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The organocatalytic conjugate addition of aldehydes to β-nitroacrylates provides direct access to β-ester-γ-nitroaldehydes and, thereby, common structural motifs of many bioactive compounds. However, the deactivation of amine-based catalysts by alkylation with the highly electrophilic nitroacrylates hampers this reaction. Here, we show that the peptide H-Mep-dPro-dGlu-NH 2 , which is reluctant to undergo alkylation, catalyzes this reaction at low catalyst loading (0.5-1 mol %) within short reaction times (15-60 min) to yield a broad range of β-ester-γ-nitroaldehydes with high stereoselectivity. Kinetic studies revealed that increased steric bulk on the β-nitroacrylate enhances the reaction rate by hindering catalyst alkylation., (© 2024 The Author(s). Chemistry - A European Journal published by Wiley-VCH GmbH.)

Peptides have been established as modular catalysts for various transformations. Still, the vast number of potential amino acid building blocks renders the identification of peptides with desired catalytic activity challenging. Here, we develop a machine-learning workflow for the optimization of peptide catalysts. First-in a hypothetical competition-we challenged our workflow to identify peptide catalysts for the conjugate addition reaction of aldehydes to nitroolefins and compared the performance of the predicted structures with those optimized in our laboratory. On the basis of the positive results, we established a universal training set (UTS) containing 161 catalysts to sample an in silico library of ∼30,000 tripeptide members. Finally, we challenged our machine learning strategy to identify a member of the library as a stereoselective catalyst for an annulation reaction that has not been catalyzed by a peptide thus far. We conclude with a comparison of data-driven versus expert-knowledge-guided peptide catalyst optimization., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Published by American Chemical Society.)

Organocatalytic conjugate addition reactions of aldehydes to fluoroalkylated nitroolefins with chiral amine catalysts offer a straightforward stereoselective path to fluoroalkylated γ-nitroaldehydes and downstream derivatives. However, amine-based catalysts suffer from deactivation by reaction with electron-poor fluoroalkylated nitroolefin. Here, we show that catalyst deactivation can be overcome by catalysts that bear an intramolecular acid for protonation and release of the alkylated catalyst through ß-elimination of the nitroolefin. NMR spectroscopic, kinetic, and molecular modeling studies provided detailed structural and mechanistic insights into the factors that control reversible catalyst alkylation and facilitate efficient catalysis.

Due to the chemical and biological relevance of amino acids, efficient methods for the recognition and separation of their enantiomers are highly sought after. Chiral receptors based on extended molecular scaffolds are typically employed for this purpose. These receptors are often effective only in specific environments and towards a narrow scope of amino acid guests. Recently we reported a simple, glucose-based macrocycle capable of enantioselective binding of a broad range of amino acid methyl esters in water. Herein we demonstrate that the same receptor can be used for chiral recognition of amino acid esters in organic solvents. We show that the binding affinity and selectivity of the receptor are highly dependent on the coordinating strength of the solvent. An in-depth analysis of the receptor's conformation and its interactions with amino acid methyl esters allowed us to propose a binding mode of amino acids to the receptor in CDCl 3 . The binding modes in CDCl 3 and D 2 O were then compared, highlighting the main interactions responsible for binding affinity and selectivity in each solvent. We envision that the insight provided by this study will facilitate the development of further amino acid receptors based on monosaccharides with improved binding affinities and both enantio- as well as chemoselectivities.

Purpose: Physiotherapists work with people with chronic conditions and can act as catalysts for behavioural change. Physiotherapy has also seen a shift to a bio-psychosocial model of health management and interdisciplinary care, which is important in the context of chronic conditions. This scoping review addressed the research question "How do physiotherapists use mental health-based interventions in their treatment of individuals with chronic conditions?" Method: The Embase, MEDLINE, PsycINFO, and CINAHL databases were searched, and a variety of study designs were included. Data were categorized and descriptively analyzed. Results: Data were extracted from 103 articles. Low back pain (43; 41.7%) and non-specified pain (16; 15.5%) were the most commonly researched chronic conditions, but other chronic conditions were also represented. Outpatient facilities were the most common setting for intervention (68; 73.1%). A total of 73 (70.9%) of the articles involved cognitive-behavioural therapy, and 41 (40.0%) included graded exercise or graded activity as a mental health intervention. Conclusions: Physiotherapists can use a variety of mental health interventions in the treatment of chronic conditions. More detailed descriptions of treatment and training protocols would be helpful for incorporating these techniques into clinical practice., (© Canadian Physiotherapy Association.)

The efficacy of adoptive cell therapy for solid tumours is hampered by the poor accumulation of the transferred T cells in tumour tissue. Here, we show that forced expression of C-X-C chemokine receptor type 6 (whose ligand is highly expressed by human and murine pancreatic cancer cells and tumour-infiltrating immune cells) in antigen-specific T cells enhanced the recognition and lysis of pancreatic cancer cells and the efficacy of adoptive cell therapy for pancreatic cancer. In mice with subcutaneous pancreatic tumours treated with T cells with either a transgenic T-cell receptor or a murine chimeric antigen receptor targeting the tumour-associated antigen epithelial cell adhesion molecule, and in mice with orthotopic pancreatic tumours or patient-derived xenografts treated with T cells expressing a chimeric antigen receptor targeting mesothelin, the T cells exhibited enhanced intratumoral accumulation, exerted sustained anti-tumoral activity and prolonged animal survival only when co-expressing C-X-C chemokine receptor type 6. Arming tumour-specific T cells with tumour-specific chemokine receptors may represent a promising strategy for the realization of adoptive cell therapy for solid tumours., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Cytoplasmic double-stranded RNA is sensed by RIG-I-like receptors (RLRs), leading to induction of type I interferons (IFN-Is), proinflammatory cytokines, and apoptosis. Here, we elucidate signaling mechanisms that lead to cytokine secretion and cell death induction upon stimulation with the bona fide RIG-I ligand 5'-triphosphate RNA (3p-RNA) in tumor cells. We show that both outcomes are mediated by dsRNA-receptor families with RLR being essential for cytokine production and IFN-I-mediated priming of effector pathways but not for apoptosis. Affinity purification followed by mass spectrometry and subsequent functional analysis revealed that 3p-RNA bound and activated oligoadenylate synthetase 1 and RNase L. RNase L-deficient cells were profoundly impaired in their ability to undergo apoptosis. Mechanistically, the concerted action of translational arrest triggered by RNase L and up-regulation of NOXA was needed to deplete the antiapoptotic MCL-1 to cause intrinsic apoptosis. Thus, 3p-RNA-induced apoptosis is a two-step process consisting of RIG-I-dependent priming and an RNase L-dependent effector phase., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Replication competent vesicular stomatitis virus (VSV) is the basis of a vaccine against Ebola and VSV strains are developed as oncolytic viruses. Both functions depend on the ability of VSV to induce adequate amounts of interferon-α/β. It is therefore important to understand how VSV triggers interferon responses. VSV activates innate immunity via retinoic acid-inducible gene I (RIG-I), a sensor for viral RNA. Our results show that VSV needs to replicate for a robust interferon response. Analysis of RIG-I-associated RNA identified a copy-back defective-interfering (DI) genome and full-length viral genomes as main trigger of RIG-I. VSV stocks depleted of DI genomes lost most of their interferon-stimulating activity. The remaining full-length genome and leader-N-read-through sequences, however, still triggered RIG-I. Awareness for DI genomes as trigger of innate immune responses will help to standardize DI genome content and to purposefully deplete or use DI genomes as natural adjuvants in VSV-based therapeutics., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Linder, Bothe, Linder, Schwarzlmueller, Dahlström, Bartenhagen, Dugas, Pandey, Thorn-Seshold, Boehmer, Koenig, Kobold, Schnurr, Raedler, Spielmann, Karimzadeh, Schmidt, Endres and Rothenfusser.)

We report a glucose-based crown ether capable of chiral recognition of a wide range of amino-acid methyl esters in aqueous environments. The enantioselectivities towards amino-acids with extended hydrophobic side chains displayed by the glucose-derived macrocycle are among the highest observed for small molecule synthetic receptors to date.

Background: The benefit of ischemic postconditioning (IPostC) might be the throttled inflow following cold ischemia. The current study investigated advantage and mechanisms of IPostC in healthy and fatty rat livers., Methods: Male SD rats received a high-fat diet to induce fatty livers. Isolated liver perfusion was performed after 24 h ischemia at 4 °C as well as in vivo experiments after 90 min warm ischemia. The so-called follow-up perfusions served to investigate the hypothesis that medium from IPostC experiments is less harmful. Lactate dehydrogenase (LDH), transaminases, different cytokines, and gene expressions, respectively, were measured., Results: Fatty livers showed histologically mild inflammation and moderate to severe fat storage. IPostC reduced LDH and TXB 2 in healthy and fatty livers and increased bile flow. LDH, TNF-α, and IL-6 levels in serum decreased after warm ischemia + IPostC. The gene expressions of Tnf, IL-6, Ccl2, and Ripk3 were downregulated in vivo after IPostC., Conclusions: IPostC showed protective effects after ischemia in situ and in vivo in healthy and fatty livers. Restricted cyclic inflow was an important mechanism and further suggested involvement of necroptosis. IPostC represents a promising and easy intervention to improve outcomes after transplantation.

Non-alcoholic fatty liver disease (NAFLD) is rising in prevalence, and a better pathophysiologic understanding of the transition to its inflammatory phenotype (NASH) is key to the development of effective therapies. To evaluate the contribution of the NLRP3 inflammasome and its downstream effectors IL-1 and IL-18 in this process, we applied the true-to-life "American lifestyle-induced obesity syndrome" (ALiOS) diet mouse model. Development of obesity, fatty liver and liver damage was investigated in mice fed for 24 weeks according to the ALiOS protocol. Lipidomic changes in mouse livers were compared to human NAFLD samples. Receptor knockout mice for IL-1 and IL-18 were used to dissect the impact of downstream signals of inflammasome activity on the development of NAFLD. The ALiOS diet induced obesity and liver steatosis. The lipidomic changes closely mimicked changes in human NAFLD. A pro-inflammatory gene expression pattern in liver tissue and increased serum liver transaminases indicated early liver damage in the absence of histological evidence of NASH. Mechanistically, Il-18r -/- - but not Il-1r -/- mice were protected from early liver damage, possibly due to silencing of the pro-inflammatory gene expression pattern. Our study identified NLRP3 activation and IL-18R-dependent signaling as potential modulators of early liver damage in NAFLD, preceding development of histologic NASH.

Pancreatic ductal adenocarcinoma is characterized by a strong immunosuppressive network with a dense infiltration of myeloid cells including myeloid-derived suppressor cells (MDSC). Two distinct populations of MDSC have been defined: polymorphonuclear MDSC (PMN-MDSC) and monocytic MDSC (M-MDSC). Several factors influence the development and function of MDSC including the transcription factor interferon regulatory factor 4 (IRF4). Here, we show that IRF4 deficiency accelerates tumor growth and reduces survival, accompanied with a dense tumor infiltration with PMN-MDSC and reduced numbers of CD8 + T cells. As IRF4 has been described to modulate myeloid cell development and function, particularly of PMN-MDSC, we analyzed its role using MDSC-specific IRF4 knockout mice with the Ly6G or LysM knock-in allele expressing Cre recombinase and Irf4 flox . In GM-CSF-driven bone marrow cultures, IRF4 deficiency increased the frequency of MDSC-like cells with a strong T cell suppressive capacity. Myeloid (LysM)-specific depletion of IRF4 led to increased tumor weight and a moderate splenic M-MDSC expansion in tumor-bearing mice. PMN cell (Ly6G)-specific depletion of IRF4, however, did not influence tumor progression or MDSC accumulation in vivo in accordance with our finding that IRF4 is not expressed in PMN-MDSC. This study demonstrates a critical role of IRF4 in the generation of an immunosuppressive tumor microenvironment in pancreatic cancer, which is independent of IRF4 expression in PMN-MDSC.

An exacerbated and unbalanced immune response may account for the severity of COVID-19, the disease caused by the novel severe acute respiratory syndrome (SARS) coronavirus 2, SARS-CoV-2. In this Viewpoint, we summarize recent evidence for the role of neutrophils in the pathogenesis of COVID-19 and propose CXCR2 inhibition as a promising treatment option to block neutrophil recruitment and activation., (© 2020 Koenig et al.)

Retinoic acid-inducible gene-I (RIG-I) is a cytoplasmic immune receptor sensing viral RNA. It triggers the release of type I interferons (IFN) and proinflammatory cytokines inducing an adaptive cellular immune response. We investigated the therapeutic potential of systemic RIG-I activation by short 5'-triphosphate-modified RNA (ppp-RNA) for the treatment of acute myeloid leukemia (AML) in the syngeneic murine C1498 AML tumor model. ppp-RNA treatment significantly reduced tumor burden, delayed disease onset and led to complete remission including immunological memory formation in a substantial proportion of animals. Therapy-induced tumor rejection was dependent on CD4 + and CD8 + T cells, but not on NK or B cells, and relied on intact IFN and mitochondrial antiviral signaling protein (MAVS) signaling in the host. Interestingly, ppp-RNA treatment induced programmed death ligand 1 (PD-L1) expression on AML cells and established therapeutic sensitivity to anti-PD-1 checkpoint blockade in vivo. In immune-reconstituted humanized mice, ppp-RNA treatment reduced the number of patient-derived xenografted (PDX) AML cells in blood and bone marrow while concomitantly enhancing CD3 + T cell counts in the respective tissues. Due to its ability to establish a state of full remission and immunological memory, our findings show that ppp-RNA treatment is a promising strategy for the immunotherapy of AML.

Improving diagnostic imaging and therapy by targeted compound delivery to pathological areas and across biological barriers is of urgent need. A lipopeptide, P-CrA-A2, composed of a highly cationic peptide sequence (A2), an N-terminally attached palmitoyl chain (P) and cryptophane molecule (CrA) for preferred uptake into blood-brain barrier (BBB) capillary endothelial cells, was generated. CrA allows reversible binding of Xe for NMR detection with hyperpolarized nuclei. The lipopeptide forms size-optimized micelles with a diameter of about 11 nm at low micromolar concentration. Their high local CrA payload has a strong and switchable impact on the bulk magnetization through Hyper-CEST detection. Covalent fixation of CrA does not impede micelle formation and does not hamper its host functionality but simplifies Xe access to hosts for inducing saturation transfer. Xe Hyper-CEST magnetic resonance imaging (MRI) allows for distinguishing BBB endothelial cells from control aortic endothelial cells, and the small micelle volume with a sevenfold improved CrA-loading density compared to liposomal carriers allows preferred cell labelling with a minimally invasive volume (≈16 000-fold more efficient than 19 F cell labelling). Thus, these nanoscopic particles combine selectivity for human brain capillary endothelial cells with great sensitivity of Xe Hyper-CEST MRI and might be a potential MRI tool in brain diagnostics., (© 2020 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: The tumor microenvironment (TME) combines features of regulatory cytokines and immune cell populations to evade the recognition by the immune system. Myeloid-derived suppressor cells (MDSC) comprise populations of immature myeloid cells in tumor-bearing hosts with a highly immunosuppressive capacity. We could previously identify RIG-I-like helicases (RLH) as targets for the immunotherapy of pancreatic cancer inducing immunogenic tumor cell death and type I interferons (IFN) as key mediators linking innate with adaptive immunity., Methods: Mice with orthotopically implanted Kras G12D p53 fl/R172H Ptf1a-Cre (KPC) pancreatic tumors were treated intravenously with the RLH ligand polyinosinic-polycytidylic acid (poly(I:C)), and the immune cell environment in tumor and spleen was characterized. A comprehensive analysis of the suppressive capacity as well as the whole transcriptomic profile of isolated MDSC subsets was performed. Antigen presentation capability of MDSC from mice with ovalbumin (OVA)-expressing tumors was investigated in T cell proliferation assays. The role of IFN in MDSC function was investigated in Ifnar1 -/- mice., Results: MDSC were strongly induced in orthotopic KPC-derived pancreatic cancer, and frequencies of MDSC subsets correlated with tumor weight and G-CSF serum levels, whereas other immune cell populations decreased. Administration of the RLH-ligand induced a IFN-driven immune response, with increased activation of T cells and dendritic cells (DC), and a reduced suppressive capacity of both polymorphonuclear (PMN)-MDSC and monocytic (M)-MDSC fractions. Whole transcriptomic analysis confirmed an IFN-driven gene signature of MDSC, a switch from a M2/G2- towards a M1/G1-polarized phenotype, and the induction of genes involved in the antigen presentation machinery. Nevertheless, MDSC failed to present tumor antigen to T cells. Interestingly, we found MDSC with reduced suppressive function in Ifnar1-deficient hosts; however, there was a common flaw in immune cell activation, which was reflected by defective immune cell activation and tumor control., Conclusions: We provide evidence that the treatment with immunostimulatory RNA reprograms the TME of pancreatic cancer by reducing the suppressive activity of MDSC, polarizing myeloid cells into a M1-like state and recruiting DC. We postulate that tumor cell-targeting combination strategies may benefit from RLH-based TME remodeling. In addition, we provide novel insights into the dual role of IFN signaling in MDSC's suppressive function and provide evidence that host-intrinsic IFN signaling may be critical for MDSC to gain suppressive function during tumor development.

Ischemia reperfusion injury (IRI) of the kidney results in interferon regulatory factor 4 (IRF4)-mediated counter-regulation of the acute inflammatory response. Beyond that, IRF4 exerts important functions in controlling the cytokine milieu, T-cell differentiation, and macrophage polarization. The latter has been implicated in tissue remodeling. It therefore remains elusive what the role of IRF4 is in terms of long-term outcome following IRI. We hypothesized that an inability to resolve chronic inflammation in Irf4 -/- mice would promote chronic kidney disease (CKD) progression. To evaluate the effects of IRF4 in chronic upon acute injury in vivo , a mouse model of chronic injury following acute IRI was employed. The expression of Irf4 increased within 10 days after IRI in renal tissue. Both mRNA and protein levels remained high up to 5 weeks upon IRI, suggesting a regulatory function in the chronic phase. Mice deficient in IRF4 display increased tubular cell loss and defective clearance of infiltrating macrophages. These phenomena were associated with increased expression of pro-inflammatory macrophage markers together with reduced expression of alternatively activated macrophage markers. In addition, IRF4-deficient mice showed defective development of alternatively activated macrophages. Hints of a residual M1 macrophage signature were further observed in human biopsy specimens of patients with hypertensive nephropathy vs. living donor specimens. Thus, IRF4 restricts CKD progression and kidney fibrosis following IRI, potentially by enabling M2 macrophage polarization and restricting a Th1 cytokine response. Deteriorated alternative macrophage subpopulations in Irf4 -/- mice provoke chronic intrarenal inflammation, tubular epithelial cell loss, and renal fibrosis in the long course after IRI in mice. The clinical significance of these finding for human CKD remains uncertain at present and warrants further studies., (Copyright © 2019 Lorenz, Moschovaki-Filippidou, Würf, Metzger, Steiger, Batz, Carbajo-Lozoya, Koziel, Schnurr, Cohen, Schmaderer, Anders, Lindenmeyer and Lech.)

Pancreatic ductal adenocarcinoma (PDAC) is a highly devastating disease with poor prognosis and rising incidence. Late detection and a particularly aggressive biology are the major challenges which determine therapeutic failure. In this review, we present the current status and the recent advances in PDAC treatment together with the biological and immunological hallmarks of this cancer entity. On this basis, we discuss new concepts combining distinct treatment modalities in order to improve therapeutic efficacy and clinical outcome - with a specific focus on protocols involving radio(chemo)therapeutic approaches.

Background: Decreased organ function following liver resection is a major clinical issue. The practical method of ischemic postconditioning (IPostC) has been studied in heart diseases, but no data exist regarding fibrotic livers., Aims: We aimed to determine whether IPostC could protect healthy, fibrotic, and cirrhotic livers from ischemia reperfusion injury (IRI)., Methods: Fibrosis was induced in male SD rats using bile duct ligation (BDL, 4 weeks), and cirrhosis was induced using thioacetamide (TAA, 18 weeks). Fibrosis and cirrhosis were histologically confirmed using HE and EvG staining. For healthy, fibrotic, and cirrhotic livers, isolated liver perfusion with 90 min of warm ischemia was performed in three groups (each with n=8): control, IPostC 8x20 sec, and IPostC 4x60 sec. additionally, healthy livers were investigated during a follow-up study. Lactate dehydrogenase (LDH) and thromboxane B 2 (TXB 2 ) in the perfusate, as well as bile flow (healthy/TAA) and portal perfusion pressure, were measured., Results: LDH and TXB 2 were reduced, and bile flow was increased by IPostC, mainly in total and in the late phase of reperfusion. The follow-up study showed that the perfusate derived from a postconditioned group had much less damaging potential than perfusate derived from the nonpostconditioned group., Conclusion: IPostC following warm ischemia protects healthy, fibrotic, and cirrhotic livers against IRI. Reduced efflux of TXB 2 is one possible mechanism for this effect of IPostC and increases sinusoidal microcirculation. These findings may help to improve organ function and recovery of patients after liver resection.

Macrocyclic host structures for generating transiently bound 129 Xe have been used in various ultra-sensitive NMR and MRI applications for molecular sensing of biochemical analytes. They are based on hyperpolarized nuclei chemical exchange saturation transfer (Hyper-CEST). Here, we tested a set of water-soluble pillar[5]arenes with different counterions in order to compare their potential contrast agent abilities with that of cryptophane-A (CrA), the most widely used host for such purposes. The exchange of Xe with such compounds was found to be sensitive to the type of ions present in solution and can be used for switchable magnetization transfer (MT) contrast that arises from off-resonant pre-saturation. We demonstrate that the adjustable MT magnitude depends on the interplay of saturation parameters and found that the optimum MT contrast surpasses the CrA CEST performance at moderate saturation power. Since modification of such water-soluble pillar[5]arenes is straightforward, these compounds can be considered a promising platform for designing various sensors that may complement the field of Xe HyperCEST-based biosensing MRI., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The rapid emergence of antibiotic-resistant bacterial "superbugs" with concomitant treatment failure and high mortality rates presents a severe threat to global health. The superbug risk is further exacerbated by chronic infections generated from antibiotic-resistant biofilms that render them refractory to available treatments. We hypothesized that efficient antimicrobial agents could be generated through careful engineering of hydrophobic and cationic domains in a synthetic semirigid polymer scaffold, mirroring and amplifying attributes of antimicrobial peptides. We report the creation of polymeric nanoparticles with highly efficient antimicrobial properties. These nanoparticles eradicate biofilms with low toxicity to mammalian cells and feature unprecedented therapeutic indices against red blood cells. Most notably, bacterial resistance toward these nanoparticles was not observed after 20 serial passages, in stark contrast to clinically relevant antibiotics where significant resistance occurred after only a few passages.

We propose a method to dynamically monitor the progress of an enzymatic reaction using NMR of hyperpolarized 129 Xe in a host-guest system. It is based on a displacement assay originally designed for fluorescence experiments that exploits the competitive binding of the enzymatic product on the one hand and a reporter dye on the other hand to a supramolecular host. Recently, this assay has been successfully transferred to NMR, using xenon as a reporter, cucurbit[6]uril as supramolecular host, and chemical exchange saturation transfer with hyperpolarized Xe (Hyper-CEST) as detection technique. Its advantage is that the enzyme acts on the unmodified substrate and that only the product is detected through immediate inclusion into the host. We here apply a method that drastically accelerates the acquisition of Hyper-CEST spectra in vitro using magnetic field gradients. This allows monitoring the dynamic progress of the conversion of lysine to cadaverine with a temporal resolution of ~30 s. Moreover, the method only requires to sample the very early onset of the reaction (<0.5% of substrate conversion where the host itself is required only at μM concentrations) at comparatively low reaction rates, thus saving enzyme material and reducing NMR acquisition time. The obtained value for the specific activity agrees well with previously published results from fluorescence assays. We furthermore outline how the Hyper-CEST results correlate with xenon T 2 measurements performed during the enzymatic reaction. This suggests that ultrafast Hyper-CEST spectroscopy can be used for dynamically monitoring enzymatic activity with NMR., (Copyright © 2017 John Wiley & Sons, Ltd.)

Checkpoint molecules such as programmed death 1 (PD-1) dampen excessive T cell activation to preserve immune homeostasis. PD-1-specific monoclonal antibodies have revolutionized cancer therapy, as they reverse tumour-induced T cell exhaustion and restore CTL activity. Based on this success, deciphering underlying mechanisms of PD-1-mediated immune functions has become an important field of immunological research. Initially described for T cells, there is emerging evidence of unconventional PD-1 expression by myeloid as well as tumor cells, yet, with cell-intrinsic functions in various animal tumor models. Here, we describe positive PD-1 antibody staining of various murine immune and tumour cells that is, unlike for T cells, not the PD-1 receptor and restricted to cells with low forward scatter characteristics. Based on flow cytometry and various approaches, including two established murine anti-PD-1 antibody clones, CRISPR/Cas9 genome editing and confocal imaging, we describe a staining pattern assigned to a nuclear antigen cross-reacting with anti-PD-1 monoclonal antibodies. Lack of PD-1 expression was further underlined by the analysis of PD-1 expression from B16-F10-derived 3D cultures and ex vivo tumours. Thus, our data provide multiple lines of evidence that PD-1 expression by non-T cells is unlikely to be the case and, taking recent data of PD-1 tumour cell-intrinsic functions into account, suggest that other antibody-mediated pathways might apply.

Western lifestyle-associated malnutrition causes steatosis that may progress to liver inflammation and mitochondrial dysfunction has been suggested as a key factor in promoting this disease. Here we have molecularly, biochemically and biophysically analyzed mitochondria from steatotic wild type and immune-compromised mice fed a Western diet (WD) - enriched in saturated fatty acids (SFAs). WD-mitochondria demonstrated lipidomic changes, a decreased mitochondrial ATP production capacity and a significant sensitivity to calcium. These changes preceded hepatocyte damage and were not associated with enhanced ROS production. Thus, WD-mitochondria do not promote steatohepatitis per se, but demonstrate bioenergetic deficits and increased sensitivity to stress signals., (Copyright © 2017 Elsevier B.V. and Mitochondria Research Society. All rights reserved.)

Inflammatory bowel disease (IBD) is associated with enhanced levels of the IL-1 family cytokines IL-1β and IL-18, which are activated by the Nlrp3 inflammasome. Here, we investigated the role of inflammasome-driven cytokine release on T cell polarization and DC differentiation in steady state and T cell transfer colitis. In vitro and in vivo data showed that IL-1β induces Th17 polarization and increases GM‑CSF production by T cells. Reduced IL-1β levels in Nlrp3-/- mice correlated with enhanced FLT3L levels and increased frequency of tolerogenic CD103+ DC. In the T cell transfer colitis model, Nlrp3 deficiency resulted in lower IL‑1β levels, reduced Th17 immunity, and less severe colitis. Unaltered IL-18 levels in both mouse strains pointed toward Nlrp3-independent processing. Importantly, cohousing revealed that the gut microbiome had no impact on the observed Nlrp3-/- phenotype. This study demonstrates that NLRP3 acts as a molecular switch of intestinal homeostasis by shifting local immune cells toward an inflammatory phenotype via IL-1β.

Volcanic ash is a heterogeneous mineral dust that is typically composed of a mixture of amorphous (glass) and crystalline (mineral) fragments. It commonly contains an abundance of the crystalline silica (SiO 2 ) polymorph cristobalite. Inhalation of crystalline silica can induce inflammation by stimulating the NLRP3 inflammasome, a cytosolic receptor complex that plays a critical role in driving inflammatory immune responses. Ingested material results in the assembly of NLRP3, ASC, and caspase-1 with subsequent secretion of the interleukin-1 family cytokine IL-1β. Previous toxicology work suggests that cristobalite-bearing volcanic ash is minimally reactive, calling into question the reactivity of volcanically derived crystalline silica, in general. In this study, we target the NLRP3 inflammasome as a crystalline silica responsive element to clarify volcanic cristobalite reactivity. We expose immortalized bone marrow-derived macrophages of genetically engineered mice and primary human peripheral blood mononuclear cells (PBMCs) to ash from the Soufrière Hills volcano as well as representative, pure-phase samples of its primary componentry (volcanic glass, feldspar, cristobalite) and measure NLRP3 inflammasome activation. We demonstrate that respirable Soufrière Hills volcanic ash induces the activation of caspase-1 with subsequent release of mature IL-1β in a NLRP3 inflammasome-dependent manner. Macrophages deficient in NLRP3 inflammasome components are incapable of secreting IL-1β in response to volcanic ash ingestion. Cellular uptake induces lysosomal destabilization involving cysteine proteases. Furthermore, the response involves activation of mitochondrial stress pathways leading to the generation of reactive oxygen species. Considering ash componentry, cristobalite is the most reactive pure-phase with other components inducing only low-level IL-1β secretion. Inflammasome activation mediated by inhaled ash and its potential relevance in chronic pulmonary disease was further evidenced in PBMC using the NLRP3 small-molecule inhibitor CP-456,773 (CRID3, MCC950). Our data indicate the functional activation of the NLRP3 inflammasome by volcanic ash in murine and human macrophages in vitro . Cristobalite is identified as the apparent driver, thereby contesting previous assertions that chemical and structural imperfections may be sufficient to abrogate the reactivity of volcanically derived cristobalite. This is a novel mechanism for the stimulation of a pro-inflammatory response by volcanic particulate and provides new insight regarding chronic exposure to environmentally occurring particles.

IL-22 has been identified as a cancer-promoting cytokine that is secreted by infiltrating immune cells in several cancer models. We hypothesized that IL-22 regulation would occur at the interface between cancer cells and immune cells. Breast and lung cancer cells of murine and human origin induced IL-22 production from memory CD4 + T cells. In the present study, we found that IL-22 production in humans is dependent on activation of the NLRP3 inflammasome with the subsequent release of IL-1β from both myeloid and T cells. IL-1 receptor signaling via the transcription factors AhR and RORγt in T cells was necessary and sufficient for IL-22 production. In these settings, IL-1 induced IL-22 production from a mixed T helper cell population comprised of Th1, Th17, and Th22 cells, which was abrogated by the addition of anakinra. We confirmed these findings in vitro and in vivo in two murine tumor models, in primary human breast and lung cancer cells, and in deposited expression data. Relevant to ongoing clinical trials in breast cancer, we demonstrate here that the IL-1 receptor antagonist anakinra abrogates IL-22 production and reduces tumor growth in a murine breast cancer model. Thus, we describe here a previously unrecognized mechanism by which cancer cells induce IL-22 production from memory CD4 + T cells via activation of the NLRP3 inflammasome and the release of IL-1β to promote tumor growth. These findings may provide the basis for therapeutic interventions that affect IL-22 production by targeting IL-1 activity., Competing Interests: The authors declare no conflict of interest.

Fast and efficient electron transfer in blue copper proteins is realized by a structural harmonization between the Cu I and Cu II complex pair ("entatic state" model). Herein, we present now a Cu I /Cu II complex pair with redox-active guanidine ligands showing almost perfect match between both redox states. By modifying the ligand electron donor strength, the redox chemistry of the copper complex can be controlled to be either metal-centered or to cross the borderline to ligand-centered. This work is the first systematic study of complexes with redox-active ligands within the concept of the entatic state., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The data presented in this article describe the fatty acid composition of chow, liver tissue and isolated liver mitochondria from mice fed for 6-24 weeks with a high caloric western diet (WD) in comparison to control diet (normal diet, ND). The fatty acid composition was measured via gas chromatography flame ionization detection (GC-FID). Moreover, WD-induced mitochondrial protein changes are presented in this work and were analyzed by mass spectrometry (LC-MS/MS). For further interpretation and discussion of the presented data please refer to the research article entitled "Mitochondrial adaptation in steatotic mice" (Einer et al., 2017) [1].

Pancreatic cancer is one of the most aggressive malignancies and has been considered poorly immunogenic for decades. However, this characterization might be over-simplistic. A more sophisticated approach is needed in order to develop new treatment strategies. In this review, we will focus on T cell exhaustion as a phenomenon of immune failure that is a useful paradigm to characterize immunosuppressive effects. Cancer creates an environment of constant antigen exposure and inflammation. In this setting, T cells transform into a differentiation state that has been termed T cell exhaustion, which is characterized by upregulation of inhibitory receptors, resulting in loss of effector function. The discovery of receptor-mediated immune checkpoints, which prevent uncontrolled T cell reactions, led to the development of a new class of antibodies termed checkpoint inhibitors. Unprecedented results in patients with metastatic melanoma and lung cancer have renewed interest in the immunotherapy of other solid tumor entities, including pancreatic cancer. Data on the efficacy of checkpoint inhibitors in pancreatic cancer are still sparse and indicate limited efficacy as single agents. Combination of checkpoint inhibitors with other immune-activating strategies or cytotoxic drugs might be a way to overcome therapy resistance in the treatment of pancreatic cancer., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Background: A number of new drugs for tumor immunotherapy have been approved in the past few years. They work by activating T cells to combat tumors., Methods: This review is based on publications on recently approved T-cell-activating drugs that were retrieved by a selective search in PubMed., Results: Randomized, controlled trials of "checkpoint" inhibitors, i.e., inhibitory antibodies for use against tumors, have shown that the cytotoxic T-lymphocyte antigen 4 (CTLA-4) inhibitor ipilimumab can prolong the survival of patients with advanced melanoma by 2 to 4 months. No data on median overall survival are yet available for the two programmed-death-1 (PD-1) inhibitors pembrolizumab und nivolumab; the endpoint "tumor response" was achieved in 24% and 32% of patients receiving these drugs, respectively. Grade 3 or 4 adverse effects occurred in 50% of patients receiving ipilimumab and in 12 to 13% of those taking either of the two PD-1-inhibitors. Nivolumab prolonged the median survival of patients with metastatic non-small-cell lung cancer from 6 to 9 months. In refractory or recurrent Philadelphia-chromosome-negative pre-B acute lymphoblastic leukemia (pre-B-ALL), treatment with the bispecific antibody construct blinatumomab led to complete remission in 43% of the patients, while grade 3, 4 or 5 toxicities occurred in 83%., Conclusion: T-cell-directed strategies have been established as a new pillar of treatment in medical oncology. As these drugs have frequent and severe adverse effects, therapeutic decision-making will have to take account not only of the predicted prolongation of survival, but also of the potential for an impaired quality of life while the patient is under treatment.

Reversibly bound Xe is a sensitive NMR and MRI reporter with its resonance frequency being influenced by the chemical environment of the host. Molecular imaging of enzyme activity presents a promising approach for disease identification, but current Xe biosensing concepts are limited since substrate conversion typically has little impact on the chemical shift of Xe inside tailored cavities. Herein, we exploit the ability of the product of the enzymatic reaction to bind itself to the macrocyclic hosts CB6 and CB7 and thereby displace Xe. We demonstrate the suitability of this method to map areas of enzyme activity through changes in magnetization transfer with hyperpolarized Xe under different saturation scenarios., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: Adoptive T cell transfer (ACT) is currently under investigation for the treatment of metastatic cancer. Recent evidence suggests that the coinhibitory PD-1-PD-L1 axis plays a major role in ACT failure. We hypothesized that a new fusion receptor reverting PD-1-mediated inhibition into CD28 costimulation may break peripheral tolerance., Methods: Different PD-1-CD28 fusion receptor constructs were created and retrovirally transduced into primary T cell receptor transgenic murine CD8(+) T cells specific for ovalbumin (OT-1). Cytokine release, proliferation, cytotoxicity, and tumor recognition were analyzed in vitro. Antitumor efficacy and mode of action were investigated in mice bearing subcutaneous tumors induced with the pancreatic carcinoma cell line Panc02 expressing the model antigen ovalbumin (Panc-OVA). For antitumoral efficacy, six to eight mice per group were used. All statistical tests are two-sided., Results: Transduction of the PD-1-CD28 receptor constructs mediated enhanced cytokine release, T cell proliferation, and T cell-induced lysis of target tumor cells. The PD-1-CD28 receptor function was dependent on two of the CD28-signaling motifs and IFN-γ release. Treatment of mice with established Panc-OVA tumors with fusion receptor-transduced OT-1 T cells mediated complete tumor regression. Mice rejecting the tumor were protected upon subsequent rechallenge with either ovalbumin-positive or -negative tumors, indicative of a memory response and epitope spreading in nine of 11 mice vs none of the six naïve mice (P < .001). Treatment efficacy was associated with accumulation of IFN-γ-producing T cells and an increased ratio of CD8(+) T cells to immunosuppressive myeloid-derived suppressor cells in the tumors., Conclusions: Transduction of T cells with this new PD-1-CD28 receptor has the potential of breaking the PD-1-PD-L1-immunosuppressive axis in ACT., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

The RIG-I-like helicase melanoma differentiation-associated protein 5 (MDA5) is an innate immune receptor for double-stranded viral RNA (dsRNA) that, upon activation, induces a Type I interferon (IFN)-driven immune response. In the present study, we demonstrate that human und murine pancreatic cancer cells express functional MDA5 and are highly sensitive to MDA5-induced cell death. Activation of MDA5 by cytosolic delivery of the synthetic dsRNA analog poly(I:C) led to phosphorylation of the transcription factor IRF3, IFNβ production and upregulation of MHC-I expression. MDA5 signaling also induced tumor cell apoptosis via the intrinsic pathway and sensitized tumor cells toward extrinsic, Fas-mediated apoptosis. Systemic treatment of orthotopic pancreatic cancer-bearing mice with the MDA5 ligand resulted in activated CD8 + T cell tumor infiltration, an increased frequency of tumor antigen-specific CD8 + T cells and an immunogenic cytokine milieu in the tumor microenvironment. These effects were paralleled by MDA5-induced pronounced tumor cell death in situ and significantly prolonged survival in two different mouse models for pancreatic cancer, an immunotherapeutic response dependent on CD8 + T cells. Treated mice were further protected from subsequent tumor challenge. In summary, we identified MDA5 as a novel therapeutic target for overcoming apoptosis resistance and tumor-mediated immunosuppression in pancreatic cancer. MDA5 ligands link innate with adaptive immune mechanisms for effective tumor control.

Sensitivity of carcinoma cells towards gemcitabine (Gem) has been linked to mitochondrial apoptotic proteins. Recently, we described synergistic efficacy of Gem-based chemoimmunotherapy and a dendritic cell (DC) tumor vaccine in a murine pancreatic carcinoma model. Here, we investigated the role of the mitochondrial proteins Bcl-2, Bcl-xL, and Bax for sensitization of pancreatic carcinoma cells toward T-cell-mediated cytotoxicity alone and in combination with Gem. Bcl-2 expression was silenced by siRNA in Panc02 pancreatic cancer cells expressing the model antigen ovalbumin (PancOVA). Tumor cells were treated with Gem and/or siRNA, and cytotoxicity induced by OVA-specific cytotoxic T lymphocytes (CTL) from OT-1 mice was assessed. Gem-induced and T-cell-induced cytotoxicity was also studied in human Colo357 pancreatic cancer cell lines overexpressing Bax or Bcl-xL. Apoptosis induction by Fas-activating antibody was measured by Annexin V staining. The in vivo capacity of Bcl-2 siRNA to augment CTL efficacy induced by DC vaccinations was assessed in C57BL/6 mice bearing PancOVA tumors. PancOVA cells treated with Bcl-2 siRNA were sensitized towards both Gem and T-cell-mediated killing; combination therapy exhibited an additive effect. Bax overexpression sensitized Colo357 cells to both Gem-mediated and T-cell-mediated cytotoxicity, whereas Bcl-xL overexpression was inhibitory. Combining Bcl-2 silencing with DC therapy improved tumor control in the PancOVA model in vivo without affecting the number of tumor-reactive CTL. In conclusion, expression of Bcl-2, Bcl-xL, and Bax in pancreatic tumor cells determines sensitivity towards both Gem-mediated and CTL-mediated toxicity. Bcl-2 silencing could be exploited therapeutically in tumor vaccine approaches.

A nanoparticulate carrier system is used to efficiently deliver a contrast agent for highly sensitive xenon Hyper-CEST MRI. The carrier system not only improves the biocompatibility and solubility of the contrast agent, it also allows selective cell targeting as demonstrated by the discrimination of human brain capillary and aortic endothelial cells., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Despite continuous progress in the understanding of deregulated pathways in pancreatic cancer cells and development of targeted therapies, therapeutic advances with clinical benefit have been scarce over the last decades. The recent success of immunotherapy for some solid cancers has fueled optimism that this approach might also work for pancreatic cancer. However, a highly immunosuppressive microenvironment mediated by tumor, stromal and immune cells creates a major hurdle for immunotherapy. Mouse models have helped to unravel critical immunosuppressive mechanisms that could serve as novel therapeutic targets. Here we review new promising strategies that alone or in combination with other modalities, such as chemotherapy or irradiation, have the potential to lead to tumor immune control and finally better clinical outcome.

RIG-I-like helicases (RLH) are cytosolic sensors for viral RNA inducing type I interferon production. We found that pancreatic cancer cells express functional RLH and are susceptible to RLH-induced apoptosis via intrinsic and extrinsic pathways. Tumor cells displayed features of immunogenic cell death resulting in dendritic cell activation, enhanced antigen cross-presentation and efficient tumor control in vivo .

Pancreatic cancer is characterized by a microenvironment suppressing immune responses. RIG-I-like helicases (RLH) are immunoreceptors for viral RNA that induce an antiviral response program via the production of type I interferons (IFN) and apoptosis in susceptible cells. We recently identified RLH as therapeutic targets of pancreatic cancer for counteracting immunosuppressive mechanisms and apoptosis induction. Here, we investigated immunogenic consequences of RLH-induced tumor cell death. Treatment of murine pancreatic cancer cell lines with RLH ligands induced production of type I IFN and proinflammatory cytokines. In addition, tumor cells died via intrinsic apoptosis and displayed features of immunogenic cell death, such as release of HMGB1 and translocation of calreticulin to the outer cell membrane. RLH-activated tumor cells led to activation of dendritic cells (DCs), which was mediated by tumor-derived type I IFN, whereas TLR, RAGE or inflammasome signaling was dispensable. Importantly, CD8α(+) DCs effectively engulfed apoptotic tumor material and cross-presented tumor-associated antigen to naive CD8(+) T cells. In comparison, tumor cell death mediated by oxaliplatin, staurosporine or mechanical disruption failed to induce DC activation and antigen presentation. Tumor cells treated with sublethal doses of RLH ligands upregulated Fas and MHC-I expression and were effectively sensitized towards Fas-mediated apoptosis and cytotoxic T lymphocyte (CTL)-mediated lysis. Vaccination of mice with RLH-activated tumor cells induced protective antitumor immunity in vivo. In addition, MDA5-based immunotherapy led to effective tumor control of established pancreatic tumors. In summary, RLH ligands induce a highly immunogenic form of tumor cell death linking innate and adaptive immunity.

Background: One bottleneck for adoptive T cell therapy (ACT) is recruitment of T cells into tumors. We hypothesized that combining tumor-specific T cells, modified with a marker antigen and a bispecific antibody (BiAb) that selectively recognizes transduced T cells and tumor cells would improve T cell recruitment to tumors and enhance therapeutic efficacy., Methods: SV40 T antigen-specific T cells from T cell receptor (TCR)-I-transgenic mice were transduced with a truncated human epidermal growth factor receptor (EGFR) as a marker protein. Targeting and killing by combined ACT and anti-EGFR-anti-EpCAM BiAb therapy was analyzed in C57Bl/6 mice (n = six to 12 per group) carrying subcutaneous tumors of the murine gastric cancer cell line GC8 (SV40(+) and EpCAM(+)). Anti-EGFR x anti-c-Met BiAb was used for targeting of human tumor-specific T cells to c-Met(+) human tumor cell lines. Differences between experimental conditions were analyzed using the Student's t test, and differences in tumor growth with two-way analysis of variance. Overall survival was analyzed by log-rank test. All statistical tests were two-sided., Results: The BiAb linked EGFR-transduced T cells to tumor cells and enhanced tumor cell lysis. In vivo, the combination of ACT and Biab produced increased T cell infiltration of tumors, retarded tumor growth, and prolonged survival compared with ACT with a control antibody (median survival 95 vs 75 days, P < .001). In human cells, this strategy enhanced recruitment of human EGFR-transduced T cells to immobilized c-Met and recognition of tyrosinase(+) melanoma cells by TCR-, as well as of CEA(+) colon cancer cells by chimeric antigen receptor (CAR)-modified T cells., Conclusions: BiAb recruitment of tumor-specific T cells transduced with a marker antigen to tumor cells may enhance efficacy of ACT., (© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Resisting cell death, reprogrammed metabolism and immune escape are fundamental traits of hard-to-treat cancers. Therapeutic improvement can be expected by designing drugs targeting all three aspects. 5'-Triphosphate RNA (ppp-RNA), a specific ligand of the pattern recognition receptor retinoic acid-inducible gene I (RIG-I), has been shown to trigger intrinsic apoptosis of malignant cells and to activate antitumor immune responses via type I interferons (IFNs). In our study, we designed a ppp-modified siRNA specifically silencing glutaminase (ppp-GLS), a key enzyme of glutaminolysis that is indispensable for many cancer types. Bifunctional ppp-GLS induced more prominent antitumor responses than RNA molecules that contained either the RIG-I ligand motif or GLS silencing capability alone. The cytopathic effect was constrained to tumor cells as nonmalignant cells were not affected. We then analyzed the mechanisms leading to the profound antitumor efficacy. First, ppp-GLS effectively induced intrinsic proapoptotic signaling. In addition, GLS silencing sensitized malignant cells to RIG-I-induced apoptosis. Moreover, disturbed glutaminolysis by GLS silencing contributed to enhanced cytotoxicity. Finally, RIG-I activation blocked autophagic degradation leading to dysfunctional mitochondria and reactive oxygen species (ROS) generation, whereas GLS silencing severely impaired ROS scavenging systems, leading to a vicious circle of ROS-mediated cytotoxicity. Taken together, ppp-GLS combines cell death induction, immune activation and glutaminase inhibition in a single molecule and has high therapeutic efficacy against cancer cells., (© 2013 UICC.)

Adjuvants are an essential component of modern vaccines and used for their ability to elicit immunity to coadministered Ags. Many adjuvants in clinical development are particulates, but how they drive innate and adaptive immune responses remains poorly understood. Studies have shown that a number of vaccine adjuvants activate inflammasome pathways in isolated APCs. However, the contribution of inflammasome activation to vaccine-mediated immunity in vivo remains controversial. In this study, we evaluated immune cell responses to the ISCOMATRIX adjuvant (IMX) in mice. Like other particulate vaccine adjuvants, IMX potently activated the NALP-3-ASC-Caspase-1 inflammasome in APCs, leading to IL-1β and IL-18 production. The IL-18R pathway, but not IL-1R, was required for early innate and subsequent cellular immune responses to a model IMX vaccine. APCs directly exposed to IMX underwent an endosome-mediated cell-death response, which we propose initiates inflammatory events locally at the injection site. Importantly, both inflammasome-related and -unrelated pathways contributed to IL-18 dependence in vivo following IMX administration. TNF-α provided a physiological priming signal for inflammasome-dependent IL-18 production by APCs, which correlated with reduced vaccine-mediated immune cell responses in TNF-α- or TNFR-deficient mice. Taken together, our findings highlight an important disconnect between the mechanisms of vaccine adjuvant action in vitro versus in vivo.

Background: Multiple studies have shown that dendritic cell (DC)-based vaccines can induce antitumor immunity. Previously, we reported that gemcitabine enhances the efficacy of DC vaccination in a mouse model of pancreatic carcinoma. The present study aimed at investigating the influence of gemcitabine on vaccine-induced anti-tumoral immune responses in a syngeneic pancreatic cancer model., Materials and Methods: Subcutaneous or orthotopic pancreatic tumors were induced in C57BL/6 mice using Panc02 cells expressing the model antigen OVA. Bone marrow-derived DC were loaded with soluble OVA protein (OVA-DC). Animals received gemcitabine twice weekly. OVA-specific CD8(+) T-cells and antibody titers were monitored by FACS analysis and ELISA, respectively., Results: Gemcitabine enhanced clinical efficacy of the OVA-DC vaccine. Interestingly, gemcitabine significantly suppressed the vaccine-induced frequency of antigen-specific CD8(+) T-cells and antibody titers. DC migration to draining lymph nodes and antigen cross-presentation were unaffected. Despite reduced numbers of tumor-reactive T-cells in peripheral blood, in vivo cytotoxicity assays revealed that cytotoxic T-cell (CTL)-mediated killing was preserved. In vitro assays revealed sensitization of tumor cells to CTL-mediated lysis by gemcitabine. In addition, gemcitabine facilitated recruitment of CD8(+) T-cells into tumors in DC-vaccinated mice. T- and B-cell suppression by gemcitabine could be avoided by starting chemotherapy after two cycles of DC vaccination., Conclusions: Gemcitabine enhances therapeutic efficacy of DC vaccination despite its negative influence on vaccine-induced T-cell proliferation. Quantitative analysis of tumor-reactive T-cells in peripheral blood may thus not predict vaccination success in the setting of concomitant chemotherapy.

We present a highly sensitive nuclear-magnetic resonance technique to study membrane dynamics that combines the temporary encapsulation of spin-hyperpolarized xenon ((129)Xe) atoms in cryptophane-A-monoacid (CrAma) and their indirect detection through chemical exchange saturation transfer. Radiofrequency-labeled Xe@CrAma complexes exhibit characteristic differences in chemical exchange saturation transfer-driven depolarization when interacting with binary membrane models composed of different molecular ratios of DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) and POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine). The method is also applied to mixtures of cholesterol and POPC. The existence of domains that fluctuate in cluster size in DPPC/POPC models at a high (75-98%) DPPC content induces up to a fivefold increase in spin depolarization time τ at 297 K. In POPC/cholesterol model membranes, the parameter τ depends linearly on the cholesterol content at 310 K and allows us to determine the cholesterol content with an accuracy of at least 5%., (Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Caged xenon has great potential in overcoming sensitivity limitations for solution-state NMR detection of dilute molecules. However, no application of such a system as a magnetic resonance imaging (MRI) contrast agent has yet been performed with live cells. We demonstrate MRI localization of cells labeled with caged xenon in a packed-bed bioreactor working under perfusion with hyperpolarized-xenon-saturated medium. Xenon hosts enable NMR/MRI experiments with switchable contrast and selectivity for cell-associated versus unbound cages. We present MR images with 10(3) -fold sensitivity enhancement for cell-internalized, dual-mode (fluorescence/MRI) xenon hosts at low micromolar concentrations. Our results illustrate the capability of functionalized xenon to act as a highly sensitive cell tracer for MRI detection even without signal averaging. The method will bridge the challenging gap for translation to in vivo studies for the optimization of targeted biosensors and their multiplexing applications., (Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Toll-like receptor (TLR) ligands hold promise for cancer immunotherapy. The isolation of intratumoral leukocytes of tumor-bearing mice is a useful technique for analyzing the immunological effects of TLR ligands on the tumor microenvironment. These isolated immune cells can be directly used for analysis (e.g., by flow cytometry) or cultured for functional in vitro studies. Here, we describe the isolation of intratumoral leukocytes by density gradient centrifugation. This technique can be used to isolate leukocytes from freshly dissected murine tumors.

Using spin hyperpolarized xenon ((129)Xe) we investigate the impact of the local molecular environment on reversible host-guest interactions. We label Xe guest atoms that are temporarily bound to cryptophane-A hosts using the Hyper-CEST technique. By varying the length of the saturation pulse and utilizing an inverse Laplace transform we can determine depolarization times for the noble gas in different local environments, in this case biomembranes possessing different fluidity. We extend this technique to magnetic resonance imaging, mapping the spatial distribution of the different biomembranes. Such decays measured in biomembranes of 200 μM 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) were characterized by mono-exponential decays with time constants of τPOPC = 3.00(-0.61)(+0.77) s and τDPPC(-4.16)(+5.19) = 22.15 s. Analyzing both environments simultaneously yielded a bi-exponential decay. This approach may give further insights into saturation transfer dynamics of reversibly bound Xe with applications extending into biomedical diagnostics.

Introduction: In lung cancer, interleukin-22 (IL-22) expression within primary tissue has been demonstrated, but the frequency and the functional consequence of IL-22 signaling have not been addressed. This study aims at analyzing the cellular effects of IL-22 on lung carcinoma cell lines and the prognostic impact of IL-22 tissue expression in lung cancer patients., Methods: Biological effects of IL-22 signaling were investigated in seven lung cancer cell lines by Western blot, flow cytometry, real-time polymerase chain reaction, and proliferation assays. Tumor tissue specimens of two cohorts with a total of 2300 lung cancer patients were tested for IL-22 expression by immunohistochemistry. IL-22 serum concentrations were analyzed in 103 additional patients by enzyme-linked immunosorbent assay., Results: We found the IL-22 receptor 1 (IL-22-R1) to be expressed in six of seven lung cancer cell lines. However IL-22 signaling was functional in only four cell lines, where IL-22 induced signal transducer activator of transcription 3 phosphorylation and increased cell proliferation. Furthermore, IL-22 induced the expression of antiapoptotic B-cell lymphoma 2, but did not rescue tumor cells from carboplatin-induced apoptosis. Cisplatin-resistant cell lines showed a significant up-regulation of IL-22-R1 along with a stronger proliferative response to IL-22 stimulation. IL-22 was preferentially expressed in small- and large-cell lung carcinoma (58% and 46% of cases, respectively). However, no correlation between IL-22 expression by immunohistochemistry and prognosis was observed., Conclusion: IL-22 is frequently expressed in lung cancer tissue. Enhanced IL-22-R1 expression and signaling in chemotherapy-refractory cell lines are indicative of a protumorigenic function of IL-22 and may contribute to a more aggressive phenotype.

Retinoic acid-inducible gene I (RIG-I) is a pattern recognition receptor that is activated by 5'-triphosphate RNA molecules to induce type I interferon secretion and apoptosis in response to viral infection. We have designed a bifunctional small-interfering RNA that combines transforming growth factor β silencing with RIG-I activation to break tumor-induced immunosuppression. This strategy showed therapeutic efficacy in a murine model of pancreatic cancer.