The impact of stroma and ionising radiation on the plasticity of dendritic cells and macrophages

The basic model of antigen-presenting cells (APC) activation describes a change of state from resting (immature) to activated (mature) upon encounter with a first signal. However, it is apparent that APC maturation is not binary but instead has a spectrum of outcomes. This likely reflects the complex and dynamic crosstalk resulting from their encounter with a wide variety of stromal cells. A deeper knowledge of these interactions, and how they are processed by APC to instruct immunity is required. This study explores the plasticity of the major APC, namely macrophage (Mφ) and dendritic cells (DC). We test the hypothesis that plasticity in APC function depends on the sequence of signals encountered during generation and activation. Human laboratory models of Mφ and DC were generated from monocytes as follows; monocyte-derived Mφ were cultured with M-CSF (M-Mφ) or GM-CSF (GM-Mφ) and DC with IL-4 and GM-CSF. These were characterised and the impact of cellular (e.g. fibroblasts (FB)) or external factors (e.g. ionising radiation (IR)) studied. Changes in microenvironment considerably alter the functional-phenotype of Mφ however it remains to be shown if their initial polarisation restricts responses. Activation of GM-Mφ elicited IL-12 and -23 whilst M-Mφ expressed IL-10. However, the classic pro-inflammatory signal interferon γ (IFNγ) did not elicit M-Mφ that secreted cytokines to the level expected of GM- Mφ. The M-Mφ displayed mixed phenotype with both pro-inflammatory cytokines (albeit at lower levels, 75% less IL-23, 21% less IL-12) on a background of high IL-10. The IL-23 response of Mφ to re-stimulation was reduced or abrogated upon pre-treatment with LPS but enhanced in Mφ conditioned by IFNγ. These findings demonstrate limitations to Mφ plasticity reflecting initial conditioning and suggest the development of innate memory with a degree of specificity in Mφ responses to external factors. We applied ionising radiation (IR) to our models at doses commonly used for radiotherapy (1-20Gy). Irradiation markedly suppressed IL-23 secretion by both Mφ and DC (p<0.01) in a dose-dependent manner without affecting viability. This was selective for IL-23 as there was no change in IL-1β, -6, -10, -27 or TNFα, and only a modest decrease in IL-12 secretion. To investigate the mechanism for IL-23 regulation we showed that IR activated Ataxia telangiectasia mutated kinase (ATM) which in turn inhibited the function of cAMP response element-binding protein (CREB) through phosphorylation of an inhibitory residue (S121). ATM activation suppressed IL-23 as inhibition of ATM with KU55933 increased its expression. Furthermore, the small-molecule CREB inhibitor C-Ci reduced IL-23 levels. As a consequence of modulation of IL-23 in APC, IR indirectly affected the generation of T-cell responses, namely Th-17. Cross-talk between APC and stromal cells was modelled by co-culture of DC with FB. The presence of FB prevented IL-23 downregulation by IR. This was dependent on TNFα and IL-1β secretion by DC which were unaffected by IR. In response, FB secreted PGE2 which resulted in a feed-back loop to enforce IL-23 secretion via activation of cAMP. Therefore, whilst IR could be seen to regulate IL-23 in a single cell culture system, this did not occur in co-cultures that simulated the interaction APC have with stroma. In conclusion, we shown that the order in which APC receive conditioning signals ultimately affects how they respond to re-challenge. This is further influenced by a mixture of micro-environmental and external factors. Understanding the molecular pathways through which these cues manifest will result in in vitro models that better reflect the biology of APC and create opportunity for intervention

Electrochemical genosensor based on disc and screen printed goldelectrodes for detection of specific DNA and RNA sequences derivedfrom Avian Influenza Virus H5N1

tThe genosensors based on thiolated ssDNA probe deposited on the two types of gold electrodes: screen-printed (miniaturized) and disc electrodes destined for determination of specific sequences of DNA andRNA derived from Avian Influenza Virus H5N1 have been proposed. The working principle of genosensor isbased on the ion-channel mechanism. The analytical signals generated upon hybridization processes wererecorded using electrochemical technique – Osteryoung square wave voltammetry in the presence of aredox active marker [Fe(CN)6]3−/4−in the sample solution. The miniaturized genosensor based on screenprinted gold electrodes was able to detect the 20-mer complementary DNA oligonucleotide sequence aswell as ∼280-mer RNA sequences containing the complementary 20-mer sequence in various positions:at 3�-terminus, at 5�-terminus and in the middle of the RNA transcript at the 1 pM concentration. Themeasuring systems were selective. Non-complementary 20-mer oligonucleotide sequence as well asRNA transcript without complementary region generated weak response. The RNA transcripts were alsotested with gold disc electrodes modified in the same manner. This device was able to detect ∼280-mer RNA sequences, but at higher concentration of 10 pM. The good discrimination of the position ofcomplementary part in the ∼280-mer RNA sequences was observed with using both types of modifiedelectrodes

Immuno-silent polymer capsules encapsulating nanoparticles for bioimaging applications

PEGylated polymer capsules encapsulating LaVO4:Tb3+, GdVO4:Tb3+, Gd2O3:Tb3+, GdF3:Tb3+, YVO4:Tb3+ and iron oxide nanoparticles are promising new fluorescence, magnetic and magnetofluorescence imaging agents. Recently, we have reported the in vitro and in vivo level toxicity profile which shows the non-toxic nature of polymer capsules encapsulating nanoparticles. However, prior to clinical use, it is essential to ensure that these agents are unlikely to activate immune responses. Herein, we investigated the immuno-compatibility of polymer capsules with dendritic cells (DC) and macrophages (MO), major antigen presenting cell (APC) subsets required for activation of innate and adaptive immunity. Capsules were efficiently internalized by both DC and MO in vitro. Importantly, despite the presence of intracellular capsules, there was no significant impact on the viability of cells. We studied the impact of different capsules on the cytokine profile of DC and MO, known to be important for the polarization of T-cell immunity. None of the capsules elicited change in cytokine secretion from DC. Furthermore, capsules did not alter the polarization of either M1 or M2 MO subsets as determined by the balance of IL-12 and IL-10 secretion. These data support the notion that PEGylated polymer capsules loaded with nanoparticles have the potential to remain immunologically silent as they do not activate APC and neither do they hinder the response of DC or MO to pathogen activating signals. These systems, therefore, exhibit promising characteristics for bioimaging applications. KEYWORDS: PEGylated polymer capsules, M1 and M2 macrophages, dendritic cells, immune respons

Barrett's esophagus in 2016: From pathophysiology to treatment

Esophageal complications caused by gastroesophageal reflux disease (GERD) include reflux esophagitis and Barrett's esophagus (BE). BE is a premalignant condition with an increased risk of developing esophageal adenocarcinoma (EAC). The carcinogenic sequence may progress through several steps, from normal esophageal mucosa through BE to EAC. A recent advent of functional esophageal testing (particularly multichannel intraluminal impedance and pH monitoring) has helped to improve our knowledge about GERD pathophysiology, including its complications. Those findings (when properly confirmed) might help to predict BE neoplastic progression. Over the last few decades, the incidence of EAC has continued to rise in Western populations. However, only a minority of BE patients develop EAC, opening the debate regarding the cost-effectiveness of current screening/surveillance strategies. Thus, major efforts in clinical and research practice are focused on new methods for optimal risk assessment that can stratify BE patients at low or high risk of developing EAC, which should improve the cost effectiveness of screening/surveillance programs and consequently significantly affect health-care costs. Furthermore, the area of BE therapeutic management is rapidly evolving. Endoscopic eradication therapies have been shown to be effective, and new therapeutic options for BE and EAC have emerged. The aim of the present review article is to highlight the status of screening/surveillance programs and the current progress of BE therapy. Moreover, we discuss the recent introduction of novel esophageal pathophysiological exams that have improved the knowledge of the mechanisms linking GERD to BE

Ultrasensitive electrochemical genosensor for direct detection of specific RNA sequences derived from avian influenza viruses present in biological samples

An electrochemical genosensor based on an epoxyphenanthroline–Fe(III)–NH2-ssDNA layer for the detection of RNA derived from Avian Influenza is presented. The biosensor preparation consists of: (I) modification of gold electrodes with aminoethanethiol, (II) modification of the self-assembled monolayer of aminoethanethiol with 5,6-epoxy-5,6-dihydro-[1,10]-phenanthroline using “click” chemistry, (III) a first step of complexation of Fe(III) by 5,6-epoxy-5,6-dihydro-[1,10]-phenanthroline, (IV) a second step of complexation of Fe(III) by 5,6-epoxy-5,6-dihydro-[1,10]-phenanthroline, (V) immobilization of the single stranded amino-DNA probe via “click” chemistry between epoxy and amino groups. The interactions between the ssDNA probe and RNA targets were explored with Osteryoung Square Wave Voltammetry. The genosensor showed a remarkable detection limit of 3 copies/μL (5 aM) for RNA extracted from A/swan/Poland/305/06 (H5N1) containing a fully complementary sequence. A linear dynamic range for this sequence was observed from 3.0×103 to 3.0×105 [copies/μl]. RNA extracted from A/mallard/Poland/446/09 (H7N7), containing a non-complementary sequence, generated a much weaker response. Moreover, the developed genosensor allows to distinguish RNA present in biological samples having 2, 3 and 4 mismatches. This biosensing approach can become a potential alternative tool for detecting RNA samples in biomedical research and early clinical diagnosis of avian influenza viruses

SLC22A3 polymorphisms do not modify pancreatic cancer risk, but may influence overall patient survival

Expression of the solute carrier (SLC) transporter SLC22A3 gene is associated with overall survival of pancreatic cancer patients. This study tested whether genetic variability in SLC22A3 associates with pancreatic cancer risk and prognosis. Twenty four single nucleotide polymorphisms (SNPs) tagging the SLC22A3 gene sequence and regulatory elements were selected for analysis. Of these, 22 were successfully evaluated in the discovery phase while six significant or suggestive variants entered the validation phase, comprising a total study number of 1,518 cases and 3,908 controls. In the discovery phase, rs2504938, rs9364554, and rs2457571 SNPs were significantly associated with pancreatic cancer risk. Moreover, rs7758229 associated with the presence of distant metastases, while rs512077 and rs2504956 correlated with overall survival of patients. Although replicated, the association for rs9364554 did not pass multiple testing corrections in the validation phase. Contrary to the discovery stage, rs2504938 associated with survival in the validation cohort, which was more pronounced in stage IV patients. In conclusion, common variation in the SLC22A3 gene is unlikely to significantly contribute to pancreatic cancer risk. The rs2504938 SNP in SLC22A3 significantly associates with an unfavorable prognosis of pancreatic cancer patients. Further investigation of this SNP effect on the molecular and clinical phenotype is warranted

Impact of HPV infection on the clinical outcome of p-CAIR trial in head and neck cancer

The purpose of the study was to analyse the influence of HPV infection on the outcome of a randomized clinical trial of conventional (CF) versus 7-days-a-week postoperative radiotherapy (p-CAIR) for squamous cell cancer of the head and neck (SCCHN). Between 2001 and 2004, 279 patients with high-risk SCC of the larynx or cancer of the oral cavity/oropharynx were randomized to receive 63 Gy in fractions of 1.8 Gy given 5 days a week or 7 days a week (Radiother Oncol 87:155–163, 2008). The presence of HPV DNA in 131 archival paraffin blocks was assessed with multiplex quantitative real-time PCR using five consensus primers for the conservative L1 region and molecular beacon probes targeting 14 high-risk HPV subtypes. Following the RT-PCR procedure, we could determine the presence and type of HPV16, HPV18 and the other 12 less frequent oncogenic subtypes. Out of 131 samples, 9 were positive for HPV infection (6.9%), all of them with HPV16 subtype. None of the 65 laryngeal tumours was HPV positive. The 5-year LRC in HPV-positive patients was 100%, compared to 58% in the HPV-negative group (p = 0.02, log-rank test). Amongst 122 patients with HPV-negative tumours, 5-year LRC was 50.3% in p-CF versus 65.2 in p-CAIR (p = 0.37). HPV infection was associated with low expression of EGFR and cyclin D. This study demonstrates a favourable outcome for HPV-positive patients with SCCHN treated with postoperative radiotherapy. While considering the small number of HPV+ tumours, the data set can be considered as hypothesis generating only, the outcome raises new questions on the necessity of aggressive postoperative treatment in HPV+ patients

Common germline variants within the CDKN2A/2B region affect risk of pancreatic neuroendocrine tumors

Pancreatic neuroendocrine tumors (PNETs) are heterogeneous neoplasms which represent only 2% of all pancreatic neoplasms by incidence, but 10% by prevalence. Genetic risk factors could have an important role in the disease aetiology, however only a small number of case control studies have been performed yet. To further our knowledge, we genotyped 13 SNPs belonging to the pleiotropic CDKN2A/B gene region in 320 PNET cases and 4436 controls, the largest study on the disease so far. We observed a statistically significant association between the homozygotes for the minor allele of the rs2518719 SNP and an increased risk of developing PNET (ORhom = 2.08, 95% CI 1.05-4.11, p = 0.035). This SNP is in linkage disequilibrium with another polymorphic variant associated with increased risk of several cancer types. In silico analysis suggested that the SNP could alter the sequence recognized by the Neuron-Restrictive Silencer Factor (NRSF), whose deregulation has been associated with the development of several tumors. The mechanistic link between the allele and the disease has not been completely clarified yet but the epidemiologic evidences that link the DNA region to increased cancer risk are convincing. In conclusion, our results suggest rs2518719 as a pleiotropic CDKN2A variant associated with the risk of developing PNETs

Stromal fibroblasts support dendritic cells to maintain IL-23/Th17 responses after exposure to ionizing radiation

Dendritic cell function is modulated by stromal cells, including fibroblasts. Although poorly understood, the signals delivered through this crosstalk substantially alter dendritic cell biology. This is well illustrated with release of TNF-0/IL-113 from activated dendritic cells, promoting PGE2 secretion from stromal fibroblasts. This instructs dendritic cells to up-regulate IL-23, a key Th17-polarizing cytokine. We previously showed that ionizing radiation inhibited IL-23 production by human dendritic cells in vitro. In the present study, we investigated the hypothesis that dendritic cell-fibroblast crosstalk over¬comes the suppressive effect of ionizing radiation to support appropriately polarized Th17 responses. Radia¬tion (1–6 Gy) markedly suppressed IL-23 secretion by activated dendritic cells (P < 0.0001) without adversely impacting their viability and consequently, inhibited the generation of Th17 responses. Cytokine suppression by ionizing radiation was selective, as there was no effect on IL-10, -6, -10, and -27 or TNF-a and only a modest (11%) decrease in IL-12p70 secretion. Coculture with fibroblasts augmented IL-23 secretion by irradiated dendritic cells and increased Th17 responses. Impor¬tantly, in contrast to dendritic cells, irradiated fibroblasts maintained their capacity to respond to TNF-0/IL-10 and produce PGE2, thus providing the key intermediary signals for successful dendritic cell-fibroblasts crosstalk. In summary, stromal fibroblasts support Th17-polarizing cytokine production by dendritic cells that would other¬wise be suppressed in an irradiated microenvironment. This has potential ramifications for understanding the immune response to local radiotherapy. These findings underscore the need to account for the impact of microenvironmental factors, including stromal cells, in understanding the control of immunity. J. Leukoc. Biol. 100: 000–000; 2016