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Colorectal cancer (CRC) screening can effectively reduce cancer-associated mortality. In Germany, individuals over the age of 50 or 55 have access to CRC screening services. However, utilization rates are persistently low, particular in the male population. This observational study investigates the effect of standard versus gender-specific invitation letters on utilization of CRC screening services. We analyzed utilization rates of individuals who were insured by a large health insurance fund in Bavaria, Germany. Persons who became eligible for CRC screening received a standard (2013-2014) or a gender-specific invitation letter (2015-2016). We compared utilization rates within 6 months after receipt of the invitation letter using billing codes of the health insurance fund. Invitation letters were sent to 49 535 individuals, of which 48.8 % were gender-specific. The overall utilization rate did not differ between recipients of the standard versus gender-specific invitation letter (11.6 % vs 11.1 %; RR: 0.97 [0.92-1.02], p = 0.19). However, uptake of screening colonoscopy was significantly higher among recipients of gender-specific invitations (2.9 % vs 3.5 %; RR: 1.21 [1.04-1.39], p = 0.01), whereas utilization of fecal occult blood tests declined (10.4 % vs 9.7 %; RR: 0.93 [0.88-0.99], p = 0.016). Gender-specific design of invitation letters can modify the patients' preference for specific CRC screening services and increase the acceptance of screening colonoscopy. Darmkrebsvorsorgeuntersuchungen können die Krebs-assoziierte Mortalität wirksam reduzieren. In Deutschland haben Personen über 50 bzw. 55 Jahren Zugang zu spezifischen Vorsorgeuntersuchungen. Die Teilnahmerate am Darmkrebsvorsorgeprogramm ist jedoch persistierend niedrig, insbesondere in der männlichen Bevölkerung. Diese Beobachtungsstudie vergleicht den Einfluss von einfachen mit geschlechtsspezifischen Einladungsschreiben auf die Inanspruchnahme von Darmkrebsvorsorgeuntersuchungen. Die Teilnahmerate am Darmkrebsvorsorgeprogramm wurde in einer Kohorte von Personen, die durch eine große, gesetzliche Krankenkasse in Bayern versichert sind, untersucht. Alle Personen, die während des Beobachtungszeitraums das 50. bzw. 55. Lebensjahr erreicht haben, erhielten entweder ein einfaches (2013–2014) oder geschlechtsspezifisches Einladungsschreiben (2015–2016). Die Inanspruchnahme von Vorsorgeuntersuchungen innerhalb von 6 Monaten nach Erhalt des Einladungsschreibens wurde verglichen. Es haben insgesamt 49 535 Personen ein Einladungsschreiben erhalten. Davon waren 48,8 % geschlechtsspezifische Einladungsschreiben. Die Teilnahmerate unterschied sich nicht zwischen Empfängern eines einfachen oder geschlechtsspezifischen Einladungsschreibens (11,6 % vs 11,1 %; RR 0,97 [0,92–1,02], p = 0,19). Die Inanspruchnahme von Vorsorgekoloskopien war jedoch signifikant höher bei Personen, die ein geschlechtsspezifisches Einladungsschreiben erhielten (2,9 % vs 3,5 %; RR 1,21 [1,04–1,39], p = 0,01). Hingegen war die Nutzung von Tests für okkultes Blut im Stuhl in der gleichen Gruppe geringer (10,4 % vs 9,7 %; RR 0,93 [0,88–0,99], p = 0,016). Geschlechtsspezifische Einladungsschreiben können die Präferenz von Patienten für spezifische Vorsorgeuntersuchungen verändern und die Inanspruchnahme von Vorsorgekoloskopien erhöhen.

GATA2 deficiency is characterized by monocytopenia, deficiency of dendritic cells, and a variable degree of lymphocytopenia affecting B cells and NK cells, leading to an enhanced risk of mycobacterial, viral, and fungal infections. Here we present a patient with a heterozygous intronic GATA2 mutation who acquired a fatal disseminated mycosis due to the black yeast-like fungus Arthrocladium fulminans following an infection with Mycobacterium sherrisii. This case illustrates that in patients with severe uncommon infections, immunodeficiency syndromes must be ruled out.

Although the oncolytic parvovirus H-1PV has entered clinical trials, predicting therapeutic success remains challenging. We investigated whether the antiviral state in tumor cells determines the parvoviral oncolytic efficacy. The interferon/interferon-stimulated genes (IFN/ISG)-circuit and its major configurator, human endogenous retroviruses (HERVs), were evaluated using qRT-PCR, ELISA, Western blot, and RNA-Seq techniques. In pancreatic cancer cell lines, H-1PV caused a late global shutdown of innate immunity, whereby the concomitant inhibition of HERVs and IFN/ISGs was co-regulatory rather than causative. The growth-inhibitory IC50 doses correlated with the power of suppression but not with absolute ISG levels. Moreover, H-1PV was not sensitive to exogenous IFN despite upregulated antiviral ISGs. Such resistance questioned the biological necessity of the oncotropic ISG-shutdown, which instead might represent a surrogate marker for personalized oncolytic efficacy. The disabled antiviral homeostasis may modify the activity of other viruses, as demonstrated by the reemergence of endogenous AluY-retrotransposons. This way of suppression may compromise the interferogenicity of drugs having gemcitabine-like mechanisms of action. This shortcoming in immunogenic cell death induction is however amendable by immune cells which release IFN in response to H-1PV.

A vaccine remains a priority in the global fight against malaria. Here, we report on a single-center, randomized, double-blind, placebo and adjuvant-controlled, dose escalation phase 1a safety and immunogenicity clinical trial of full-length Plasmodium falciparum merozoite surface protein 1 (MSP1) in combination with GLA-SE adjuvant. Thirty-two healthy volunteers were vaccinated at least three times with MSP1 plus adjuvant, adjuvant alone, or placebo (24:4:4) to evaluate the safety and immunogenicity. MSP1 was safe, well tolerated and immunogenic, with all vaccinees sero-converting independent of the dose. The MSP1-specific IgG and IgM titers persisted above levels found in malaria semi-immune humans for at least 6 months after the last immunization. The antibodies were variant- and strain-transcending and stimulated respiratory activity in granulocytes. Furthermore, full-length MSP1 induced memory T-cells. Our findings encourage challenge studies as the next step to evaluate the efficacy of full-length MSP1 as a vaccine candidate against falciparum malaria (EudraCT 2016-002463-33).

Background and Aim Participation rates in the German colorectal cancer screening program are low. Starting in 2013, a large health insurance plan in Bavaria, Germany, is sending an additional invitation letter to insured individuals when they turn 50 or 55 years and become eligible for participation in the program. The letter provides detailed information on colorectal cancer screening. We assessed the impact of the invitation letter on utilization rates. Methods Insurance claims data of a total of 48 343 individuals who had turned 50 or 55 years between 2012 to 2014 were reviewed for utilization rates of screening colonoscopy and fecal blood tests. Utilization rates 1 year prior (2012) and 2 years after introduction of the invitation letter (2013 and 2014) were compared. Furthermore, providers of colorectal cancer screening were determined. Results Within 6 months after turning 50 or 55 years, 8.8 – 10.2 % of all insured individuals participated in colorectal cancer screening, with the majority being females. After the introduction of the invitation letter, a moderate increase in participation rates could be observed (increase to 109 % [RR 101.7 – 117.3 %, p = 0.02] in 2014). The uptake rate of screening colonoscopy was significantly higher in recipients of the letter (increase to 138.4 % [RR 110.4 – 173.8 %, p = 0.0043] in 2013 and to 149 % [RR 119.5 – 186.3 %, p = 0.0003] in 2014). Furthermore, a significantly higher proportion of general practitioners and gastroenterologists provided colorectal cancer screening in individuals receiving the invitation letter. Conclusions Introduction of an invitation letter can improve participation rates for colorectal cancer screening.

SARS-CoV-2 infection can lead to severe acute respiratory distress syndrome with the need of invasive ventilation. Pulmonary herpes simplex-1 (HSV-1) reactivation in invasively ventilated patients is a known phenomenon. To date very little is known about the frequency and the predisposing factors of HSV-1 reactivation in COVID-19. Therefore, we evaluated our cohort of invasively ventilated COVID-19 patients with severe pneumonia for HSV-1 in respiratory specimens and combined these results with functional immunomonitoring of the peripheral blood. Tracheal secretions and bronchial lavages were screened by PCR for HSV-1 positivity. Comprehensive immunophenotyping and quantitative gene expression analysis of Interferon-stimulated genes (IFI44L, MX1, RSAD2, ISIG15 and IFIT1) and IL-1 beta were performed in whole blood. Time course of infection beginning at symptom onset was grouped into three phases (“early” phase 1: day 1–10, “middle” phase 2: day 11–30 and “late” phase 3: day 31–40). Pulmonary HSV-1 reactivation was exclusively observed in the later phases 2 and 3 in 15 of 18 analyzed patients. By FACS analysis a significant increase in activated CD8 T cells (CD38+HLADR+) in phase 2 was found when compared with phase 1 (p+HLADR+ CD8 T cells.

Gene expression regulated by the transcription factor NFAT (nuclear factor of activated T-cells) has been proposed for monitoring the pharmacodynamic effect of calcineurin inhibitors. We aimed to correlate the pharmacokinetics of tacrolimus with the suppression of NFAT-regulated gene expression. Tacrolimus trough (Csubtrough/sub) and peak concentrations (Csubpeak/sub) were measured by LC-MS. The effect on NFAT-regulated gene expression at trough (Esubtrough/sub) and at peak levels (Esubpeak/sub) were determined by qRT-PCR. The pharmacodynamic concentration producing the half-maximum effect (CEsub50/sub) and the Hill coefficient (H) were estimated from Esubtrough/suband from Esubpeak/sub. Ten stable kidney transplant recipients on triple immunosuppression with prednisolone, mycophenolate, and tacrolimus were analyzed. Median age was 58 years, median time since transplant was 84 months, and median serum creatinine was 249 µmol/L. The immunosuppressive effect on NFAT-regulated genes at trough concentrations was 38% (Esubtrough/sub), and the effect at peak concentrations was 59% (Esubpeak/sub) of maximum immunosuppression (Esubmax/sub). The pharmacodynamic parameters of the action of tacrolimus were estimated with the Hill coefficient H at 1.5 and the CE50 at 6.7 ng/mL. Accordingly, the pharmacodynamic threshold concentration was estimated at 0.9 ng/mL and the ceiling concentration at 48 ng/mL, indicating a wide span between target trough and peak levels. The low Hill coefficient indicates concentration-dependent pharmacodynamics of tacrolimus on NFAT transcripts. Therefore, the extension of the administration interval to 24 hours is not likely to jeopardize the immunosuppressive effect of the prolonged-release tacrolimus preparations. .

Pancreatic ductal adenocarcinoma (PDAC) displays a marked fibro-inflammatory microenvironment in which infiltrated immune cells fail to eliminate the tumor cells and often&mdash, rather paradoxically&mdash, promote tumor progression. Of special interest are tumor-promoting T cells that assume a Th17-like phenotype because their presence in PDAC tissue is associated with a poor prognosis. In that context, the role of IL-21, a major cytokine released by Th17-like cells, was assessed. In all tissue samples (n = 264) IL-21+ immune cells were detected by immunohistochemistry and high density of those cells was associated with poor prognosis. In the majority of patients (221/264), tumor cells expressed the receptor for IL-21 (IL-21R) and also a downstream target of IL-21, Blimp-1 (199/264). Blimp-1 expression closely correlated with IL-21R expression and multivariate analysis revealed that expression of both IL-21R and Blimp-1 was associated with shorter survival time of the patients. In vitro data using pancreatic tumor cells lines provided a possible explanation: IL-21 activated ERK and STAT3 pathways and upregulated Blimp-1. Moreover, IL-21 increased invasion of tumor cell lines in a Blimp-1-dependent manner. As an in vivo correlate, an avian xenograft model was used. Here again Blimp-1 expression was significantly upregulated in IL-21 stimulated tumor cells. In summary, our data showed an association of IL-21+ immune cell infiltration and IL-21 receptor expression in PDAC with poor survival, most likely due to an IL-21-mediated promotion of tumor cell invasion and enhanced colony formation, supporting the notion of the tumor-promoting abilities of the tumor microenvironment.

Introduction: Cytomegalovirus (CMV) reactivation occurs in seronegative patients after solid organ transplantation (SOT) particularly from seropositive donors and can be lethal. Generation of CMV-specific T cells helps to prevent CMV reactivation. Therefore, we initiated a clinical phase I CMVpp65 peptide vaccination trial for seronegative end-stage renal disease patients waiting for kidney transplantation. Methods: The highly immunogenic nonamer peptide NLVPMVATV derived from CMV phosphoprotein 65(CMVpp65) in a water-in-oil emulsion (Montanide™) plus imiquimod (Aldara™) as an adjuvant was administered subcutaneously four times biweekly. Clinical course as well as immunological responses were monitored using IFN-γ ELISpot assays and flow cytometry for CMV-specific CD8+ T cells. Results: Peptide vaccination was well tolerated, and no drug-related serious adverse events were detected except for Grade I–II local skin reactions. Five of the 10 patients (50%) mounted any immune response (responders) and 40% of the patients presented CMV-specific CD8+ T cell responses elicited by these prophylactic vaccinations. No responders experienced CMV reactivation in the 18 months post-transplantation, while all non-responders reactivated. Conclusion: CMVpp65 peptide vaccination was safe, well tolerated, and clinically encouraging in seronegative end-stage renal disease patients waiting for kidney transplantation. Further studies with larger patient cohorts are planned.

We tested the hypothesis that proliferative activity of hematopoietic stem cells has impact on survival in newly diagnosed patients with myelodysplastic syndrome (MDS) and secondary acute myeloid leukemia (AML). RNA expression profiles of CD34(+) cells were analyzed in 125 MDS patients and compared to healthy controls. Prognostic impact on overall survival (OS) of mRNA proliferation signatures established for solid tumor cells was analyzed retrospectively. For validation on the protein level, immunofluorescence and immunohistochemistry analyses in bone marrow (BM) biopsies were performed, and an independent cohort of 223 MDS and secondary AML patients was investigated. Lower proliferative activity correlated with the expression of cyclin-dependent kinase inhibitor 1C (CDKN1C) and with shorter OS (p

The pathogenesis of intrahepatic biliary stricture formation in patients with primary sclerosing cholangitis (PSC) or after liver transplantation (LTx) remains elusive. CD14 receptor signaling is a key mediator of the innate immune system; its common genetic variant is associated with alcoholic liver disease. PSC and LTx cohort patients and primary biliary cirrhosis (PBC) control patients were genotyped for the CD14 -260C>T (rs2569190) polymorphism, and genotypes were correlated with long-term clinical outcome. Biliary tissue, bile, and whole blood of PSC patients and healthy controls were screened for markers of the innate immune system and bacterial infection. In 121 PSC patients, the CD14 -260C>T genotype was associated with development of dominant bile duct strictures (P = 0.02). In 365 LTx patients, TT carriers (4.1%) were protected against the formation of nonanastomotic biliary strictures versus CC/CT patients (12.6%; P = 0.01). Chemokine ligand 8 (P = 0.04) and chemokine receptor 6 (P = 0.004) were up-regulated in biliary tissue of PSC patients with the TT versus the CC/CT genotype. Lipopolysaccharide whole-blood stimulation resulted in a significant change in interleukin (IL)-8 (P = 0.05) and IL-12p40 levels (P = 0.04) in healthy control subjects carrying the TT genotype. TT PSC patients were protected against Gram-negative bacterial biliary infection (TT: 0% vs. CC/CT: 22.5%; P = 0.02). Serum-soluble CD14 levels correlated with the CD14 -260C>T genotype (P = 0.02), representing an independent risk indicator of survival in PSC patients (hazard ratio, 0.40; 95% confidence interval, 0.19-0.86; P =0.01). Conclusions: The function of the innate immune response by CD14 is crucial during biliary infection and stricture formation. The benefits of CD14 signaling modification should be addressed in future studies. (Hepatology 2016;64:843-852)

Item does not contain fulltext BACKGROUND: Cytotoxic T-lymphocyte antigen 4 (CTLA-4) is a negative immune regulator. Heterozygous CTLA4 germline mutations can cause a complex immune dysregulation syndrome in human subjects. OBJECTIVE: We sought to characterize the penetrance, clinical features, and best treatment options in 133 CTLA4 mutation carriers. METHODS: Genetics, clinical features, laboratory values, and outcomes of treatment options were assessed in a worldwide cohort of CTLA4 mutation carriers. RESULTS: We identified 133 subjects from 54 unrelated families carrying 45 different heterozygous CTLA4 mutations, including 28 previously undescribed mutations. Ninety mutation carriers were considered affected, suggesting a clinical penetrance of at least 67%; median age of onset was 11 years, and the mortality rate within affected mutation carriers was 16% (n = 15). Main clinical manifestations included hypogammaglobulinemia (84%), lymphoproliferation (73%), autoimmune cytopenia (62%), and respiratory (68%), gastrointestinal (59%), or neurological features (29%). Eight affected mutation carriers had lymphoma, and 3 had gastric cancer. An EBV association was found in 6 patients with malignancies. CTLA4 mutations were associated with lymphopenia and decreased T-, B-, and natural killer (NK) cell counts. Successful targeted therapies included application of CTLA-4 fusion proteins, mechanistic target of rapamycin inhibitors, and hematopoietic stem cell transplantation. EBV reactivation occurred in 2 affected mutation carriers after immunosuppression. CONCLUSIONS: Affected mutation carriers with CTLA-4 insufficiency can present in any medical specialty. Family members should be counseled because disease manifestation can occur as late as 50 years of age. EBV- and cytomegalovirus-associated complications must be closely monitored. Treatment interventions should be coordinated in clinical trials.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive neoplasm characterized by a marked fibro-inflammatory microenvironment, the presence of which can promote both cancer induction and growth. Therefore, selective manipulation of local cytokines is an attractive, although unrealized, therapeutic approach. T cells possess a unique mechanism of p38 mitogen-activated protein kinase (MAPK) activation downstream of T cell receptor (TCR) engagement through the phosphorylation of Tyr323 (pY323). This alternative p38 activation pathway is required for pro-inflammatory cytokine production. Here we show in human PDAC that a high percentage of infiltrating pY323(+) T cells was associated with large numbers of tumor necrosis factor (TNF)-α- and interleukin (IL)-17-producing CD4(+) tumor-infiltrating lymphocytes (TILs) and aggressive disease. The growth of mouse pancreatic tumors was inhibited by genetic ablation of the alternative p38 pathway, and transfer of wild-type CD4(+) T cells, but not those lacking the alternative pathway, enhanced tumor growth in T cell-deficient mice. Notably, a plasma membrane-permeable peptide derived from GADD45-α, the naturally occurring inhibitor of p38 pY323(+) (ref. 7), reduced CD4(+) TIL production of TNF-α, IL-17A, IL-10 and secondary cytokines, halted growth of implanted tumors and inhibited progression of spontaneous KRAS-driven adenocarcinoma in mice. Thus, TCR-mediated activation of CD4(+) TILs results in alternative p38 activation and production of protumorigenic factors and can be targeted for therapeutic benefit.

Introduction Calcineurin inhibitors are critical-dose drugs with a narrow therapeutic range and optimal monitoring strategies are discussed in terms of safety and efficacy. A new pharmacodynamic monitoring tool - assessing the expression of nuclear factor of activated T-cells (NFAT)-regulated genes - has been established to directly measure the functional effect of cyclosporine A (CsA) in an individual patient. Until now, only sparse data on NFAT-regulated gene expression within the early post-transplant period have been available. Method Altogether 80 de novo renal transplant patients were enrolled in this non-interventional cohort-study. Immunosuppression consisted of interleukin (IL)-2 receptor antagonist induction, CsA, mycophenolic acid and steroids. Expression of NFAT-regulated genes (IL-2, granulocyte-macrophage colony stimulating-factor (GM-CSF), interferon-γ (IFN-γ)) was determined by qRT-PCR (real-time reverse transcription-PCR) at CsA C0 (prior to CsA intake) and C2 (2 hours after CsA intake) at regular follow-up visits within 6 months after transplantation. Results The median age of all patients was 47.9 ± 13.7 years (54 male). Residual NFAT-regulated gene expression showed a high interindividual variability. Inversely to reduction of CsA doses, NFAT-regulated genes increased from 1.78 ± 1.33% to 8.04 ± 7.36% in month 1 to month 6. Despite comparable CsA C0 levels, NFAT-regulated gene expression was significantly less inhibited in patients with treated biopsy-proven acute rejections (2.9 ± 2.2% vs. 2.0 ± 1.7%, p = 0.047). Patients with very low residual expression of NFAT-regulated genes were at an increased risk for early infectious episodes. Residual expression of IFN-γ and GM-CSF genes correlated significantly with clinical outcomes. Conclusion NFAT-regulated gene expression is highly inhibited in the early post-transplant period in renal allograft recipients on CsA treatment. High residual NFAT-regulated gene expression was related to acute rejection episodes and low residual expression with infectious complications. Thus, NFAT-monitoring has the potential to support pharmacokinetic monitoring during the early post-transplant period.

Targeting early molecular events in intestinal inflammation may represent a useful therapeutic strategy for maintaining remission in inflammatory bowel disease. Recently, we established an intestinal organ culture model (LEL model), which allows to study the initiation of an intestinal inflammatory response in human tissue. In this model, EDTA-mediated depletion of epithelial cells of colonic mucosa results in an instantaneous inflammatory response in resident lamina propria cells, which shows features of intestinal inflammation in vivo. Furthermore, activated immune cells emigrate from the lamina propria onto the luminal side of the basement membrane. Here, we standardize the LEL model and explore its suitability for drug testing. To this end, human mucosal punches of defined surface area were prepared, depleted of epithelial cells, and cultured at an optimized ratio of medium volume/punch area. The intra-assay variability of measurements of inflammatory parameters ranged from 13% for cell migration to 19% for secretion and 30% for tissue gene expression, respectively, of the inflammatory mediators IL-8 and IL-6. Importantly, known suppressive effects of dexamethasone, a drug employed for the treatment of inflammatory bowel diseases, on leucocyte migration, IL8, IL6, and TNF-α production as well as CD86 surface expression by myeloid cells were observed in this model. In conclusion, the present results suggest that the LEL model may represent a useful human experimental system not only for studying initial activation mechanisms in intestinal inflammation but also for evaluating drug compounds for the treatment of mucosal inflammation.

// Verena M. Throm 1 , David Mannle 1 , Thomas Giese 2 , Andrea S. Bauer 3 , Matthias M. Gaida 4 , Juergen Kopitz 4 , Thomas Bruckner 5 , Konstanze Plaschke 1 , Svetlana P. Grekova 1 , Klaus Felix 1 , Thilo Hackert 1 , Nathalia A. Giese 1 and Oliver Strobel 1 1 European Pancreas Centre/EPZ, Department of General, Visceral and Transplantation Surgery, University Hospital Heidelberg, Heidelberg, Germany 2 Institute of Immunology, University Hospital Heidelberg, Heidelberg, Germany 3 Department of Functional Genomics, DKFZ, Heidelberg, Germany 4 Institute of Pathology, University Hospital Heidelberg, Heidelberg, Germany 5 Institute of Medical Biometry and Informatics/IMBI, University Hospital Heidelberg, Heidelberg, Germany Correspondence to: Nathalia A. Giese, email: nathalia.giese@med.uni-heidelberg.de Keywords: SLURP1; CHRNA7; pancreatic cancer; nicotine Received: March 13, 2017 Accepted: December 05, 2017 Published: January 24, 2018 ABSTRACT Smoking is associated with increased risk and poorer prognosis of pancreatic ductal adenocarcinoma (PDAC). Nicotine acts through cholinergic nicotinic receptors, preferentially α7 (CHRNA7) that also binds the endogenous ligand SLURP1 (Secreted Ly-6/uPAR-Related Protein 1). The clinical significance of SLURP1 and its interaction with nicotine in PDAC are unclear. We detected similar levels of SLURP1 in sera from healthy donors and patients with chronic pancreatitis or PDAC; higher preoperative values were associated with significantly better survival in patients with resected tumors. Pancreatic tissue was not a source of circulating SLURP1 but contained diverse CHRNA7-expressing cells, preferentially epithelial and immune, whereas stromal stellate cells and a quarter of the tumor cells lacked CHRNA7. The CHRNA7 mRNA levels were decreased in PDAC, and CHRNA7 high -PDAC patients lived longer. In CHRNA7 high COLO357 and PANC-1 cultures, opposing activities of SLURP1 (anti-malignant/CHRNA7-dependent) and nicotine (pro-malignant/CHRNA7-infidel) were exerted without reciprocally interfering with receptor binding or downstream signaling. These data suggested that the ligands act independently and abolish each other’s effects through a mechanism resembling functional antagonism. Thus, SLURP1 might represent an inborn anti-PDAC defense being sensitive to and counteracting nicotine. Boosting SLURP1-CHRNA7 interaction might represent a novel strategy for treatment in high-risk individuals, i.e., smokers with pancreatic cancer.

Steroid-resistant nephrotic syndrome (SRNS) causes 15% of chronic kidney disease cases. A mutation in 1 of over 40 monogenic genes can be detected in approximately 30% of individuals with SRNS whose symptoms manifest before 25 years of age. However, in many patients, the genetic etiology remains unknown. Here, we have performed whole exome sequencing to identify recessive causes of SRNS. In 7 families with SRNS and facultative ichthyosis, adrenal insufficiency, immunodeficiency, and neurological defects, we identified 9 different recessive mutations in SGPL1, which encodes sphingosine-1-phosphate (S1P) lyase. All mutations resulted in reduced or absent SGPL1 protein and/or enzyme activity. Overexpression of cDNA representing SGPL1 mutations resulted in subcellular mislocalization of SGPL1. Furthermore, expression of WT human SGPL1 rescued growth of SGPL1-deficient dpl1. yeast strains, whereas expression of disease-associated variants did not. Immunofluorescence revealed SGPL1 expression in mouse podocytes and mesangial cells. Knockdown of Sgpl1 in rat mesangial cells inhibited cell migration, which was partially rescued by VPC23109, an S1P receptor antagonist. In Drosophila, Sply mutants, which lack SGPL1, displayed a phenotype reminiscent of nephrotic syndrome in nephrocytes. WT Sply, but not the disease-associated variants, rescued this phenotype. Together, these results indicate that SGPL1 mutations cause a syndromic form of SRNS.

Next-generation DNA sequencing has accelerated the genetic characterization of many human primary immunodeficiency diseases (PIDs). These discoveries can be lifesaving for the affected patients and also provide a unique opportunity to study the effect of specific genes on human immune function. In the past 18 months, a number of independent groups have begun to define novel PIDs caused by defects in the caspase recruitment domain family, member 11 (CARD11) –B-cell chronic lymphocytic leukemia/lymphoma 10 (BCL10) –mucosa-associated lymphoid tissue lymphoma translocation gene 1 (MALT1 [CBM]) signalosome complex. The CBM complex forms an essential molecular link between the triggering of cell-surface antigen receptors and nuclear factor κB activation. Germline mutations affecting the CBM complex are now recognized as the cause of novel combined immunodeficiency phenotypes, which all share abnormal nuclear factor κB activation and dysregulated B-cell development as defining features. For this "Current perspectives" article, we have engaged experts in both basic biology and clinical immunology to capture the worldwide experience in recognizing and managing patients with PIDs caused by CBM complex mutations.

Bacterial infections of bones remain a serious complication of endoprosthetic surgery. These infections are difficult to treat, because many bacterial species form biofilms on implants, which are relatively resistant towards antibiotics. Bacterial biofilms elicit a progressive local inflammatory response, resulting in tissue damage and bone degradation. In the majority of patients, replacement of the prosthesis is required. To address the question of how the local inflammatory response is linked to bone degradation, tissue samples were taken during surgery and gene expression of the macrophage inflammatory proteins MIP1α(CCL3) and MIP2α(CXCL2) was assessed by quantitative RT-PCR. MIPs were expressed predominantly at osteolytic sites, in close correlation with CD14 which was used as marker for monocytes/macrophages. Colocalisation of MIPs with monocytic cells could be confirmed by histology. In vitro experiments revealed that, aside from monocytic cells, also osteoblasts were capable of MIP production when stimulated with bacteria; moreover, CCL3 induced the differentiation of monocytes to osteoclasts. In conclusion, the multifunctional chemokines CCL3 and CXCL2 are produced locally in response to bacterial infection of bones. In addition to their well described chemokine activity, these cytokines can induce generation of bone resorbing osteoclasts, thus providing a link between bacterial infection and osteolysis.

Our aim was to establish and characterize a novel pancreatic ductal adenocarcinoma cell line from a patient in whom the origin of the invasive carcinoma could be traced back to the intraductal papillary mucinous neoplasm (IPMN) precursor lesion.The primary patient-derived tumor was propagated in immunocompromised mice for 2 generations and used to establish a continuous in vitro culture termed ASAN-PaCa. Transplantation to fertilized chicken eggs confirmed the tumorigenic potential in vivo. Molecular analyses included karyotyping, next-generation genomic sequencing, expression analysis of marker proteins, and mucin-profiling.The analysis of marker proteins confirmed the epithelial nature of the established cell line, and revealed that the expression of the mucin MUC1 was higher than that of MUC2 and MUC5AC. ASAN-PaCa cells showed rapid in vitro and in vivo growth and multiple chromosomal aberrations. They harbored mutations in KRAS (Q61H), TP53 (Y220C), and RNF43 (I47V and L418M) but lacked either IPMN-specific GNAS or presumed pancreatic ductal adenocarcinoma-driving mutations in KRAS (codons 12/13), SMAD, and CDKN2A genes.ASAN-PaCa cell line represents a novel preclinical model of pancreatic adenocarcinoma arising in the background of IPMN, and offers an opportunity to study how further introduction of known driver mutations might contribute to pancreatic carcinogenesis.

Purpose: Environmental conditions in lymph node proliferation centers protect chronic lymphocytic leukemia (CLL) cells from apoptotic triggers. This situation can be mimicked by in vitro stimulation with CD40 ligand (CD40L) and interleukin 4 (IL-4). Our study investigates the impact of the drug leflunomide to overcome apoptosis resistance of CLL cells. Experimental Design: CLL cells were stimulated with CD40L and IL-4 and treated with fludarabine and the leflunomide metabolite A771726. Results: Resistance to fludarabine-mediated apoptosis was induced by CD40 activation alone stimulating high levels of BCL-XL and MCL1 protein expression. Apoptosis resistance was further enhanced by a complementary Janus-activated kinase (JAK)/STAT signal induced by IL-4. In contrast, CLL proliferation required both a CD40 and a JAK/STAT signal and could be completely blocked by pan-JAK inhibition. Leflunomide (A771726) antagonized CD40L/IL-4–induced proliferation at very low concentrations (3 μg/mL) reported to inhibit dihydroorotate dehydrogenase. At a concentration of 10 μg/mL, A771726 additionally attenuated STAT3/6 phosphorylation, whereas apoptosis of CD40L/IL-4–activated (“resistant”) CLL cells was achieved with higher concentrations (IC50: 80 μg/mL). Apoptosis was also effectively induced by A771726 in clinically refractory CLL cells with and without a defective p53 pathway. Induction of apoptosis involved inhibition of NF-κB activity and loss of BCL-XL and MCL1 expression. In combination with fludarabine, A771726 synergistically induced apoptosis (IC50: 56 μg/mL). Conclusion: We thus show that A771726 overcomes CD40L/IL-4–mediated resistance to fludarabine in CLL cells of untreated as well as clinically refractory CLL cells. We present a possible novel therapeutic principle for attacking chemoresistant CLL cells. Clin Cancer Res; 18(2); 417–31. ©2011 AACR.

Oncolytic viruses with their capacity to specifically replicate in and kill tumor cells emerged as a novel class of cancer therapeutics. Rat oncolytic parvovirus (H-1PV) was used to treat different types of cancer in preclinical settings and was lately successfully combined with standard gemcitabine chemotherapy in treating pancreatic ductal adenocarcinoma (PDAC) in rats. Our previous work showed that the immune system and particularly the release of interferon-gamma (IFNγ) seem to mediate the anticancer effect of H-1PV in that model. Therefore, we reasoned that the therapeutic properties of H-1PV can be boosted with IFNγ for the treatment of late incurable stages of PDAC like peritoneal carcinomatosis. Rats bearing established orthotopic pancreatic carcinomas with peritoneal metastases were treated with a single intratumoral (i.t.) or intraperitoneal (i.p.) injection of 5 x 10⁸ plaque forming units of H-1PV with or without concomitant IFNγ application. Intratumoral injection proved to be more effective than the intraperitoneal route in controlling the growth of both the primary pancreatic tumors and peritoneal carcinomatosis, accompanied by migration of virus from primary to metastatic deposits. Concomitant i.p. treatment of H-1PV with recIFNγ resulted in improved therapeutic effect yielding an extended animal survival, compared with i.p. treatment with H-1PV alone. IFNγ application enhanced the H-1PV-induced peritoneal macrophage and splenocyte responses against tumor cells while causing a significant reduction in the titers of H1-PV-neutralising antibodies in ascitic fluid. Thus, IFNγ co-application together with H-1PV might be considered as a novel therapeutic option to improve the survival of PDAC patients with peritoneal carcinomatosis.

The potential advantage of using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) methodology to detect metastasis in sentinel lymph nodes (SLNs) of breast cancer (BC) patients was evaluated in this prospective study. We measured the expression of relevant gene transcripts in SLNs using an innovative algorithm and compared the results of single-marker assays versus multi-marker assays with conventional histological detection methods. SLNs from women aged ≥ 18 years diagnosed with unilateral BC were examined by haematoxylin-eosin staining and immunohistochemistry and analysed for transcripts of several relevant genes using qRT-PCR (learning group). Four candidate panels of expressed transcript combinations with high sensitivity and specificity were selected for further investigation. The candidate panels were then validated using SLNs from a second group of BC patients (validation group). In the learning group, 74/314 SLN sections from 150 patients were positive for metastasis by histology. The transcripts analysed showed the following individual sensitivities/specificities: cytokeratin 19 (CK19) 94.6%/97.9%; mammaglobin 1 (MGB1) 82.4%/91.7%; mammaglobin 2 (MGB2) 82.4%/96.7%; carcinoembryonic antigen (CEA) 71.6%/97.5%; EPCAM (epithelial cell adhesion molecule) 91.9%/97.1%; and NY-BR-1 82.4%/93.8%. The optimal panel based on the predefined criteria comprised four markers: CK19, MGB1, EPCAM, and NY-BR-1, of which ≥ 2 had to be positive (95.9% sensitivity, 95.0% specificity, 85.5% positive predictive value (PPV), and 98.7% negative predictive value (NPV)). Overall concordance with histology was 95.2%. In the validation group, 84/315 SLN sections from 235 patients were histologically positive, and panel sensitivity, specificity and overall accuracy were 88.1, 95.2 and 93.3%, respectively, at the SLN section level. In conclusion, molecular staging using expression patterns of relevant transcripts in SLNs could serve as a useful complement to standard diagnostic work-up in BC patients. The proposed flexible multi-parametric approach does not improve the overall accuracy compared with the single-marker approach. However, it overcomes several limitations of the previously reported molecular assays for SLN diagnosis.

Pharmacokinetic monitoring of CsA is unsatisfactory, because at comparable CsA blood concentrations, the frequency and severity of adverse effects vary considerably among patients. We have therefore recently developed a precise, reliable, and robust whole-blood pharmacodynamic assay that measures the suppression of CsA-target genes in T lymphocytes. Because of the different characteristics of CsA pharmacokinetics in children and the higher propensity for infectious complications, this assay requires validation in the pediatric patient population. We therefore quantified in a prospective study of 45 pediatric renal transplant recipients the residual expression of NFAT-regulated genes in lymphocytes by RT-PCR and correlated these findings with the frequency of recurrent infections in the maintenance period post-transplant. Patients with recurrent infections showed a significantly stronger inhibition of NFAT-regulated gene expression (18.2%) than patients without recurrent infections (31.7%; p = 0.012). This difference was specific, because various PK parameters of CsA and the concomitant immunosuppressive therapy were comparable between patients. Multivariate regression analysis showed that patient age and residual NFAT-regulated gene expression were the only independent determinants of recurrent infections. By ROC curve analysis, a cutoff value of 23% residual NFAT-regulated gene expression had the highest sensitivity (71.1%) and specificity (65.4%) for the discrimination of patients with and without recurrent infections. Pharmacodynamic monitoring of CsA by measurement of residual NFAT-regulated gene expression in T lymphocytes has the potential to identify over-immunosuppressed pediatric renal transplant recipients and is therefore a useful tool for the optimization of CsA therapy.

Herpes simplex virus type 1 (HSV-1) is one of the most frequent and successful human pathogens. It targets immature dendritic cells (iDCs) to interfere with the antiviral immune response. The mechanisms underlying apoptosis of HSV-1-infected iDCs are not fully understood. Previously, we have shown that HSV-1-induced apoptosis of iDCs is associated with downregulation of the cellular FLICE-inhibitory protein (c-FLIP), a potent inhibitor of caspase-8-mediated apoptosis. In this study, we prove that HSV-1 induces degradation of c-FLIP in a proteasome-independent manner. In addition, by using c-FLIP-specific small interfering RNA (siRNA) we show for the first time that downregulation of c-FLIP expression is sufficient to drive uninfected iDCs into apoptosis, underlining the importance of this molecule for iDC survival. Surprisingly, we also observed virus-induced c-FLIP downregulation in epithelial cells and many other cell types that do not undergo apoptosis after HSV-1 infection. Microarray analyses revealed that HSV-1-encoded latency-associated transcript (LAT) sequences, which can substitute for c-FLIP as an inhibitor of caspase-8-mediated apoptosis, are much less abundant in iDCs as compared to epithelial cells. Finally, iDCs infected with an HSV-1 LAT knockout mutant showed increased apoptosis when compared to iDCs infected with the corresponding wild-type HSV-1. Taken together, our results demonstrate that apoptosis of HSV-1-infected iDCs requires both c-FLIP downregulation and diminished expression of viral LAT.

Long-term immunosuppression causes a significantly increased risk for the development of malignancies in transplanted patients. A link between immunosuppression and incidence of cancer is well documented and involves the effect of immunosuppression on antitumor surveillance and antiviral adaptive immune responses. Using a recently described pharmacodynamic assay, a strong correlation between the incidence of malignancies and the individual degree of immunosuppression after cyclosporin A treatment in patients with kidney transplants was observed. The availability of a quantitative and quick laboratory test for the assessment of the individual functional activity of immunocompetent cells crucial for transplant rejection, defense against viral infection and tumor surveillance, along with the ability to adjust doses of immunosuppressive agents such that patients are largely protected against malignant disease and/or viral infection while maintaining a stable allograft function, represents an enormous breakthrough in transplantation medicine and advances our attempts to individualize treatment in transplanted patients.

Long-term immunosuppression causes a significantly increased risk for the development of malignancies in transplanted patients. A link between immunosuppression and incidence of cancer is well documented and involves the effect of immunosuppression on anti-tumor surveillance and antiviral adaptive immune responses. We present a 67-year-old patient with a history of recurrent non-melanoma skin cancer. After adjustment of immunosuppressive therapy under close pharmacodynamic control, the development of new malignant lesions could be prevented. The availability of a quantitative, quick laboratory test for an assessment of the individual functional activity of immunocompetent cells that are crucial for transplant rejection, defense against viral infection, and tumor surveillance along with the ability to adjust doses of immunosuppressive agents such that patients are largely protected against malignant disease and/or viral infection are important. NFAT-regulated gene expression measured in peripheral blood allowed us to predict "safe" immunosuppression. Thus patients could maintain a stable allograft function. This represents a breakthrough in transplantation medicine and advances our attempts to individualize treatment in transplanted patients.

Human cytomegalovirus (HCMV) is a ubiquitous pathogen with a predilection for dendritic cells (DCs). Latently infected myeloid progenitor cells develop into actively infected DCs with impaired functionality, allowing dissemination and transfer of virus throughout the body. However, the viral genes expressed in DCs and their effect on the cellular transcriptome are currently unknown. We investigated human DCs infected with HCMV by using SuperSAGE, allowing us to analyse the transcriptomes of both host and pathogen simultaneously. A small number of viral transcripts were expressed strongly and rapidly post-infection. However, only two were of the immediate-early class, including one with an unknown function. The viral genes expressed reflected the cellular milieu, with the majority having a known or suspected immune-evasion function. Several viral genes identified lack a known function and may fulfil specialized roles within DCs. The cellular response to infection included a strong interferon response, induction of cytokine and anti-apoptotic genes and alterations in genes involved in antigen presentation. We demonstrated the validity of our approach by showing that novel changes first seen in the transcriptome were reflected in the phenotype of HCMV-infected DCs. Delineation of the transcriptional changes underlying the phenotype of HCMV-infected DCs allows a better understanding of how a herpesvirus infects DCs and pinpoints linkages between phenotype and specific viral genes.

The chemokine fractalkine induces migration of inflammatory cells into inflamed tissues, thereby aggravating inflammatory tissue damage and fibrosis. Furthermore, fractalkine increases neuropathic pain through glial activation, which can be diminished by blocking of its receptor, CX3CR1, through neutralizing antibodies. As chronic pancreatitis (CP) is characterized by tissue infiltration of inflammatory cells, fibrosis, pancreatic neuritis and severe pain, the roles of fractalkine and CX3CR1 were investigated in CP (n=61) and normal pancreas (NP, n=21) by QRT-PCR, western blot and immunohistochemistry analyses. Their expression correlated with the severity of pancreatic neuritis, fibrosis, intrapancreatic nerve fiber density and hypertrophy, pain, CP duration and with the amount of inflammatory cell infiltrate immuno-positive for CD45 and CD68. To investigate the influence of fractalkine on pancreatic fibrogenesis, human pancreatic stellate cells (hPSCs) were isolated from patients with CP, incubated with fractalkine and then Collagen-1 and alpha-smooth muscle actin (alpha-SMA) expressions were measured. CX3CR1, but not fractalkine, mRNA was overexpressed in CP. In contrast, the protein levels of both CX3CR1 and fractalkine were upregulated. Neuro-immunoreactivity for fractalkine and CX3CR1 was strongest in patients suffering from severe pain and pancreatic neuritis. Long-term suffering from CP was noticeably related to increased neural immunoreactivity of fractalkine. Furthermore, fractalkine and CX3CR1 mRNA overexpressions were associated with enhanced lymphocyte and macrophage infiltration. Advanced fibrosis was associated with increased fractalkine expression, whereas in vitro fractalkine had no significant impact on collagen-1 and alpha-SMA expressions in hPSCs. Therefore, pancreatic fractalkine expression appears to be linked to visceral pain and to the recruitment of inflammatory cells into the pancreatic tissue and nerve fibers, with subsequent pancreatic neuritis. However, pancreatic fibrogenesis is probably indirectly influenced by fractalkine. Taken together, these novel findings suggest that CX3CR1 represents a potential novel therapeutic target to reduce inflammation and modulate pain in CP.

Systemic and local inflammatory processes have a key, mainly detrimental role in the pathophysiology of ischemic stroke. Currently, little is known about endogenous counterregulatory immune mechanisms. We examined the role of the key immunomodulators CD4(+)CD25(+) forkhead box P3 (Foxp3)(+) regulatory T lymphocytes (T(reg) cells), after experimental brain ischemia. Depletion of T(reg) cells profoundly increased delayed brain damage and deteriorated functional outcome. Absence of T(reg) cells augmented postischemic activation of resident and invading inflammatory cells including microglia and T cells, the main sources of deleterious cerebral tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma), respectively. Early antagonization of TNF-alpha and delayed neutralization of IFN-gamma prevented infarct growth in T(reg) cell-depleted mice. Intracerebral interleukin-10 (IL-10) substitution abrogated the cytokine overexpression after T(reg) cell depletion and prevented secondary infarct growth, whereas transfer of IL-10-deficient T(reg) cells in an adoptive transfer model was ineffective. In conclusion, T(reg) cells are major cerebroprotective modulators of postischemic inflammatory brain damage targeting multiple inflammatory pathways. IL-10 signaling is essential for their immunomodulatory effect.

We investigated the expression pattern and clinical significance of the costimulatory ligands B7-1, B7-2, B7-H1, and B7-DC, and their counter-receptors CTLA-4 and PD-1 in pancreatic cancer. Gene expression of all examined costimulatory molecules was significantly upregulated in pancreatic cancer tissues. B7-1, B7-2, B7-H1, and B7-DC protein was detectable in pancreatic cancer cells. Only the expression of B7-H1 significantly correlated with postoperative survival (p

Introduction: Among elderly allograft recipients non-melanoma skin cancer (NMSC) is the most common malignancy. We have previously shown that malignancies are associated with a higher intensity of ciclosporin A (CsA)-induced immunosuppression. Method: Fifty-five long-term elderly renal transplant patients with a stable transplant function had regular skin examinations. The expression of the nuclear factor of activated T cells (NFAT)-regulated genes (interleukin-2, granulocyte-macrophage colony stimulating-factor, interferon-γ) was determined by real-time PCR at CsA trough levels and two h after oral intake. Results: The CsA dose was 2.0 mg/kg (0.95–3.50), with CsA trough level (C0) level 97 μg/L (33–157) and CsA two-h level (C2) 538 μg/L (350–1228). NMSC was diagnosed in 14/55 patients (25.4%). A total of 85.7% of allograft recipients with NMSC were male (p

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.

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 (P0.0001). 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.

Background & Aims: The functional involvement of the endocannabinoid system in modulation of pancreatic inflammation, such as acute pancreatitis, has not been studied to date. Moreover, the therapeutic potential of cannabinoids in pancreatitis has not been addressed. Methods: We quantified endocannabinoid levels and expression of cannabinoid receptors 1 and 2 (CB1 and CB2) in pancreas biopsies from patients and mice with acute pancreatitis. Functional studies were performed in mice using pharmacological interventions. Histological examination, serological, and molecular analyses (lipase, myeloperoxidase, cytokines, and chemokines) were performed to assess disease pathology and inflammation. Pain resulting from pancreatitis was studied as abdominal hypersensitivity to punctate von Frey stimuli. Behavioral analyses in the open-field, light-dark, and catalepsy tests were performed to judge cannabinoid-induced central side effects. Results: Patients with acute pancreatitis showed an up-regulation of cannabinoid receptors and elevated levels of endocannabinoids in the pancreas. HU210, a synthetic agonist at CB1 and CB2, abolished abdominal pain associated with pancreatitis and also reduced inflammation and decreased tissue pathology in mice without producing central, adverse effects. Antagonists at CB1- and CB2-receptors were effective in reversing HU210-induced antinociception, whereas a combination of CB1- and CB2-antagonists was required to block the anti-inflammatory effects of HU210 in pancreatitis. Conclusions: In humans, acute pancreatitis is associated with up-regulation of ligands as well as receptors of the endocannabinoid system in the pancreas. Furthermore, our results suggest a therapeutic potential for cannabinoids in abolishing pain associated with acute pancreatitis and in partially reducing inflammation and disease pathology in the absence of adverse side effects.

Background Neuropeptides, such as substance P (SP), are mediators of neurogenic inflammation and play an important role in inflammatory disorders. To further investigate the role of the SP pathway in inflammatory bowel disease (IBD), we analyzed the following in normal intestinal tissue specimens and in tissue specimens from patients with Crohn's disease (CD) and ulcerative colitis (UC): neurokinin receptor-1 (NK-1R); its isoforms (NK-1R-L and NK-1R-S); its ligand SP, encoded by preprotachykinin-A (PPT-A); and the SP-degradation enzyme, neutral endopeptidase (NEP). Methods Real-time quantitative reverse transcription–polymerase chain reaction was used to simultaneously determine the expression of NK-1R-L, NK-1R-S, and PPT-A. Protein levels of NK-1R and NEP were determined by immunoblot analysis. Results In noninflamed small-bowel tissue samples of CD patients, PPT-A mRNA expression was significantly increased, whereas there was no difference between inflamed or noninflamed UC and normal intestinal tissue samples. Examining subgroups of diverse intestinal segments from CD and UC samples with various levels of inflammation revealed no differences in NK-1R-L and NK-1R-S mRNA expression, whereas there was a tendency toward overall lower NK-1R-S mRNA copy numbers. Immunoblot analysis showed upregulation of NK-1R protein levels in cases of IBD, with more pronounced enhancement in cases of CD than in UC. For NEP, there were no differences in protein levels in normal, CD, and UC intestinal tissues. Comments These observations suggest a contribution of SP and its receptor, NK-1R, in the local inflammatory reaction in IBD and particularly in ileal CD. Moreover, significant upregulation of PPT-A mRNA in the noninflamed ileum of these patients suggests an influence of inflamed intestines on their healthy counterparts.

Background: Concentrations of proinflammatory cytokines are increased in the intestinal mucosa of patients with active Crohn's disease (CD). In a prospective study we investigated whether cytokines can predict long-term remission (>6 months) in patients with steroid-refractory CD receiving treatment with infliximab or cyclophosphamide, followed by azathioprine or methotrexate. Methods: Cytokine transcripts were quantified using real-time polymerase chain reaction (PCR) in mucosal biopsies from 19 patients with active, steroid-refractory CD before and 8 weeks after initiation of therapy. Patients were treated with cyclophosphamide (monthly treatment of 750 mg cyclophosphamide intravenously) or infliximab (5 mg/kg body weight) and were followed until relapse of the disease. Statistical analysis was performed to identify predictive factors to discriminate between patients with or without long-term remission. Results: Seventeen out of 19 patients achieved remission of the disease, two patients were nonresponders, while six out of 17 patients exhibited an early recurrence. Pretreatment TNF-α, IL-18, MRP-14, and IL-8 transcripts were significantly correlated with long-term remission. While several cytokines, most importantly MMP-1, determined after 8 weeks were able to predict patients achieving long-term remission, only a decrease of TNF-α levels after 8 weeks was predictive. Overall, statistical analysis identified lower pretreatment TNF-α levels as the strongest predictor of long-term remission among baseline variables. Conclusions: Quantification of mucosal TNF-α transcripts prior to therapy allows identification of patients achieving long-term remission upon immunosuppression with infliximab or cyclophosphamide. Real-time PCR might have considerable potential in the analysis of disease activity and subsequent clinical management of patients with immunosuppressive therapies. (Inflamm Bowel Dis 2007;13:65–70)

Neural alterations and aberrantly expressed nerve-specific factors promoting tumor progression are known to contribute to pancreatic cancer's extremely poor prognosis. Despite hints that axon guidance factor semaphorin 3A (SEMA3A) may function as a tumor inhibitor, its clinical importance and therapeutic potential have not yet been explored. The present study investigated the role of SEMA3A and its receptors—plexins A1–A4 (PLXNA1–A4) and neuropilin-1 (NRP1)—in pancreatic cancer. QRT-PCR and immunohistochemical analyses revealed overexpression of SEMA3A, NRP1 and PLXNA1 in metaplastic ducts, malignant cells and nerves of cancerous specimens, and showed that elevated levels of corresponding mRNA (6.8-fold, 2.0-fold and 1.5-fold, respectively) clearly correlated with negative clinicopathological manifestations such as shorter survival (SEMA3A and PLXNA1) and a lesser degree of tumor differentiation (NRP1) in Stages I–III patients. High SEMA3A expression in pancreata of Stage IV M1 patients and in peritoneal metastases, and consequent functional studies indicated that poor clinical outcome might be related to the ability of SEMA3A to promote dissemination and invasiveness of pancreatic cancer cells through activation of multiple pathways involving Rac1, GSK3b or p42/p44 MAPK, but not E- to N-cadherin switch, MMP-9 or VEGF induction. Thus, this study is the first to quantify expression of the SEMA3A system in human malignancy and to show that overexpression of SEMA3A by nerves and transformed cells leads to a SEMA3A-rich environment which may favor malignant activities of tumor cells. Furthermore, negative clinicopathological correlations suggest that SEMA3A might represent a novel intervention target but not a treatment option for pancreatic cancer patients. © 2007 Wiley-Liss, Inc.

Bone sialoprotein (BSP) is an acidic glycoprotein that plays an important role in cancer cell growth, migration and invasion. The expression, localization and possible function of BSP in chronic pancreatitis (CP) and pancreatic ductal adenocarcinoma (PDAC) were analyzed by QRT-PCR, laser capture microdissection, DNA microarray analysis, immunoblotting, radioimmunoassays and immunohistochemistry as well as cell growth, invasion, scattering, and adhesion assays. BSP mRNA was detected in 40.7% of normal, in 80% of CP and in 86.4% of PDAC samples. The median BSP mRNA levels were 6.1 and 0.9copies/microl cDNA in PDAC and CP tissues, respectively, and zero copies/microl cDNA in normal pancreatic tissues. BSP was weakly present in the cytoplasm of islet cells and ductal cells in 20% of normal pancreatic tissues. BSP was localized in the tubular complexes of both CP and PDAC, as well as in pancreatic cancer cells. Five out of 8 pancreatic cancer cell lines expressed BSP mRNA. Recombinant BSP (rBSP) inhibited Capan-1 and SU8686 pancreatic cancer cell growth, with a maximal effect of -46.4+/-12.0% in Capan-1 cells and -45.7+/-14.5% in SU8686 cells. rBSP decreased the invasion of SU8686 cells by -59.1+/-11.2% and of Capan-1 cells by -13.3+/-3.8% (P

Adrenomedullin (ADM) is synthesized by different types of cells and acts by binding calcitonin receptor-like receptor (CRLR) and members of the receptor activity-modifying protein (RAMP) family. In this study, the expression and functional role of ADM and its signaling components were investigated in pancreatic adenocarcinoma (PDAC). By QRT-PCR, median mRNA levels of ADM and CRLR were 1.5- and 2.4-fold higher, respectively, in PDAC tissues compared to normal pancreatic tissues. By immunohistochemistry, ADM, CRLR, RAMP1 and RAMP2, but not RAMP3, were expressed in pancreatic cancer cells. ADM serum levels were significantly increased in PDAC patients compared to healthy controls and chronic pancreatitis (CP) patients, with an area under the ROC curve of 0.83 and 0.98, respectively. At a cut-off level of 30.6 ng/ml, the specificity of ADM to differentiate PDAC from controls and CP patients was 85.5 and 83.6%, with a sensitivity of 80 and 100%. All 5 evaluated pancreatic cancer cells lines expressed ADM, CRLR, RAMP1 and RAMP2, whereas RAMP3 was expressed in only 1/5 pancreatic cancer cell lines. ADM was strongly induced by hypoxia and significantly increased invasiveness in 3/5 human pancreatic cancer cells. Blocking of CRLR decreased invasiveness in 4/5 human pancreatic cancer cells. In addition, rADM slightly up-regulated vascular endothelial growth factor secretion in 3/5 cell lines. In conclusion, ADM is induced by hypoxia and over-expressed in PDAC and might therefore serve as a potential tumor marker. Furthermore, ADM increases invasiveness of some pancreatic cancer cells and might influence angiogenesis, suggesting that blocking this pathway might have a therapeutic potential.

Chronic pancreatitis as an inflammatory process characterized by morphological changes, pancreatic dysfunction, and pain. During pancreatic injury and repair the Notch signaling pathway is reinstated. The current study analyzed this pathway in chronic pancreatitis and characterized its influence on fibrogenesis. Real-time quantitative PCR and immunohistochemistry were used for expression studies. Notch activation was determined by a specific luciferase-HES-1-reporter gene constructs. Cells were stimulated with alcohol, glucose, bile acids, and steroids. Notch-2, -3, and -4 mRNA, were overexpressed in chronic pancreatitis specimens. The ligands Jagged-1, -2, and Delta-1 were highly overexpressed. Jagged-1 and Notch receptors were observed in nerves, regenerating exocrine cells, and endocrine cells. Delta staining was present in ductal but not in acinus cells and not in nerves. Activation of Notch signaling was detectable upon cell stimulation with glucose, steroids, and bile acids. High glucose levels were further associated with increased collagen-I production. The Notch pathway is reactivated during chronic pancreatitis. Among the stimuli activating the Notch pathway are steroids, high glucose levels, and bile acids. These findings suggest a possible role of the Notch pathway during pancreatic regeneration since Jagged-1 inhibits inducible collagen-1 production, suggesting a new mechanism of tissue repair in this disease.

BACKGROUND: At present it is unclear which dose and consecutive blood levels of cyclosporine A (CsA) are optimal with respect to immunosuppressive efficacy and drug specific side effects at the level of individual patients. Several pharmacodynamic measures of CsA effects have been proposed, but have not become clinical routine yet. Besides the lack of practicability, the biological relevance of these assays has not been determined so far. METHODS: Residual expression of nuclear factor of activated T-cells (NFAT)-regulated genes two hours after drug intake was used as molecular pharmacodynamic marker to assess CsA effects on lymphocytes and correlated with the frequency of recurrent infections and malignancies in patients with five or more years of follow-up posttransplantation. RESULTS: Recurrent infectious complications were observed in 44% and malignancies in 20% of the 133 patients studied. Patients with a strong suppression of NFAT-regulated genes by CsA--as judged by a residual level of transcription of less than 15% after drug intake--develop more frequent infections (53% vs. 29%; P = 0.005) and malignancies (22% vs. 4%; P = 0.002). The lack of correlation between the incidence of these complications and CsA blood concentration might point to the interindividual differences in the sensitivity towards calcineurin inhibition. CONCLUSION: The data presented here reveal a clear relation between the frequency of infectious and malignant complications and the degree of suppression of NFAT-regulated genes by CsA in transplanted patients. Therefore, pharmacodynamic monitoring of CsA efficacy in transplanted patients might be a useful tool to adjust immunosuppressive therapy in individual patients.

To analyze the role of Artemin in pancreatic ductal adenocarcinoma (PDAC) in terms of expression, influence on cancer cell behavior, and pain correlation.PDAC is characterized by prominent local nerve alterations and perineural invasion, which frequently affects the extrapancreatic nerve plexus, causing severe pain and precluding curative resection. Artemin, a neurotrophic protein controlling growth, regeneration, and survival of neurons was analyzed to highlight the neuro-cancer interactions in PDAC.Artemin and its receptors (GFRalpha3/RET) were studied in PDAC tissues and normal pancreas by Western blot analysis and immunohistochemistry. RNA expression was analyzed in pancreatic tissues (normal, cancer) and pancreatic cancer cell lines by QRT-PCR. To evaluate whether Artemin influences cancer cell proliferation and invasion, MTT-growth and Matrigel-invasion assays were used. In addition, the tissue expression of Artemin was correlated with pain in PDAC.Artemin and GFRalpha3/RET were both detected at enhanced levels in PDAC compared with normal pancreas, localizing predominantly in hypertrophic nerves and arterial walls, as well as in cancer cells of primary and metastatic lesions. The levels of Artemin and GFRalpha3 did not correlate with pain in PDAC patients. However, Artemin promoted pancreatic cancer cell invasion up to 5-fold, without affecting cancer cell proliferation.Artemin expression was not associated with pain in PDAC. However by increasing cancer cell invasion, Artemin seems to influence neural invasion and thereby contribute to cancer cell spreading along pancreatic nerves.

The EGF family of ligands and receptors plays an important role in the pathogenesis of pancreatic ductal adenocarcinoma (PDAC) and contributes to its aggressiveness. A number of molecular approaches have been developed to block these pathways, and studies have already proven the clinical benefit of this concept in PDAC. In the present study, we sought to determine the potential role of heregulins (HRGs), a family of EGF-like growth factors, in PDAC. Quantitative RT-PCR analysis revealed that HRGs as well as its signaling ErbB receptors were present in 4 of 4 human pancreatic cancer cell lines (PCCL). HRG-β1 stimulated the growth of 3 of 4 PCCL, whereas HRG-α1 inhibited cell growth in 3 of 4 cell lines. Responses towards HRGs could in part be predicted by ErbB2 and ErbB3 expression levels. HRGs induced phosphorylation of different ErbB receptors as well as activation of MAPK, p38MAPK, JNK and PI3K in a cell- and ligand-specific manner. In vivo, HRG was upregulated in pancreatic cancer tissues and localized predominantly in the cancer cells. High HRG-β levels but not HRG-α levels were associated with decreased patient survival. In conclusion, HRG is expressed by pancreatic cancer cells and influences pancreatic cancer cell growth and patient survival. © 2006 Wiley-Liss, Inc.

Background/objectives Meaningful profiling of pancreatic cancer samples is particularly challenging due to their complex cellular composition. Beyond tumor cells, surgical biopsies contain desmoplastic stroma with infiltrating inflammatory cells, adjacent normal parenchyma, and "non-pancreatic tissues". The risk of misinterpretation rises when the heterogeneous cancer tissues are sub-divided into smaller fragments for multiple analytic procedures. Pre-analytic histological evaluation is the best option to characterize pancreatic tissue samples. Our aim was to develop a complement or alternative procedure to determine the cellular composition of pancreatic cancerous biopsies, basing on intra-analytic molecular annotation. A standard process for sample stratification at a molecular level does not yet exist. Particularly in the case of retrospective or data depository-based studies, when hematoxylin-eosin stained sections are not available, it supports the correct interpretation of expression profiles. Methods A five-gene transcriptional signature ( RNA CellStrat) was defined that allows cell type-specific stratification of pancreatic tissues. Testing biopsy material from biobanks with this procedure demonstrated high correspondence of molecular (qRT-PCR and microarray) and histologic (hematoxylin-eosin stain) evaluations. Results Notably, about a quarter of randomly selected samples (tissue fragments) were exposed as inappropriate for subsequent clinico-pathological interpretation. Conclusions Via immediate intra-analytical procedure, our RNA-based stratification RNA CellStrat increases the accuracy and reliability of the conclusions drawn from diagnostic and prognostic molecular information.

Background and Purpose: The mechanism behind aggressive development of cachexia in patients suffering from pancreatic cancer is not well understood. In this study, we investigated which factors are associated with the cachectic status of the patients and evaluated cachexia-promoting capacity of cancer and inflammatory cells. Experimental Design: DNA microarray analysis and quantitative reverse transcription-PCR were used to screen for cachexia-associated factors in pancreatic specimens obtained from noncachectic and cachetic patients diagnosed with pancreatic ductal adenocarcinoma. The expression pattern of the most prominently altered cachexia-associated factor, interleukin-6 (IL-6), was further analyzed in patients sera by ELISA, in pancreatic specimens by immunohistochemistry, and in a coculture system by quantitative reverse transcription-PCR using pancreatic cancer cell lines T3M4 (IL-6 positive) and Panc-1 (IL-6 negative) and peripheral blood mononuclear cells (PBMC) obtained from donors and noncachectic and cachectic patients. Results: Among numerous analyzed factors, IL-6 was significantly overexpressed in pancreatic specimens and elevated in serum of cachectic patients. The coculture system revealed that pancreatic cancer T3M4 cells but not Panc-1 cells were able to stimulate IL-6 exclusively in cachectic PBMC (by 14-fold) and this triggering was reduced by half in the presence of IL-6-neutralizing antibodies. Conclusion: IL-6 represents a prominent cachexia-associated factor in pancreatic cancer. IL-6 overexpression in cachectic patients is related to the ability of certain tumors to sensitize PBMC and induce cytokine expression in cachectic PBMC.

Objective: We sought to examine the expression and functional role of osteonectin in primary and metastatic pancreatic ductal adenocarcinoma (PDAC). Background: The glycoprotein osteonectin plays a vital role in cell–matrix interactions and is involved in various biologic processes. Overexpression of osteonectin is present in malignant tumors and correlates with disease progression and poor prognosis. Methods: Expression of osteonectin was analyzed by quantitative polymerase chain reaction and immunohistochemistry in pancreatic tissues and by enzyme-linked immunosorbent assay in the serum of patients and donors. Recombinant osteonectin and specific antisense oligonucleotides were used to examine the effects of osteonectin on induction of target genes, and on proliferation and invasiveness of pancreatic cancer cells. Results: There was a 31-fold increase in osteonectin mRNA levels in PDAC and a 16-fold increase in chronic pancreatitis as compared with the normal pancreas (P 0.01). By immunohistochemistry, faint immunoreactivity was detected in the normal pancreas. In contrast, strong staining of the cancer cells was observed in addition to extensive osteonectin immunoreactivity in surrounding fibroblasts and in the extracellular matrix. In metastatic tissues, strong immunoreactivity was observed in fibroblasts and in extracellular matrix surrounding metastatic cancer cells, whereas the signal was absent in most tumor cells. In vitro studies showed that osteonectin was able to inhibit cancer cell growth while promoting invasiveness of pancreatic tumor cells. Conclusion: Osteonectin is markedly overexpressed in pancreatic cancer and has the potential to increase the invasiveness of pancreatic cancer cells. (Ann Surg 2005;242: 224 –234)

Background and aims: Conflicting results exist about the presence of Mycobacterium avium subspecies paratuberculosis (MAP) specific IS 900 DNA in Crohn’s disease (CD) tissues. Therefore, we examined IS 900 in a large number of gut samples from patients with CD (n = 100) and ulcerative colitis (UC, n = 100), and in non-inflamed control tissues (nIBD, n = 100). We hypothesised that IS 900 DNA detection might be associated with distinct clinical phenotypic characteristics in CD. Methods: The prevalence of MAP DNA in surgically resected tissues was examined using a mechanical-enzymatic disruption technique and nested IS 900 specific polymerase chain reaction (PCR). CD patients were stratified according to the criteria of the Vienna classification and other clinical characteristics. Results: IS 900 PCR detection rate was significantly higher in CD tissue samples (52%) than in UC (2%) or nIBD (5%) specimens (p 900 DNA was detected in samples from both diseased small bowel (47%) as well as from the colon (61%). No firm association between MAP specific IS 900 detection rates and clinical phenotypic characteristics in CD could be established. However, corticosteroid medication constituted a factor which tended to have a negative influence on IS 900 DNA detection rates in CD (p Conclusions: The presence of MAP specific IS 900 DNA is a predominant feature of CD. Therapeutic intervention against MAP might represent a potential target for disease mitigation in Crohn’s disease.

Background & Aims: Regulatory CD25+ T cells (Treg) are effective in the prevention and down-regulation of inflammatory bowel disease (IBD) in animal models. Functional Treg cells are characterized by the expression of the transcription factor FOXP3 and show a CD4+CD25high phenotype in humans. The aim of this study was to determine whether disease activity in IBD correlates with changes in frequency of Treg cells and their distribution in the intestinal mucosa. Methods: Treg cells were analyzed from peripheral blood and from biopsy specimens of IBD patients, inflammatory controls, and healthy volunteers by flow cytometry (CD4+CD25high), immunochemistry (FOXP3), and real-time PCR (FOXP3). Regulatory properties of purified peripheral CD4+CD25high Treg cells were determined by their suppressive effect on the proliferation of CD4+CD25− T cells. Results: In peripheral blood, CD4+CD25high T cells from IBD patients retain their suppressive activity. CD4+CD25high and FOXP3+ Treg cells are increased during remission but decreased during active disease. This contrasts with their strong increase in peripheral blood of patients with acute diverticulitis. Different than peripheral blood, inflamed IBD mucosa contains an increased number of CD4+CD25high T cells, FOXP3+ T cells, and transcripts for FOXP3 compared with noninflamed mucosa. However, the increase of FOXP3+ T cells in IBD lesions is significantly lower compared with inflammatory controls. Conclusions: The frequency of CD4+CD25+ Treg cells varies with IBD activity. Active IBD is not associated with a functional defect but with a contraction of the peripheral blood Treg pool and an only moderate expansion in intestinal lesions. Thus, compensatory mechanisms, numerically, are not successfully achieved in these diseases.

Altered expression of apoptosis-regulating genes plays an important role in the aggressive growth behavior and chemoresistance of pancreatic ductal adenocarcinoma. In the present study, the hypoxia-inducible proapoptotic gene, BNIP3, was analysed in terms of expression, effect on patient survival, and chemo-responsiveness in pancreatic cancer cell lines. cDNA microarray, real-time light cycler quantitative polymerase chain reaction, laser-capture microdissection, and immunohistochemistry analyses were used to evaluate BNIP3 expression in normal and diseased pancreatic specimens. Modulation of BNIP3 expression was achieved using specific siRNA molecules. The effect of chemotherapeutic agents on pancreatic cancer cells was assessed utilizing 3-(4,5-methylthiazol-2-yl)-2,5-diphenyl-tetrazolium-bromide assays. BNIP3 mRNA levels were 3.0- and 6.3-fold lower in chronic pancreatitis and pancreatic cancer compared to the normal pancreas, respectively. Microdissection analysis confirmed the reduction of BNIP3 expression in pancreatic cancer cells compared to normal duct cells. By immunohistochemistry, BNIP3 was predominantly expressed in the acinar cells of the normal and diseased pancreas. Interestingly, while BNIP3 was undetectable in the cancer cells of 59% of the cases, 75-100% of PanIN2/3 lesions displayed BNIP3 immunoreactivity. Loss of BNIP3 expression correlated with poorer survival of patients (8 vs 14 months for BNIP3 negative vs positive tumors). Hypoxia induced BNIP3 expression in four out of eight pancreatic cancer cell lines, while it was absent under normoxic and hypoxic conditions in the remaining four. Downregulation of BNIP3 resulted in increased resistance to 5-fluoro-uracil and gemcitabine. In conclusion, loss of BNIP3 expression occurs late in pancreatic cancer, contributes to resistance to chemotherapy, and correlates with a worsened prognosis.