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Cytokines released during chronic inflammatory diseases induce pro-inflammatory properties in polymorphonuclear neutrophils (PMNs). Here, we describe the development of a subgroup of human PMNs expressing CCR5, termed CCR5

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.

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.

Aims Calcineurin inhibitors (CNI) have a small therapeutic window, and drug monitoring is required. Pharmacokinetic monitoring does not correlate sufficiently with clinical outcome. Therefore, the expression of nuclear factor of activated T cells (NFAT)-regulated genes in the peripheral blood has been suggested as a potentially useful immune monitoring tool to optimize CNI therapy. NFAT-regulated gene expression (RGE) was evaluated in renal allograft recipients as predictive biomarker to detect patients at risk of acute rejection or infections. Methods NFAT-RGE (interleukin-2, interferon-gamma, granular-macrophage colony-stimulating factor) was evaluated by quantitative real-time polymerase chain reaction in whole blood samples at day 7, day 14, month 1, 3, and 6 after transplantation in 64 de novo renal allograft recipients from 3 European centres. Immunosuppression consisted of tacrolimus (Tac), mycophenolic acid, and corticosteroids. Results Tac concentrations (C0 and C1.5) correlated inversely with NFAT-RGE (P < .01). NFAT-RGE showed a high interindividual variability (1-61%). Patients with high residual gene expression (NFAT-RGE >= 30%) were at the increased risk of acute rejection in the following months (35 vs. 5%, P = .02), whereas patients with low residual gene expression (NFAT-RGE

Purpose Biallelic pathogenic NBAS variants manifest as a multisystem disorder with heterogeneous clinical phenotypes such as recurrent acute liver failure, growth retardation, and susceptibility to infections. This study explores how NBAS-associated disease affects cells of the innate and adaptive immune system. Methods Clinical and laboratory parameters were combined with functional multi-parametric immunophenotyping methods in fifteen NBAS-deficient patients to discover possible alterations in their immune system. Results Our study revealed reduced absolute numbers of mature CD56dim natural killer (NK) cells. Notably, the residual NK cell population in NBAS-deficient patients exerted a lower potential for activation and degranulation in response to K562 target cells, suggesting an NK cell–intrinsic role for NBAS in the release of cytotoxic granules. NBAS-deficient NK cell activation and degranulation was normalized upon pre-activation by IL-2 in vitro, suggesting that functional impairment was reversible. In addition, we observed a reduced number of naïve B cells in the peripheral blood associated with hypogammaglobulinemia. Conclusion In summary, we demonstrate that pathogenic biallelic variants in NBAS are associated with dysfunctional NK cells as well as impaired adaptive humoral immunity.

Patients after polytrauma suffer from posttraumatic immune system dysregulation and multiple organ dysfunction. Genome-wide microarray profiling in monocytes revealed a regulatory network of inflammatory markers around the transcription factor AP-1 in severely injured patients. Recent research focuses on the role of neutrophils in posttraumatic inflammation. The aim of this study was, therefore, to evaluate the impact of this inflammatory network in neutrophils.Blood sampling and neutrophil separation were performed on admission of the patient and at 6 h, 12 h, 24 h, 48 h, and 72 h after trauma. Neutrophil expression levels of the target genes c-Jun, c-Fos, BCL2A, MMP-9, TIMP-1, ETS-2, IL-1β, and MIP-1β were quantified by RT-qPCR. Patients were assorted into groups according to distinct clinical parameters like massive transfusion (10 RBC units/24 h), injury severity (ISS), 90-d survival, and the presence of traumatic brain injury (defined by ICI on head CT). Statistics were calculated by Mann-Whitney Rank-Sum Test, Receiver Operating Curves, and binary multiple logistic regression.Forty severely injured patients (mean ISS 36 ± 14) were included. BCL2A, MMP-9, TIMP-1, and ETS2 levels showed a significant correlation to 90-d-survival in the early posttraumatic period (6 h-24 h). Furthermore, differential BCL2A, IL-1β, MIP-1β, and MMP-9 regulation was observed in patients requiring massive transfusion. We could further show a significant TIMP-1 response in trauma PMN associated with traumatic brain injury.This study of seriously injured patients highlights very early posttraumatic transcriptional changes in PMNs, which were clearly associated with posttraumatic events and outcomes.

A new immune monitoring tool which assesses the expression of nuclear factor of activated T cells (NFAT)-regulated genes measures the functional effects of cyclosporine A. This is the first prospective randomized controlled study to compare standard pharmacokinetic monitoring by cyclosporine trough levels to NFAT-regulated gene expression (NFAT-RE).Expression of the NFAT-regulated genes was determined by qRT-PCR at cyclosporine trough and peak level. Cardiovascular risk was assessed by change of pulse wave velocity from baseline to month 6. Clinical follow-up was 12 months.In total, 55 stable kidney allograft recipients were enrolled. Mean baseline residual NFAT-RE was 13.1 ± 9.1%. Patients in the NFAT-RE group showed a significant decline in pulse wave velocity from baseline to month 6 versus the standard group (-1.7 ± 2.0 m/s vs 0.4 ± 1.4 m/s, P0.001). Infections occurred more often in the standard group compared with the immune monitoring group. No opportunistic infections occurred with NFAT-RE monitoring. At 12 months of follow-up, renal function was significantly better with NFAT-RE versus standard monitoring (Nankivell glomerular filtration rate: 68.5 ± 17.4 mL/min vs 57.2 ± 19.0 mL/min; P = 0.009).NFAT-RE as translational immune monitoring tool proved efficacious and safe in individualizing cyclosporine therapy, with the opportunity to reduce the cardiovascular risk and improve long-term renal allograft function.

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. .

Analysis of residual gene expression of the nuclear factor of activated T cell (NFAT)-regulated genes has been developed as a pharmacodynamic biomarker to monitor therapy with calcineurin inhibitors. The availability of commercial primer sets (Search-LC) and the well-established assay protocol makes this biomarker a promising candidate to be used clinically in different laboratories. However, implementation of the method in routine practice requires analytical robustness and comparable results across laboratories. Therefore, a protocol originally established at the Institute of Immunology, Heidelberg was verified at the Institute of Laboratory Medicine, Klinikum Stuttgart, and a comparison study was conducted between the 2 laboratories.For the analytical verification, whole blood samples of healthy individuals were incubated with tacrolimus in vitro. Linearity, imprecision, and limit of quantification, as well as sample stability, were investigated. For interlaboratory comparison, samples of patients under cyclosporine A therapy were analyzed in Heidelberg and then reanalyzed in Stuttgart within 24 hours.Tacrolimus (6.25-50 mcg/L) decreased the expression of NFAT-regulated genes in vitro dose dependently (15%-89%). Within- and between-assay coefficient of variations (n = 6 each) were17%. The limit of quantification was200 cDNA copies for each of the interleukin-2, interferon-γ, and granulocyte-macrophage colony-stimulating factor genes. Samples were stable for 24 hours. Interlaboratory comparison using patient samples correlated well (r = 0.951) but showed an inconsistent bias depending on the magnitude of residual gene expression.The assay can be set up with a satisfactory analytical performance in a routine molecular biological laboratory and shows comparable results between laboratories. The reproducibility of the NFAT-regulated gene expression assay across laboratories can facilitate the implementation of this assay for pharmacodynamic routine monitoring of calcineurin inhibitors in different centers.

OBJECTIVE CD8+ T cells contribute to rheumatoid arthritis (RA) by releasing proinflammatory and cytolytic mediators, even in a challenging hypoxic and nutrient-poor microenvironment such as the synovial membrane. This study was undertaken to explore the mechanisms through which CD8+ T cells meet their metabolic demands in the blood and synovial membrane of patients with RA. METHODS Purified blood CD8+ T cells from patients with RA, patients with psoriatic arthritis (PsA), and patients with spondyloarthritis (SpA), as well as healthy control subjects, and CD8+ T cells from RA synovial membrane were stimulated in medium containing 13 C-labeled metabolic substrates in the presence or absence of metabolic inhibitors, under conditions of normoxia or hypoxia. The production of metabolic intermediates was quantified by 1 H-nuclear magnetic resonance. The expression of metabolic enzymes, transcription factors, and immune effector molecules was assessed at both the messenger RNA (mRNA) and protein levels. CD8+ T cell functional studies were performed. RESULTS RA blood CD8+ T cells met their metabolic demands through aerobic glycolysis, production of uniformly 13 C-enriched lactate in the RA blood (2.6 to 3.7-fold higher than in patients with SpA, patients with PsA, and healthy controls; P < 0.01), and induction of glutaminolysis. Overexpression of Warburg effect-linked enzymes in all RA CD8+ T cell subsets maintained this metabolic profile, conferring to the cells the capacity to proliferate under hypoxia and low-glucose conditions. In all RA CD8+ T cell subsets, lactate dehydrogenase A (LDHA) was overexpressed at the mRNA level (P < 0.03 versus controls; n = 6 per group) and protein level (P < 0.05 versus controls; n = 17 RA patients, n = 9 controls). In RA blood, inhibition of LDHA with FX11 led to reductions in lipogenesis, migration and proliferation of CD8+ T cells, and CD8+ T cell effector functions, while production of reactive oxygen species was increased by 1.5-fold (P < 0.03 versus controls). Following inhibition of LDHA with FX11, RA CD8+ T cells lost their capacity to induce healthy B cells to develop a proinflammatory phenotype. Similar metabolic alterations were observed in RA CD8+ T cells from the synovial membrane. CONCLUSION Remodeling glucose and glutamine metabolism in RA CD8+ T cells by targeting LDHA activity can reduce the deleterious inflammatory and cytolytic contributions of these cells to the development of autoimmunity.

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.

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.

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.

Atherosclerosis is a chronic inflammatory disease. Lesion progression is primarily mediated by cells of the monocyte/macrophage lineage. IL-17A is a proinflammatory cytokine, which modulates immune cell trafficking and is involved inflammation in (auto)immune and infectious diseases. But the role of IL-17A still remains controversial. In the current study, we investigated effects of IL-17A on advanced murine and human atherosclerosis, the common disease phenotype in clinical care. The 26-wk-old apolipoprotein E–deficient mice were fed a standard chow diet and treated either with IL-17A mAb (n = 15) or irrelevant Ig (n = 10) for 16 wk. Furthermore, essential mechanisms of IL-17A in atherogenesis were studied in vitro. Inhibition of IL-17A markedly prevented atherosclerotic lesion progression (p = 0.001) by reducing inflammatory burden and cellular infiltration (p = 0.01) and improved lesion stability (p = 0.01). In vitro experiments showed that IL-17A plays a role in chemoattractance, monocyte adhesion, and sensitization of APCs toward pathogen-derived TLR4 ligands. Also, IL-17A induced a unique transcriptome pattern in monocyte-derived macrophages distinct from known macrophage types. Stimulation of human carotid plaque tissue ex vivo with IL-17A induced a proinflammatory milieu and upregulation of molecules expressed by the IL-17A–induced macrophage subtype. In this study, we show that functional blockade of IL-17A prevents atherosclerotic lesion progression and induces plaque stabilization in advanced lesions in apolipoprotein E–deficient mice. The underlying mechanisms involve reduced inflammation and distinct effects of IL-17A on monocyte/macrophage lineage. In addition, translational experiments underline the relevance for the human system.

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.

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.

Autophagy pathways promote the growth of pancreatic ductal adenocarcinoma (PDAC), but the critical role is yet to be determined. Transcription factor EB (TFEB) centrally controls lysosomal and autophagy biogenesis. This study aimed to explore the role of TFEB for autophagy regulation in PDAC. We found that TFEB expression was significantly elevated in human PDAC samples (n=45), and localized to the cytoplasm and nucleus in 11 of 15 cases. In primary PDAC cell lines, TFEB nuclear expression was evident even under basal conditions, and further nuclear enrichment was achieved by starvation. Transient RNA interference reduced TFEB expression to 11-23%, but starvation-induced accumulation of the lipidated, autophagosome-associated LC3-II and the autophago-to-lysosome route was maintained after TFEB silencing. Likewise, gemcitabine treatment of the cancer cells augmented apoptosis and LC3-II as an indicator of autophagy, regardless of the TFEB expression levels. Moreover, the interplay of oncogenic KRAS with TFEB and autophagy was investigated. KRAS silencing caused PDAC cell apoptosis and a reciprocal increase in TFEB expression. This inverse correlation could be confirmed in published data sets of genetically engineered mouse models and human PDAC samples using the the Pubmed GEO and BioPortal databases, and was independent of KRAS mutation status. In conclusion, the central autophagy regulator TFEB is expressed and active in PDAC, but autophagy is sustained after TFEB knockdown, suggesting alternative bypass signaling. TFEB is dispensable for gemcitabine-induced cell death, but inversely correlated with KRAS expression.

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 0.001). In multivariable analysis, higher CDKN1C expression was associated with worse OS (p = 0.02). On the BM level, a total of 84 (38%) patients showed CDKN1C protein expression before start of treatment. Patient, disease and treatment characteristics did not differ between CDKN1C-positive and -negative patients. Positive CDKN1C BM status was associated with shorter OS in multivariable analysis (HR 1.54, p = 0.04). There was an interaction between CDKN1C BM status and subsequent treatment with negative impact on OS being most pronounced in patients receiving conventional cytotoxic chemotherapy (n = 83, 2-year OS 30% versus 58%, p = 0.002). In conclusion, low-proliferative phenotype and CDKN1C expression were associated with shorter OS. CDKN1C protein expression in the BM of newly diagnosed, treatment-naïve MDS and secondary AML patients was identified as a prognostic factor for poor survival in patients treated with antiproliferative chemotherapy.

BACKGROUND Developmental regulation of the pharmacodynamics of cyclosporin A (CsA) has been suggested by in vitro studies. However, these results have not yet been reproduced in the complexity of an in vivo immune system, because reliable biomarkers of CsA effects have not been available. METHODS Gene expression of interleukin-2 (IL-2), interferon (IFN)-γ, and granulocyte macrophage colony stimulating factor (GM-CSF) in peripheral blood from stable pediatric (N = 31) and adult renal transplant recipients (N = 153) (age range 6.5-78 years) was measured by quantitative real-time polymerase chain reaction before (C0) and 2 hours (C2) after oral CsA intake. To control for the effect of varying CsA concentrations, an index was calculated as a measure of individual CsA sensitivity. RESULTS The CsA sensitivity of IL-2 gene expression in pediatric patients was 3.9% higher than in middle-aged adults and 5.2% higher than in seniors, indicating stronger immunosuppression at a given CsA blood concentration in younger patients. For the entire patient cohort, there was a statistically significant inverse correlation between the CsA sensitivity of IL-2 and chronological age (r = 0.142, P < 0.0001). Also, the CsA sensitivity of IFN-γ (r = 0.131, P < 0.0001) and GM-CSF (r = 0.036, P < 0.01) were inversely correlated with chronological age. Multiple linear regression analysis revealed that age was a highly significant (P = 0.0027) independent predictor for residual gene expression of IL-2, but not of IFN-γ and GM-CSF. CONCLUSIONS An increased sensitivity of IL-2 to suppression by CsA was found in pediatric renal transplant recipients in vivo compared with adults. Hence, there seems to be an effect of human development on CsA pharmacodynamics, which, besides the effect of age on pharmacokinetics, should also be considered for the design of treatment regimens of CsA and potentially other calcineurin inhibitors in the pediatric patient population.

Summary 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.

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.

CD40Ligand (CD40L) represents a strong endogenous danger signal associated with chronic inflammatory disease. CD40L induces activation of antigen-presenting cells (APCs) such as DCs, monocytes, B-cells and endothelial cells. However, CD40 activation alone, whilst inducing IL-10 production, is insufficient to induce interleukin (IL)-12p70 release in human APCs suggesting that additional cytokine signals (e.g. GM-CSF, IL-4 or IFN-γ) are required for the induction of a pro-inflammatory cytokine profile. We demonstrate that IFN-γ-induced Janus kinase 1 (JAK1) enhances CD40-induced IL-12p70 release whilst simultaneously inhibiting IL-10 synthesis, resulting in a pro-inflammatory phenotype of CD40L-activated dendritic cells (DCs). JAK2 mediated enhancing effects on IL-12p70 but did not inhibit IL-10 release, whereas Tyk2 mediated inhibitory effects on IL-12p70 release in this system. The mechanism by which complementary IFN-γ/JAK activities affect IL-12p70 production involves STAT1 activation and de novo induction of interferon-responsive factors (IRF)-1 and IRF-8. Simultaneously, JAK1 was unique in inhibiting IL-10 synthesis via STAT1 and IRF-8 with both transcription factors binding to the IL-10 promoter. We demonstrate that CD40- and JAK/STAT/IRF-signalling pathways are strictly complementary for the induction of a pro-inflammatory cytokine profile in human APCs. This suggests that a number of CD40 effects in chronic inflammatory diseases might be weakened by targeting JAK/STAT.

The efficacy of cancer-immunotherapy with complement-activating monoclonal antibodies is limited by over-expression of one or more membrane-bound complement regulatory proteins (mCRPs: CD46, CD55, CD59) on the surface of neoplastic cells. In this study we designed small interfering RNAs (siRNAs) for posttranscriptional gene knock down of CD46, CD55 and CD59 aiming at to sensitize tumor cells to complement attack and thereby to better exploit complement for tumor cell destruction. Tumor cell lines of different origin, such as Du145 (prostate), BT474 (breast) and K562 (erythroleukemia) were selected for the study. FACS-analysis demonstrated that siRNA anti-CD46(301) reduced CD46 protein expression up to 80%, siRNA anti-CD55(255) diminished CD55 protein expression up to 49%, and CD59 protein expression was inhibited up to 82% by siRNA anti-CD59(1339). Time course experiments revealed a long-lasting silencing effect with >50% complement regulator inhibition up to day 13. Upon mCRP knock down, complement-dependent cytotoxicity (CDC) was augmented by 20-30% for CD46, by up to 24% for CD55 and by up to 55% for CD59. The combined inhibition of all three inhibitors further enhanced CDC by up to 66%. Dependent on the cell line, CD46 and CD55 downregulation increased significantly C3 ospsonization, which is known to support cell-mediated defense mechanisms. mCRP blocking antibodies were only partly able to further augment the tumor cells' susceptibility to complement lysis. Thus, siRNA-induced inhibition of complement regulator expression clearly sensitizes malignant cells to complement attack and, if specifically targeted to the tumor, appears suited as adjuvant to improve antibody-based cancer immunotherapy.

Virulent varicella-zoster virus (VZV) can spread in immunocompetent humans, resulting in symptoms mostly of the skin. In contrast, vaccine Oka (V-Oka), the attenuated VZV vaccine strain, only rarely causes clinical reactions. The mechanisms underlying these pathogenetic differences are unclear. In this study, we comparatively analyzed the ability of virulent VZV and V-Oka to modulate instruction of dendritic cells (DCs) by innate signals. DCs isolated from normal human skin were susceptible to infection with VZV and V-Oka. Moreover, inflammatory DCs, which play a crucial role in the stimulation of Th1 immune responses, accumulated in herpes zoster lesions. Infection of inflammatory DCs generated in vitro with virulent VZV or V-Oka resulted in upregulation of CD1c. Upon coculture with CD1c-restricted innate cells, DCs developed a mature phenotype whether infected with virulent VZV or V-Oka. Intriguingly, a striking difference was detected on the functional level. The release of IFN-γ and IL-12, the signature cytokines of Th1 responses, was enhanced by V-Oka but blocked by virulent VZV. V-Oka and virulent VZV efficiently synergized with CD40L, eliminating the possibility that CD40 signaling was a target of VZV-associated immune evasion. Instead, virulent VZV selectively interfered with signaling through TLR2, which is known to sense VZV. Thus, virulent VZV subverts Th1-promoting instruction of human DCs by blocking TLR2-mediated innate signals that prime IL-12 production by DCs. Taken together, our results demonstrate a novel immune-evasion mechanism of virulent VZV that has been lost during the attenuation process leading to the VZV vaccine strain.

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.

Inflammatory liver disorders are often associated with a potentially tissue damaging complement activation directly at the main site of complement protein synthesis. As hepatocytes may be the primary target of complement attack, we investigated the expression and protective capacity of soluble and membrane-bound complement regulatory proteins in primary human hepatocytes (PHH). Isolated PHHs were analyzed for their basal and cytokine-induced complement regulator expression by cytofluorometry, rtPCR, confocal laser microscopy and ELISA. Susceptibility to complement-mediated cell lysis was investigated with cytotoxicity tests. In contrast to previous reports, PHHs expressed CD46, CD55, CD59, soluble CD59 (sCD59) and factor H (fH), but not CD35. A low basal expression of CD55 was strongly enhanced by IFN-gamma, IL-1 beta and TNF-alpha. The expression of CD59 could be augmented by IL-1 beta, IL-6 and TNF-alpha but was suppressed by IFN-gamma. CD46 expression was not significantly altered. PHHs synthesized fH and sCD59 and fH was detected on PHH surface after exposure to IL-1 beta. Inhibition experiments revealed that CD59 was most effective in protecting PHHs from complement attack. These data clearly indicate that PHHs are protected by multiple complement regulatory proteins, which are controlled by proinflammatory cytokines. CD59 appears to be pivotal in protecting PHHs against complement-mediated lysis.

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 arginine-hydrolyzing enzyme arginase is constitutively expressed by human polymorphonuclear granulocytes (PMN). Upon PMN cell death arginase is liberated and depletes arginine in the microenvironment. This amino acid depletion suppresses T cell proliferation and cytokine secretion and emerges as a key mechanism of immunosuppression during chronic inflammation and tumor growth. Here we show that PMN arginase also severely impairs key functions of primary human NK cells as well as IL-2-activated NK cells. In the absence of arginine, NK cell proliferation and IL-12/IL-18-induced secretion of IFN-γ are severely diminished. In contrast, NK cell viability, granule exocytosis, and cytotoxicity are independent of extracellular arginine. The mechanism of NK cell suppression by arginine depletion is posttranscriptional since mRNA transcript frequency is unaffected upon NK cell activation in the absence of arginine. Finally, we demonstrate that human purulent exudate ex vivo inhibits NK cell functions exclusively due to liberated arginase. Arginase inhibitors are therefore promising pharmacological agents to treat unwanted suppression of the innate (NK cell) as well as the adaptive (T cell) immune system.

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.

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

The nonpolymorphic CD1 molecules present lipid antigens to T cells. In myeloid DC humans express five different CD1 proteins (CD1a-e; the corresponding CD1 genes are designated CD1A-E). A role for CD1d-restricted NKT cells in the control of virus infections has been delineated from clinical observations, mouse models and viral evasion mechanisms targeting CD1d. How NKT cells are activated by virus infections is unclear. We found that human myeloid DC differentially regulate CD1 antigen presentation in response to viral danger signals. Stimulation with type I IFN, viral TLR ligands or viruses strongly enhanced the number of CD1D transcripts in human myeloid DC but diminished the abundance of CD1A, CD1B and CD1E mRNA. These changes on the transcriptional level were mirrored by altered cellular distribution and increased surface expression of CD1d. As a consequence NKT cells were activated and showed a Th1-like response. Moreover, NKT cell activation in PBMC exposed to viral danger signals was dependent on human plasmacytoid DC which produce large amounts of IFN-alpha. In conclusion, our data indicate that viral danger signals trigger NKT cell activation by enhancing CD1d de novo synthesis through increasing the abundance of CD1D mRNA in human myeloid DC.

Summary 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 3β (PI3-kinase/AKT/GSK-3β) 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.

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.

Although it is recognized that neurogenic influences contribute to progression of chronic inflammatory diseases, the molecular basis of neuroimmune interactions in the pathogenesis of chronic pancreatitis (CP) is not well defined. Here we report that responsiveness of peripheral blood mononuclear cells (PBMC) to the neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) is altered in CP. Expression of PACAP and its receptors in human CP was analyzed with quantitative RT-PCR, laser-capture microdissection, and immunohistochemistry. Regulation of PACAP expression was studied in coculture systems using macrophages and acinar cells. Responsiveness of donor and CP PBMC to PACAP was determined based on cytokine profiles and NF-kappaB activation of LPS- or LPS+PACAP-exposed cells. Although donor and CP PBMC responded equally to LPS, PACAP-mediated counteraction of LPS-induced cytokine response was switched from inhibiting TNF-alpha to decreasing IL-1beta and increasing IL-10 secretion. The change of PACAP-mediated anti-inflammatory pattern was associated with altered activation of NF-kappaB: compared with LPS alone, a combination of LPS and PACAP had no effect on NF-kappaB p65 nuclear translocation in CP PBMC, whereas NF-kappaB was significantly decreased in donor PBMC. According to laser-capture microdissection and coculture experiments, PBMC also contributed to generation of a PACAP-rich intrapancreatic environment by upregulating PACAP expression in macrophages encountering apoptotic pancreatic acini. The nociceptive status of CP patients correlated with pancreatic PACAP levels and with IL-10 bias of PACAP-exposed CP PBMC. Thus the ability of PBMC to produce and to respond to PACAP might influence neuroimmune interactions that regulate pain and inflammation in CP.

Recently, we established a pharmacodynamic assay to monitor immunosuppressive effectiveness of cyclosporine A (CsA) in patients on standard CsA regimen. The aim of the present study was to extend this correlation to reduced CsA regimen and to compare pharmacodynamic and kinetic parameters to allow prediction of rejections and infections. In 53 heart allograft recipients, nuclear factor of activated T cells (NFAT)-regulated gene expression was quantified at trough (C0) and 2-h post-CsA dose (C2). Gene expression at C2 was calculated relative to C0 (residual gene expression, RGE) or relative to a healthy reference group (absolute gene expression, AGE). RGE correlated with CsA C2-levels in bimodal fashion: above 575 ng/ml correlation was seen with flat regression gradient. Below 575 ng/ml, correlation was excellent with markedly steeper gradient. At C0 in the low-C2 group (575 ng/ml), AGE remained unchanged, whereas in the high-C2 group (575 ng/ml) AGE was markedly reduced. In both groups, AGE at C2 was strongly inhibited. In patients contracting infection during follow-up, RGE was lower than in those without infections independent of CsA levels. CsA-monitoring by quantitation of NFAT-regulated gene expression is feasible with standard and reduced CsA regimens. It correlates better with the incidence of infections than measurement of CsA concentrations and might help in avoiding over-immunosuppression.

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.

B cells possess functional characteristics of innate immune cells, as they can present Ag to T cells and can be stimulated with microbial molecules such as TLR ligands. Because crude preparations of Staphylococcus aureus are frequently used as polyclonal B cell activators and contain potent TLR2 activity, the scope of this study was to analyze the impact of S. aureus-derived TLR2-active substances on human B cell activation. Peripheral B cells stimulated with chemically modified S. aureus cell wall preparations proliferated in response to stimulation with crude cell wall preparations but failed to be activated with pure peptidoglycan, indicating that cell wall molecules other than peptidoglycan are responsible for B cell proliferation. Subsequent analysis revealed that surface protein A (SpA), similar to BCR cross-linking with anti-human Ig, sensitizes B cells for the recognition of cell wall-associated TLR2-active lipopeptides (LP). In marked contrast to TLR7- and TLR9-triggered B cell stimulation, stimulation with TLR2-active LP and SpA or with crude cell wall preparations failed to induce IgM secretion, thereby revealing qualitative differences in TLR2 signaling compared with TLR7/9 signaling. Notably, combined stimulation with SpA plus TLR2 ligands induced vigorous proliferation of a defined B cell subset that expressed intracellular IgM in the presence of IL-2. Conclusion: S. aureus triggers B cell activation via SpA-induced sensitization of B cells for TLR2-active LP. Combined SpA and TLR2-mediated B cell activation promotes B cell proliferation but fails to induce polyclonal IgM secretion as seen after TLR7 and TLR9 ligation.

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)