analysis. Compared to the control group, blood of NSCLC patients contained more neutrophils resulting in a higher neutrophil-to-lymphocyte ratio (NLR), but a lower number of blood DC, in particular of plasmacytoid DC (pDC), natural killer (NK) cells and naive CD4+ and CD8+ T cells. Furthermore, a higher frequency of CD4+ regulatory T cells (Treg) and HLA-DRlow monocytes was detected, and smoking had a significant impact on these values. HLA-DRlow monocytes were positively correlated to the number of neutrophils, monocytes and NLR, but negatively associated with the number of pDC and naive CD4+ T cells. The frequency of Treg, HLA-DRlow monocytes and naive CD4+ and CD8+ T cells as well as the ratios of CD4/HLA-DRlow monocytes and HLA-DRlow monocytes/pDC correlated with patient's overall survival. Next to Treg, HLA-DRlow monocytes and naive T cells represent prognostic markers for NSCLC patients and might be useful for monitoring of patients' responses to immunotherapies in future studies.

Reduced residual gene expression of nuclear factor of activated T cells-regulated genes correlates with the risk of cytomegalovirus infection after liver transplantation.

BACKGROUND: Pharmacokinetic monitoring of calcineurin inhibitors (CNIs) is unsatisfactory because, at comparable blood concentrations, side effects vary considerably. We recently confirmed the applicability of a pharmacodynamic (PD) assay that measures the suppression of CNI target genes, specifically the suppression of nuclear factor of activated T cells (NFAT)-regulated genes in liver transplant (LT) recipients. The aim of this prospective study was to prove the clinical reliability of this assay. Therefore, we quantified the residual gene expression (RGE) of NFAT-regulated genes and evaluated the association between the RGE of NFAT-regulated genes and the incidence of cytomegalovirus (CMV) infection. PATIENTS AND METHODS: In 20 LT recipients, 10 patients on cyclosporine (CsA) and 10 patients on tacrolimus (Tac) therapy, who presented with CMV infection, the RGEs of interleukin-2, interferon-γ (IFNγ), and granulocyte-monocyte colony-stimulating factor were measured and compared with the RGEs of these cytokines in 40 healthy dose-matched LT controls. RESULTS: CsA-treated CMV patients demonstrated a lower RGE of all NFAT-regulated genes compared with controls (30 ± 17 vs. 44 ± 20, P = 0.067). For IFNγ, the level of significance was reached (26 ± 17 vs. 43 ± 17, P = 0.0125). Daily CsA dosage, CsA baseline (C0 ) and 2 h (C2 ) concentrations were comparable (CsA dosage 169 mg/day vs. 165 mg/day; CsA C0 94 µg/L vs. 85 µg/L; CsA C2 389 µg/L vs. 381 µg/L). In addition, Tac-treated CMV patients demonstrated a lower RGE of all NFAT-regulated genes compared with controls (68 ± 25 vs. 84 ± 22, P = 0.0769). Analogous to CsA-treated CMV patients, the level of significance was reached for IFNγ (61 ± 24 vs. 88 ± 29, P = 0.0154). Daily Tac dosage and Tac 1.5 h concentrations (C1.5 ) were comparable in both groups (Tac dosage 4 mg/day vs. 4 mg/day; Tac C1.5 8 µg/L vs. 10 µg/L), whereas Tac C0 concentrations were significantly higher in controls (Tac C0 4 µg/L vs. 6 µg/L, P = 0.0276). CONCLUSION: Measuring the RGE of NFAT-regulated genes is appropriate to assess the risk of infections in LT recipients. Measuring the RGE of IFNγ is particularly suitable to assess the risk of CMV infection. PD monitoring of CNIs in LT recipients is an approach to individualize immunosuppression, which may help to reduce infectious complications.

The novel c-Met inhibitor cabozantinib overcomes gemcitabine resistance and stem cell signaling in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDA) is one of the most lethal malignancies. Cancer stem cells (CSCs), which are not targeted by current therapies, may be the reason for pronounced therapy resistance. A new treatment option in phase II trials is cabozantinib that inhibits the pancreatic CSC surface marker and tyrosine kinase receptor c-Met. The purpose of this study was to evaluate the effect of cabozantinib to stem-like features and therapy resistance. Established PDA cell lines, a gemcitabine-resistant subclone, non-malignant pancreatic ductal cells and primary spheroidal cultures from patient tumors were analyzed by MTT-assay, flow cytometry, colony and spheroid formation assays, western blotting, qRT-PCR, antibody protein array, immunohistochemistry and morphological features. Cabozantinib inhibited viability and spheroid formation and induced apoptosis in malignant cells with minor effects in non-malignant cells. After long-term cabozantinib treatment, PDA cells had altered anti- and pro-apoptotic signaling, but still responded to cabozantinib, as apoptosis only slightly decreased and viability only slightly increased suggesting a low resistance-inducing potential of cabozantinib. In parallel, c-Met expression and the pluripotency transcription factor SOX2 were downregulated, which might counteract development of full therapy resistance in long-term treated subclones. In single-treatment studies, cabozantinib increased efficacy of gemcitabine. Most importantly, cabozantinib strongly induced apoptosis and reduced viability in PDA cell lines, which are completely resistant toward gemcitabine. In primary, CSC-enriched spheroidal cultures cabozantinib downregulated CSC markers SOX2, c-Met and CD133 and induced apoptosis. These findings suggest that the clinical use of cabozantinib may be more effective than current chemotherapeutics.

Human mucosal CD4+ T cells but not blood CD4+ T cells respond vigorously towards CD28 engagement.

Human lamina propria T lymphocytes (LPT) possess functional properties profoundly different from those of peripheral blood T lymphocytes (PBT). While they are characterized by a low proliferative response to T cell receptor (TCR)/CD3 stimulation in vitro their responsiveness to activation through the 'co-stimulatory' CD2-receptor is enhanced when compared to PBT. In this study, we demonstrate that engagement of another co-stimulatory receptor on both LPT and PBT, namely CD28, by a single monoclonal antibody (mAb), respectively, strongly activates the former but not the latter through a PI3-kinase dependent signalling pathway leading to the production of inflammatory cytokines such as interleukin (IL)-2, tumour necrosis factor (TNF)-α, interferon (IFN)-γ and granulocyte-macrophage colony-stimulating factor (GM-CSF). In addition to the high sensitivity of LPT to CD2 stimulation, this finding supports the notion that 'non-specific/innate' mechanisms to activate T lymphocytes play a predominant role vis-à-vis'TCR driven/adaptive' responses in the intestinal mucosa. Furthermore, it suggests that results from preclinical tests for therapeutic antibodies performed with human blood derived T cells are probably insufficient to predict reactivities of tissue-resident immune cells, which--given their quantitative predominance--may critically determine the in-vivo response to such compounds.

Immunogenicity of SEREX-identified antigens and disease outcome in pancreatic cancer.

Despite spontaneous or vaccination-induced immune responses, pancreatic cancer remains one of the most deadly immunotherapy-resistant malignancies. We sought to comprehend the spectrum of pancreatic tumor-associated antigens (pTAAs) and to assess the clinical relevance of their immunogenicity. An autologous SEREX-based screening of a cDNA library constructed from a pancreatic T3N0M0/GIII specimen belonging to a long-term survivor (36 months) revealed 18 immunogenic pTAA. RT-PCR analysis displayed broad distribution of the identified antigens among normal human tissues. PNLIPRP2 and MIA demonstrated the most distinct pancreatic cancer-specific patterns. ELISA-based screening of sera for corresponding autoantibodies revealed that although significantly increased, the immunogenicity of these molecules was not a common feature in pancreatic cancer. QRT-PCR and immunohistochemistry characterized PNLIPRP2 as a robust acinar cell-specific marker whose decreased expression mirrored the disappearance of parenchyma in the diseased organ, but was not related to the presence of PNLIPRP2 autoantibodies. Analyses of MIA-known to be preferentially expressed in malignant cells-surprisingly revealed an inverse correlation between intratumoral gene expression and the emergence of autoantibodies. MIA(high) patients were autoantibody-negative and had shorter median survival when compared with autoantibody-positive MIA(low) patients (12 vs. 34 months). The observed pTAA spectrum comprised molecules associated with acinar, stromal and malignant structures, thus presenting novel targets for tumor cell-specific therapies as well as for approaches based on the bystander effects. Applying the concept of cancer immunoediting to interpret relationships between gene expression, antitumor immune responses, and clinical outcome might better discriminate between past and ongoing immune responses, consequently enabling prognostic stratification of patients and individual adjustment of immunotherapy.

TNF-alpha antibody treatment in refractory collagenous sprue: report of a case and review of the literature.

Collagenous sprue (CS) is a rare disease characterized by celiac type small bowel malabsorption that is resistant to gluten free diet (GFD) and is associated with a poor prognosis. Our aim was to assess the properties of the monoclonal TNF-alpha antibody infliximab in a patient with high-dose steroid refractory CS. A 27-year-old man developed watery diarrhea with weight loss and abdominal pain. Duodenal biopsies showed a subtotal villous atrophy with an extensive subepithelial layer of collagenous fibers. An apparent GFD did not reduce symptoms. High dose steroid treatment (75 mg prednisone) in combination with azathioprine (150 mg) reduced diarrhea but did not induce complete remission. Based on strongly elevated mucosal TNF-alpha transcript concentrations we introduced infliximab (5 mg/kg body weight) into therapy. After two applications the patient's symptoms quickly improved. During the following year no recurrence of diarrhea has been observed. This case suggests that infliximab is an effective treatment in complicated cases of collagenous sprue.

Ex vivo chemosensitivity testing and gene expression profiling predict response towards adjuvant gemcitabine treatment in pancreatic cancer.

Efficacy of chemotherapy for pancreatic cancer may be improved by tailoring it to individual chemosensitivity profiles. Identification of nonresponders before initiation of treatment may help to avoid side effects. In this study, primary pancreatic cancer cells were isolated from 18 patients undergoing pancreaticoduodenectomy for pancreatic cancer. Eight commonly used pancreatic cancer cell lines were used as controls. Ex vivo chemosensitivity for gemcitabine, 5-fluorouracil, mitomycin-C, cisplatinum, oxaliplatinum, paclitaxel and a combination of gemcitabine with oxaliplatinum or mitomycin-C was determined using a cellular ATP-based tumour chemosensitivity assay (ATP-TCA). Quantitative real-time-polymerase chain reaction was performed to determine RNA expression levels of genes implicated in chemoresistance. Chemosensitivity towards cytotoxic agents was highly variable in primary pancreatic cancer cells and pancreatic cancer cell lines. ATP-TCA results for gemcitabine correlated to the tissue expression of human equilibrative nucleoside transporter-1 (hENT1). Time to relapse in patients with gemcitabine-sensitive tumours was significantly higher than in patients with chemoresistant pancreatic cancers (P=0.01; 71 vs 269 days). Furthermore, time to relapse in gemcitabine-treated patients was related to hENT1 expression (P=0.0067). Thus, chemosensitivity testing using ATP-TCA in pancreatic cancer is feasible and correlated with time to relapse in gemcitabine-treated patients. This suggests that ATP-TCA testing could be used as a decision-making tool in the adjuvant treatment of pancreatic cancer.

Activity of nuclear factor of activated T cells is independent of the number of peripheral lymphocytes in FTY720-treated patients.

UNLABELLED: We studied the inhibition of nuclear factor of activated T-cell (NFAT)-regulated gene expression (interleukin-2 [IL-2], interferon-gamma [IFN-gamma], granulocyte-macrophage colony-stimulating factor [GMCSF]) in cyclosporine (CsA)-treated de novo patients with and without lymphopenia due to FTY720. MATERIALS AND METHODS: Sixteen CsA-treated de novo renal transplant recipients received either FTY720 (n = 8) or mycophenolic acid (MPA; n = 8) in combination with low-dose steroids. Expressions of IL-2, INF-gamma, and GMCSF were measured at 1 (visit 1), 2 (visit 2), and 14 (visit 3) months postoperatively using peripheral lymphocytes obtained at 0 hour versus 2 hours after CsA intake. Gene expression was assessed by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: CsA 0- and 2-hour levels were comparable in both groups. Absolute NFAT-regulated gene expression was significantly lower among FTY720-treated patients at visits 1 and 2. Median residual NFAT-regulated gene expression was 5.9% in the FTY720 group and 4.2% in the MPA-treated group at visit 1, increasing to 7.2% and 7.0%, respectively, at visit 3. One borderline rejection occurred in the MPA group. Median serum creatinine was 1.5 mg/dL among FTY720 and 1.8 mg/dL among MPA patients. CONCLUSIONS: Despite significantly lower expression of NFAT-regulated genes, the relative reduction in NFAT gene expression was comparable in both groups. The absolute number of lymphocytes was not relevant for this immune response. In addition, gene expression increased to comparable levels after FTY720 was switched to MPA. The relative residual gene expression increased with reduction in CsA dose.

Abnormal crosstalk between pancreatic acini and macrophages during the clearance of apoptotic cells in chronic pancreatitis.

In chronic pancreatitis (CP), both the progressive loss of acinar parenchyma and aggressive fibro-inflammatory reactions ultimately lead to irreversible organ destruction. Dying cells are normally removed by macrophages and elimination is associated with anti-inflammatory cytokine switch. We investigated whether defective clearance of damaged acini by macrophages such as compromised phagocytosis or altered cytokine reaction occurs in CP and thus represents a causative link between acinar loss and fibro-inflammation. In a checkerboard-like co-culture system, we assessed normal and CP macrophages for their phagocytic and cytokine responses to dying pancreatic acinar cells of normal or CP origin by FACS, confocal microscopy, QRT-PCR, and ELISA. In CP, phagocytosis of apoptotic acini by macrophages was not impaired; however, the associated cytokine responses were gradually perturbed. Most interestingly, only normal acini suppressed TGFbeta1 expression and accumulation specifically in normal macrophage cultures, while CP acini lost this ability. Both types of apoptotic acini induced pro-inflammatory cytokine bursts of varying strength in both types of macrophages; however, the most significant difference (more than 50-fold higher expression of IL-1beta, IL-6, and IL-8) was evident between CP/CP and normal/normal combinations, indicating that acinar and macrophage alterations synergistically lead to the ultimate CP-specific bias. In combination with in situ data comparing circulating inflammatory cells to pancreatic resident ones, our results indicate that cytokine expression in inflammatory cells undergoes spatiotemporal modulation, most likely through a successive interplay of acinar, stromal, and circulating factors. Thus, clearance of injured pancreatic acini by macrophages is associated with a unique cytokine reaction which may constitute a basis for progression of SAPE (sentinel acute pancreatitis event) to the irreversible fibro-inflammation in CP.

Up-regulation of the phosphoinositide 3-kinase pathway in human lamina propria T lymphocytes.

Human intestinal lamina propria T lymphocytes (LPT), when investigated ex vivo, exhibit functional properties profoundly different from those of peripheral blood T lymphocytes (PBT). One prominent feature represents their enhanced sensitivity to CD2 stimulation when compared to PBT. Given that LPT are hyporesponsive to T cell receptor (TCR)/CD3 stimulation, an alternative activation mode, as mimicked by CD2 triggering in vitro, may be functional in mucosal inflammation in vivo. This study provides insight into signalling events associated with the high CD2 responsiveness of LPT. When compared to PBT, LPT show an increased activation of the phosphoinositide 3/protein kinase B/glycogen synthase kinase 3beta (PI3-kinase/AKT/GSK-3beta) pathway in response to CD2 stimulation. Evidence is provided that up-regulation of this pathway contributes to the enhanced CD2-induced cytokine production in LPT. Given the importance of TCR-independent stimulation for the initiation of intestinal immune responses analysis of signalling pathways induced by 'co-stimulatory' receptors may provide valuable information for therapeutic drug design.

Association between suppressors of cytokine signalling, T-helper type 1/T-helper type 2 balance and allergic sensitization in children.

BACKGROUND: Suppressors of cytokine signalling (SOCS) family members have been shown to play an important role in the balance of cytokines that determine the onset of T-helper type 1 (Th1)- and Th2-mediated immune responses. In particular, for cytokine-induced Src-homology 2 protein (CIS), SOCS1, SOCS3 and SOCS5, a role in the regulation of T cell differentiation has been discussed. However, only few data exist so far in the human system. OBJECTIVES: The aim of the present study was to analyse the relationship between these suppressors and Th1/Th2 regulation as well as allergic sensitizations within a population-based study. METHODS: Within the Lifestyle-Immune system-Allergy plus cohort study, mRNA was prepared from blood samples of 6-year-old children for the analysis of cytokines, transcription factors for T cell regulation and SOCS molecule expression by quantitative real-time polymerase chain reaction. In addition, total and specific IgE concentrations have been measured by the Pharmacia CAP System. A complete data set from 248 children was available. Results Among the SOCS molecules investigated, only SOCS1 showed a strong positive correlation to allergic sensitizations. In addition, an up-regulated SOCS1 expression correlated with down-regulated T-box expressed in T cells (Tbet) and higher expression levels of GATA-binding protein 3 (GATA-3) and IL-4. No association between SOCS1 and forkhead box P3 (FOXP3) was observed. For SOCS3, SOCS5 and CIS, a contradictory picture was found. The expression of these SOCS molecules was positively correlated with Tbet and FOXP3 and (with the exception of CIS) negatively with IL-4. CONCLUSIONS: Our data suggest that SOCS3, SOCS5 and CIS, which correlate with an up-regulated Th1 and regulatory T cell activity, are without relevance for the allergic status. In contrast, SOCS1 might be involved in the development of a Th2-skewed immune response and subsequent allergic sensitizations.

Regulatory CD4+ CD25+ T cells prevent thymic dysfunction in experimental chronic colitis.

Chronic colitis in T-cell deficient Tg epsilon26 mice develops due to a dysfunction of the thymus which generates colitogenic T cells after bone marrow (BM) transplantation. Regulatory CD4+ CD25+ T cells have been shown to prevent colitis in this model by normalizing the peripheral T-cell pool. We tested the hypothesis that T-cell normalization takes place in the thymus. Tg epsilon26 mice were transplanted with BM (BM-->Tg epsilon26 mice) and consequently received either CD4+ CD25+ or CD4+ CD25- cells from syngenic wild type mice. Furthermore, untransplanted Tg epsilon26 mice received CD4+ CD25+ or CD4+ CD25- cells or complete mesenteric lymph node cells. Transfer of regulatory. CD4+ CD25+ cells normalized the total number of thymocytes and the percentage and number of double positive CD4+ CD8+ cells in transplanted mice while percentage of single positive CD4+ and CD8+ thymocytes in BM-->Tg epsilon26 mice was reduced upon CD4+ CD25+ transfer. Timing of CD4+ CD25+ cell injection was important as transfer later than 7 days after BM transplantation failed to prevent abnormal thymic T-cell distribution in BM-->Tg epsilon26 mice. Isolated CD4+ CD25+ cell transfer without preceding BM transplantation failed to reconstitute thymic architecture. Differences of thymic cell composition could not be exclusively explained by presence or absence of colitis, respectively, because 19 days after BM transplantation when both groups showed no histological signs of colitis, animals transferred with CD4+ CD25+ T cells had a significantly higher percentage and number of CD4+ CD25+ thymocytes and CD4+ Foxp3+ cells than BM-->Tg epsilon26 mice. In conclusion, early CD4+ CD25+ cotransfer prevents thymic dysfunction which underlies immune-mediated bowel inflammation in BM-->Tg epsilon26 mice.

Down-regulation of CD55 and CD46 expression by anti-sense phosphorothioate oligonucleotides (S-ODNs) sensitizes tumour cells to complement attack.

Overexpression of one or more membrane-bound complement regulatory proteins (mCRPs) protects tumour cells against complement-mediated clearance by the autologous humoral immune response and is also considered as a barrier for successful immunotherapy with monoclonal anti-tumour antibodies. Neutralization of mCRPs by blocking antibodies, enzymatic removal or cytokine-mediated down-regulation has been shown to sensitize tumour cells to complement attack. In our study we applied, for the first time, anti-sense phosphorothioate oligonucleotides (S-ODNs) to knock down the expression of the mCRPs CD55 and CD46 with the aim of exploiting complement more effectively for tumour cell damage. Potent anti-sense oligonucleotides against CD55 and CD46 were identified by screening various target sequences (n = 10) for each regulator. S-ODN anti-CD55(687) reduced CD55 protein expression up to 84% and CD46 protein expression was inhibited up to 76% by S-ODN anti-CD46(85). Reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed a similar reduction of the CD55 and CD46 mRNA levels, which argues for an RNAse H-dependent anti-sense mechanism. T47D, A549 and PC3 cells, representing breast, lung and prostate carcinoma, were used for functional studies. Dependent on the particular cell line, anti-sense-based inhibition of mCRP expression enhanced complement-dependent cytolysis (CDC) up to 42% for CD55 and up to 40% for CD46, and the combined inhibition of both regulators yielded further additive effects in T47D cells. C3 opsonization of CD55/CD46-deficient tumour cells was also clearly enhanced upon mCRP suppression. Due to the clinical applicability of S-ODNs, the anti-sense approach described in this study may offer an additional alternative to improve the efficacy of antibody- and complement-based cancer immunotherapy.

Regulation and functional role of the Runt-related transcription factor-2 in pancreatic cancer.

Recent evidence suggests that Runt-related transcription factors play a role in different human tumours. In the present study, the localisation of the Runt-related transcription factor-2 (Runx2), its transcriptional activity, as well as its regulation of expression was analysed in human pancreatic ductal adenocarcinoma (PDAC). Quantitative real-time PCR and immunohistochemistry were used for Runx2 expression and localisation analysis. Runt-related transcription factor-2 expression was silenced using specific siRNA oligonucleotides in pancreatic cancer cells (Panc-1) and immortalised pancreatic stellate cells (IPSCs). Overexpression of Runx2 was achieved using a full-length expression vector. TGF-beta1, BMP2, and other cytokines were assessed for their potential to regulate Runx2 expression. There was a 6.1-fold increase in median Runx2 mRNA levels in PDAC tissues compared to normal pancreatic tissues (P<0.0001). Runt-related transcription factor-2 was localised in pancreatic cancer cells, tubular complexes, and PanIN lesions of PDAC tissues as well as in tumour-associated fibroblasts/stellate cells. Coculture of IPSCs and Panc-1 cells, as well as treatment with TGF-beta1 and BMP2, led to increased Runx2 expression in Panc-1 cells. Runt-related transcription factor-2 overexpression was associated with decreased MMP1 release as well as decreased growth and invasion of Panc-1 cells. These effects were reversed by Runx2 silencing. In conclusion, Runx2 is overexpressed in PDAC, where it is regulated by certain cytokines such as TGF-beta1 and BMP2 in an auto- and paracrine manner. In addition, Runx2 has the potential to regulate the transcription of extracellular matrix modulators such as SPARC and MMP1, thereby influencing the tumour microenvironment.

Interleukin-18 is increased only in a minority of patients with active Crohn's disease.

BACKGROUND AND AIMS: It has been suggested that Crohn's Disease (CD) is associated with an elevated T helper 1 response as manifested by increased production of interleukin-18 (IL-18). Local concentrations of neutralizing IL-18 binding proteins (IL-18 bp) may counteract biological functions of mature IL-18 in mucosal inflammation. Therefore, we investigated the IL-18/IL-18 bp system in a large group of patients with active inflammatory bowel disease (IBD) to identify patients that could respond theoretically to IL-18 neutralizing treatment strategies. PATIENT/METHODS: IL-18 and IL-18 bp messenger RNA (mRNA) expression in colonic mucosa from patients with active CD (n = 72), active ulcerative colitis (UC; n = 32), and non-IBD controls (infectious colitis or diverticulitis; n = 19) and normal, non-diseased controls (n = 20) were measured by reverse-transcribed real-time polymerase chain reaction. Mature IL-18 protein and IL-18 bp expression in inflamed mucosa were assessed by Western blotting. RESULTS/FINDINGS: Although IL-18 mRNA was increased in some patients with CD, the increase was not statistically significant. Densitometric evaluation of IL-18/alpha-actin ratio in patients with active CD (n = 20) and patients with UC (n = 10) demonstrated an increased ratio of IL-18 protein in CD when compared to UC (1.04 vs 0.72 [median]). On closer inspections, only 7/20 CD patients had an increased IL-18 protein expression in inflamed areas compared to noninflamed mucosa. INTERPRETATION/CONCLUSION: IL-18 expression in active CD is heterogeneous, only a minority of patients expresses elevated levels. Further treatment strategies targeting IL-18 expression in active CD should be concentrated on this subgroup of patients.

Association of neuropeptides with Th1/Th2 balance and allergic sensitization in children.

BACKGROUND: Among neurogenic factors, the neuropeptides have an important regulatory influence on immune system activity and may lead to allergic sensitization. OBJECTIVE: The aim of our study was to investigate the relationship of the neuropeptides vasoactive intestinal peptide (VIP), somatostatin (SOM) and substance P (SP) on modulation of Th1/Th2 balance and allergic sensitization in children. METHODS: Within the LISAplus (Life style-Immune system-Allergy) study, blood samples of 321 six-year-old children were analysed for concentration of neuropeptides, Th1 and Th2 cytokines, transcription factors for T cell regulation and suppressors of cytokine signalling. In addition, samples were screened for specific IgE against inhalant and food allergens. RESULTS: Children with high SOM values showed a Th2 polarization and a reduced expression of FOXP3, the marker for regulatory T cells. High (VIP) levels correlated inversely with the expression of T cell transcription factors (Tbet and SOCS3). In contrast, elevated levels of SP were associated with reduced GATA3 and SOCS3 expression and with increased IFN-gamma concentrations. Allergic sensitization was more prevalent in children with higher SOM and VIP concentrations but not associated with SP levels. CONCLUSION: Our data reveal an association between neuropeptides and modulatory effects on immune cells in vivo, especially on Th1/Th2 balance with a correlation to allergic sensitization in children. We suggest that elevated SOM and VIP concentrations and the inducing factors should be considered as allergy risk factors.

The ECM proteoglycan decorin links desmoplasia and inflammation in chronic pancreatitis.

BACKGROUND: Recurrent inflammation in chronic pancreatitis (CP) is not well understood. AIMS: To investigate whether decorin, an extracellular matrix (ECM) proteoglycan with macrophage modulating activity, is a pathogenic factor allowing diseased pancreatic stroma to sustain inflammation by affecting the cytokine profile of accumulating inflammatory cells. METHODS: Decorin was examined in 18 donors and 32 patients with CP by quantitative reverse transcription polymerase chain reaction (QRT-PCR), western blotting, and immunohistochemistry of pancreatic specimens. QRT-PCR was used to assess cytokine expression in donor peripheral blood mononuclear cells (PBMC), exposed or not to decorin in vitro, and to compare it with the cytokine profile of circulating and resident mononuclear cells (MNC) of patients with CP. RESULTS: In CP, desmoplasia is associated with overexpression of decorin in the growing ECM and enlarged pancreatic nerves. In culture, exposure of MNC to decorin stimulated expression of the MNC recruiting chemokine MCP-1. In biopsies, MNC infiltrates in decorin rich CP tissue showed a 300-fold upregulation of MCP-1 compared with decorin free peripheral blood, whereas no difference was found in basal MCP-1 expression in PBMC of patients versus donors. This effect was specific for MCP1-other inflammatory cytokines, such as interleukin 1beta and tumour necrosis factor alpha, were not affected. CONCLUSION: Decorin is a molecular marker of desmoplasia in CP, and excessive decorin may allow fibrotic masses to nourish and protract inflammation by deregulating the process of MNC accumulation and activation. These data provide a molecular basis for surgical resection of diseased tissue as a treatment option in CP.