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24 Z -Masticadienonic acid (MNA) and 24 Z -isomasticadienonic acid (IMNA) are the major triterpenic acids in Chios Mastic Gum (CMG), a resin derived from Pistacia lentiscus var. Chia . Despite their promising pharmacological potential, limited information is available due to the complexity of isolating them in pure form. This study developed a chemo-selective method for isolating MNA and IMNA and investigated their chemical transformation through isomerization of the external double bond and A-ring contraction of the triterpene scaffold. A rapid method for isolating MNA from CMG was first established, followed by a high-yield acid-catalyzed procedure to obtain both 24 Z and 24 E isomers of IMNA. Additionally, a basic catalyzed isomerization of IMNA led to the formation of two new compounds with A-ring contraction, which could serve as novel scaffolds for the design of new triterpene analogs. The mixture of MNA/IMNA, along with the individual compounds and their semi-synthetic analogs, exhibited significant anti-inflammatory activity. Notably, 24 E -isomasticadienonic acid and 24 Z -2-hydroxy-3-oxotirucalla-1,8,24-trien-26-oic acid, a previously unreported compound, significantly reduced the mRNA expression levels of Tnf , Il6 , and Nfkb1 in RAW 264.7 macrophage cells.

Background: To estimate the protection that coronavirus disease 2019 (COVID-19) vaccine doses conferred to hospitalized patients with COVID-19 against adverse outcomes and longer length of stay during the Omicron BA.2 and BA.5 subvariant epidemics in Greece., Methods: The study was conducted from November 2022 to May 2023. Multivariable logistic and negative binomial regression models were applied to estimate the association between any adverse outcomes and length of stay with the number of COVID-19 vaccine doses., Results: We studied 962 patients (median age: 78 years; mean length of stay: 9.2 days), of whom 847 (88.0%) had ≥ 1 comorbidity. Of these, 39 (4.0%) were admitted to the intensive care unit, 44 (4.6%) received invasive mechanical ventilation, and 110 (11.4%) died in hospital. There were 184 (19.1%) unvaccinated patients, 125 (13.0%) with one or two vaccine doses, and 653 (67.9%) with ≥ 3 doses. In multivariable analyses, patients with ≥ 3 doses had lower odds of experiencing any adverse outcomes (adjusted odds ratio: 0.57; 95% confidence interval [CI]: 0.37-0.86) compared with unvaccinated patients. On average, patients with one or two doses and those with ≥ 3 had decreased length of hospital stay (-1.5 days [95% CIs: -2.6 to -0.4] and -2.8 days [95% CIs: -4.1 to -1.4], respectively] compared with unvaccinated patients. Other characteristics consistently associated with adverse outcomes and longer length of stay included older age, having three or more comorbidities compared with none, and being admitted to the hospital two or more weeks post-diagnosis., Conclusions: A history of ≥ 3 vaccine doses conferred significant protection against any adverse outcome and longer length of stay in hospitalized patients with COVID-19., (© 2024 The Author(s).)

Regulatory T cells (Tregs) are essential mediators of tolerance mitigating aberrant immune responses. While naturally occurring Treg (nTreg) development and function are directed by epigenetic events, induced Treg (iTreg) identity and mechanisms of action remain elusive. Mirroring the epigenetic circuits of nTregs, we and others have used hypomethylation agents (HAs) to ex vivo convert T cells into iTregs (HA-iTregs) and further showed that the suppressive properties of the HA-iTregs are predominantly confined in an emergent population, which de novo expresses the immunomodulatory molecule HLA-G, consequently providing a surface marker for isolation of the suppressive HA-iTreg compartment (G + cells). We isolated the HA-induced G + cells and their G - counterparts and employed high-throughput RNA-sequencing (RNA-seq) analyses to uncover the G + -specific transcriptomic changes guiding T cells toward a regulatory trajectory upon their exposure to HA. We found a distinct transcriptional upregulation of G + cells accompanied by enrichment of immune-response-related pathways. Although single-cell RNA-seq profiling revealed regulatory G + cells to have molecular features akin to nTregs, when assessed in conjunction with the comparative transcriptomic analysis and profiling of secreted cytokines against the non-suppressive G - cells, FOXP3 and other T-helper signatures appear to play a minor role in their suppressive phenotype. We found an ectopic expression of IDO-1 and CCL17/22 in G + cells, denoting that in vitro exposure of T cells to HA may well unlock myeloid suppressor genes. This report provides transcriptional data shaping the molecular identity of a highly purified and potent HA-iTreg population and hints toward ectopic myeloid-specific molecular mechanisms mediating HA-iTreg function., 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 © 2023 Lysandrou, Stamou, Kefala, Pierides, Kyriakou, Savvopoulos, Christofi, Papadopoulou, Yannaki, Costeas and Spyridonidis.)

Aim: We estimated vaccine effectiveness (VE) of full (booster) vaccination against severe outcomes in hospitalized COVID-19 patients during the Delta and Omicron waves., Methods: The study extended from November 15, 2021 to April 17, 2022. Full vaccination was defined as a primary vaccination plus a booster ≥ 6 months later., Results: We studied 1138 patients (mean age: 66.6 years), of whom 826 (72.6 %) had ≥ 1 comorbidity. Of the 1138 patients, 75 (6.6 %) were admitted to intensive care unit (ICU), 64 (5.6 %) received mechanical ventilation, and 172 (15.1 %) died. There were 386 (33.9 %) fully vaccinated, 172 (15.1 %) partially vaccinated, and 580 (51 %) unvaccinated patients. Unvaccinated patients were absent from work for longer periods compared to partially or fully vaccinated patients (mean absence of 20.1 days versus 12.3 and 17.3 days, respectively; p-value = 0.03). Compared to unvaccinated patients, fully vaccinated patients were less likely to be admitted to ICU [adjusted relative risk (ARR: 0.49; 95 % CI: 0.29-0.84)], mechanically ventilated (ARR: 0.43; 95 % CI: 0.23-0.80), and die (ARR: 0.57; 95 % CI: 0.42-0.78), while they were hospitalized for significantly shorter periods (ARR: 0.79; 95 % CI: 0.70-0.89). The adjusted full VE was 48.8 % (95 % CI: 42.7 %-54.9 %) against ICU admission, 55.4 % (95 % CI: 52.0 %-56.2 %) against mechanical ventilation, and 22.6 % (95 % CI: 7.4 %-34.8 %) against death. For patients with ≥ 3 comorbidities, VE was 56.2 % (95 % CI: 43.9 %-67.1 %) against ICU admission, 60.2 % (95 % CI: 53.7 %-65.4 %) against mechanical ventilation, and 43.9 % (95 % CI: 19.9 %-59.7 %) against death., Conclusions: Full (booster) COVID-19 vaccination conferred protection against severe outcomes, prolonged hospitalization, and prolonged work absenteeism., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Polypharmacy in type 2 diabetes is an issue of major concern as the prescription of multiple medi-cations for the management of diabetes-associated comorbidities can lead to drug-to-drug interactions, which can pose serious risks to patients' health. Within this context, the development of bioanalytical methods for monitoring the therapeutic levels of antidiabetic drugs is notably useful to ensure patients' safety. In the present work, a liquid chromatography-mass spectrometry method for the quantitation of pioglitazone, repaglinide, and nateglinide in human plasma is described. Sample preparation was performed by fabric phase sorptive extraction (FPSE), and hydrophilic interaction liquid chromatography (HILIC) was implemented for the chromatographic separation of the analytes, using a ZIC®-cHILIC analytical column (150 × 2.1 mm, 3 µm) under isocratic elution. The mobile phase consisted of 10 mM ammonium formate aqueous solution (pH = 6.5)/ acetonitrile, 10/90 v/v, and was pumped at a flow rate of 0.2 mL min -1 . Design of Experiments was used during the development of the sample preparation method to gain deeper insight into the effect of various experimental parameters on extraction efficiency, their potential interactions and to optimize the recovery rates of the analytes. The linearity of the assay was assessed over the ranges of 25 to 2000, 6.25 to 500, and 125 to 10000 ng mL -1 for pioglitazone, repaglinide, and nateglinide, respectively. The presented method was fully validated and can be used for the therapeutic monitoring of the targeted analytes in human plasma samples., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Background: It is not known whether intensive care unit (ICU) patients' family members realistically assess patients' health status., Objectives: The aim was to investigate the agreement between family and intensivists' assessment concerning changes in patient health, focusing on family members' resilience and their perceptions of decision making., Methods: For each ICU patient, withdrawal criteria were assessed by intensivists while family members assessed the patient's health development and completed the Connor-Davidson Resilience Scale and the Self-Compassion Scale. Six months after ICU discharge, follow-up contact was established, and family members gave their responses to two hypothetical scenarios., Results: 162 ICU patients and 189 family members were recruited. Intensivists' decisions about whether a patient met the withdrawal criteria had 75,9% accuracy for prediction of survival. Families' assessments were statistically independent of intensivists' opinions, and resilience had a significant positive effect on the probability of agreement with intensivists. Six months after discharge, family members whose relatives were still alive were significantly more likely to consider that the family or patient themselves should be involved in decision-making., Conclusions: Resilience is related to an enhanced probability of agreement of the family with intensivists' perceptions of patients' health progression. Family attitudes in hypothetical scenarios were found to be significantly affected by the patient's actual health progression.

A series of Mn(II) complexes of 5-nitro-salicyladehyde or substituted 2-hydroxy-phenones (HL) were synthesized in the absence or presence of a N,N'-donor co-ligand such as 2,2'-bipyridine, 1,10-phenanthroline, or 2,2'-bipyridylamine. The resultant coordination compounds were formulated as [Mn(L) 2 (CH 3 OH) 2 ] (1-3) and [Mn(L) 2 (N,N'-donor)] (4-14), respectively, and characterized by diverse techniques. The crystal structures of three complexes were determined by single-crystal X-ray crystallography. Diverse techniques were employed to study the interaction of the complexes with calf-thymus DNA and showed intercalation as the most possible mode of their tight interaction. The affinity of the complexes for bovine serum albumin was investigated by fluorescence emission spectroscopy in order to calculate the binding constants which suggested a tight and reversible binding., (Copyright © 2022 Elsevier Inc. All rights reserved.)

The field of metagenomics has rapidly expanded to become the go-to method for complex microbial community analyses. However, there is currently no straightforward route from metagenomics to traditional culture-based methods of strain isolation, particularly in (bacterio)phage biology, leading to an investigative bottleneck. Here, we describe a method that exploits specific phage receptor binding protein (RBP)-host cell surface receptor interaction enabling isolation of phage-host combinations from an environmental sample. The method was successfully applied to two complex sample types-a dairy-derived whey sample and an infant fecal sample, enabling retrieval of specific and culturable phage hosts. IMPORTANCE PhRACS aims to bridge the current divide between in silico genetic analyses (i.e., phageomic studies) and traditional culture-based methodology. Through the labeling of specific bacterial hosts with fluorescently tagged recombinant phage receptor binding proteins and the isolation of tagged cells using flow cytometry, PhRACS allows the full potential of phageomic data to be realized in the wet laboratory.

In healthy hosts the gut microbiota is restricted to gut tissues by several barriers some of which require MyD88-dependent innate immune sensor pathways. Nevertheless, some gut taxa have been reported to disseminate to systemic tissues. However, the extent to which this normally occurs during homeostasis in healthy organisms is still unknown. In this study, we recovered viable gut bacteria from systemic tissues of healthy wild type (WT) and MyD88 -/- mice. Shotgun metagenomic-sequencing revealed a marked increase in the relative abundance of L. johnsonii in intestinal tissues of MyD88 -/- mice compared to WT mice. Lactobacillus johnsonii was detected most frequently from multiple systemic tissues and at higher levels in MyD88 -/- mice compared to WT mice. Viable L. johnsonii strains were recovered from different cell types sorted from intestinal and systemic tissues of WT and MyD88 -/- mice. L. johnsonii could persist in dendritic cells and may represent murine immunomodulatory endosymbionts.

Rewiring of host cytokine networks is a key feature of inflammatory bowel diseases (IBD) such as Crohn's disease (CD). Th1-type cytokines-IFN-γ and TNF-α-occupy critical nodes within these networks and both are associated with disruption of gut epithelial barrier function. This may be due to their ability to synergistically trigger the death of intestinal epithelial cells (IECs) via largely unknown mechanisms. In this study, through unbiased kinome RNAi and drug repurposing screens we identified JAK1/2 kinases as the principal and nonredundant drivers of the synergistic killing of human IECs by IFN-γ/TNF-α. Sensitivity to IFN-γ/TNF-α-mediated synergistic IEC death was retained in primary patient-derived intestinal organoids. Dependence on JAK1/2 was confirmed using genetic loss-of-function studies and JAK inhibitors (JAKinibs). Despite the presence of biochemical features consistent with canonical TNFR1-mediated apoptosis and necroptosis, IFN-γ/TNF-α-induced IEC death was independent of RIPK1/3, ZBP1, MLKL or caspase activity. Instead, it involved sustained activation of JAK1/2-STAT1 signalling, which required a nonenzymatic scaffold function of caspase-8 (CASP8). Further modelling in gut mucosal biopsies revealed an intercorrelated induction of the lethal CASP8-JAK1/2-STAT1 module during ex vivo stimulation of T cells. Functional studies in CD-derived organoids using inhibitors of apoptosis, necroptosis and JAKinibs confirmed the causative role of JAK1/2-STAT1 in cytokine-induced death of primary IECs. Collectively, we demonstrate that TNF-α synergises with IFN-γ to kill IECs via the CASP8-JAK1/2-STAT1 module independently of canonical TNFR1 and cell death signalling. This non-canonical cell death pathway may underpin immunopathology driven by IFN-γ/TNF-α in diverse autoinflammatory diseases such as IBD, and its inhibition may contribute to the therapeutic efficacy of anti-TNFs and JAKinibs., (© 2021. The Author(s).)

Exopolysaccharide (EPS) is a bacterial extracellular carbohydrate moiety which has been associated with immunomodulatory activity and host protective effects of several gut commensal bacteria. Bifidobacterium breve are early colonizers of the human gastrointestinal tract (GIT) but the role of EPS in mediating their effects on the host has not been investigated for many strains. Here, we characterized EPS production by a panel of human B. breve isolates and investigated the effect of EPS status on host immune responses using human and murine cell culture-based assay systems. We report that B. breve EPS production is heterogenous across strains and that immune responses in human THP-1 monocytes are strain-specific, but not EPS status-specific. Using wild type and isogenic EPS deficient mutants of B. breve strains UCC2003 and JCM7017 we show that EPS had strain-specific divergent effects on cytokine responses from murine bone marrow derived macrophages (BMDMs) and dendritic cells (BMDCs). The B. breve UCC2003 EPS negative (EPS - ) strain increased expression of cytokine genes ( Tnfa, Il6, Il12a , and Il23a ) relative to untreated BMDCs and BMDCs treated with wild type strain. B. breve UCC2003 and JCM7017 EPS - strains increased expression of dendritic cell (DC) activation and maturation marker genes ( Cd80, Cd83 , and Cd86 ) relative to untreated BMDCs. Consistent with this, BMDCs co-cultured with B. breve UCC2003 and JCM7017 EPS - strains engineered to express OVA antigen activated OVA-specific OT-II CD4 + T-cells in a co-culture antigen-presentation assay while EPS proficient strains did not. Collectively, these data indicate that B. breve EPS proficient strains use EPS to prevent maturation of DCs and activation of antigen specific CD4 + T cells responses to B. breve . This study identifies a new immunomodulatory role for B. breve EPS and suggests it may be important for immune evasion of adaptive immunity by B. breve and contribute to host-microbe mutualism., Competing Interests: OH is employed by the company Luminex Corporation which manufactures the Amnis ImageStream flow cytometer used in this study. The remaining 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 Hickey, Stamou, Udayan, Ramón-Vázquez, Esteban-Torres, Bottacini, Woznicki, Hughes, Melgar, Ventura, Van Sinderen, Rossini and Nally.)

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has rapidly spread globally, becoming a huge public health challenge. Even though the vast majority of patients are asymptomatic, some patients present with pneumonia, acute respiratory distress syndrome (ARDS), septic shock, and death. It has been shown in several studies that the severity and clinical outcomes are related to dysregulated antiviral immunity and enhanced and persistent systemic inflammation. Corticosteroids have been used for the treatment of COVID-19 patients, as they are reported to elicit benefits by reducing lung inflammation and inflammation-induced lung injury. Dexamethasone has gained a major role in the therapeutic algorithm of patients with COVID-19 pneumonia requiring supplemental oxygen or on mechanical ventilation. Its wide anti-inflammatory action seems to form the basis for its beneficial action, taming the overwhelming "cytokine storm". Amid a plethora of scientific research on therapeutic options for COVID-19, there are still unanswered questions about the right timing, right dosing, and right duration of the corticosteroid treatment. The aim of this review article was to summarize the data on the dexamethasone treatment in COVID-19 and outline the clinical considerations of corticosteroid therapy in these patients.

The complexity of oxytocin-mediated functions is strongly associated with its modulatory effects on other neurotransmission systems, including the serotonin (5-hydroxytryptamine, 5-HT) system. Signalling between oxytocin (OT) and 5-HT has been demonstrated during neurodevelopment and in the regulation of specific emotion-based behaviours. It is suggested that crosstalk between neurotransmitters is driven by interaction between their specific receptors, particularly the oxytocin receptor (OTR) and the 5-hydroxytryptamine 2C receptor (5-HTR 2C ), but evidence for this and the downstream signalling consequences that follow are lacking. Considering the overlapping central expression profiles and shared involvement of OTR and 5-HTR 2C in certain endocrine functions and behaviours, including eating behaviour, social interaction and locomotor activity, we investigated the existence of functionally active OTR/5-HTR 2C heterocomplexes. Here, we demonstrate evidence for a potential physical interaction between OTR and 5-HTR 2C in vitro in a cellular expression system using flow cytometry-based FRET (fcFRET). We could recapitulate this finding under endogenous expression levels of both receptors via in silico analysis of single cell transcriptomic data and ex vivo proximity ligation assay (PLA). Next, we show that co-expression of the OTR/5-HTR 2C pair resulted in a significant depletion of OTR-mediated Gαq-signalling and significant changes in receptor trafficking. Of note, attenuation of OTR-mediated downstream signalling was restored following pharmacological blockade of the 5-HTR 2C . Finally, we demonstrated a functional relevance of this novel heterocomplex, in vivo, as 5-HTR 2C antagonism increased OT-mediated hypoactivity in mice. Overall, we provide compelling evidence for the formation of functionally active OTR/5-HTR 2C heterocomplexes, adding another level of complexity to OTR and 5-HTR 2C signalling functionality. This article is part of the special issue on Neuropeptides., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Proteins of the BCL-2 family are evolutionarily conserved modulators of apoptosis that function as sensors of cellular integrity. Over the past three decades multiple BCL-2 family members have been identified, many of which are now fully incorporated into regulatory networks governing the mitochondrial apoptotic pathway. For some, however, an exact role in cell death signalling remains unclear. One such 'orphan' BCL-2 family member is BCL-G (or BCL2L14). In this study we analysed gastrointestinal expression of human BCL-G in health and disease states, and investigated its contribution to inflammation-induced tissue damage by exposing intestinal epithelial cells (IEC) to IFN-γ and TNF-α, two pro-inflammatory mediators associated with gut immunopathology. We found that both BCL-G splice variants - BCL-G S (short) and BCL-G L (long) - were highly expressed in healthy gut tissue, and that their mRNA levels decreased in active inflammatory bowel diseases (for BCL-G S ) and colorectal cancer (for BCL-G S/L ). In vitro studies revealed that IFN-γ and TNF-α synergised to upregulate BCL-G S/L and to trigger apoptosis in colonic epithelial cell lines and primary human colonic organoids. Using RNAi, we showed that synergistic induction of IEC death was STAT1-dependent while optimal expression of BCL-G S/L required STAT1, NF-κB/p65 and SWI/SNF-associated chromatin remodellers BRM and BRG1. To test the direct contribution of BCL-G to the effects of IFN-γ and TNF-α on epithelial cells, we used RNAi- and CRISPR/Cas9-based perturbations in parallel with isoform-specific overexpression of BCL-G, and found that BCL-G was dispensable for Th1 cytokine-induced apoptosis of human IEC. Instead, we discovered that depletion of BCL-G differentially affected secretion of inflammatory chemokines CCL5 and CCL20, thus uncovering a non-apoptotic immunoregulatory function of this BCL-2 family member. Taken together, our data indicate that BCL-G may be involved in shaping immune responses in the human gut in health and disease states through regulation of chemokine secretion rather than intestinal apoptosis.

Chronic obstructive pulmonary disease (COPD) is a chronic inflammatory disorder with a course that is not uniform for all COPD patients. Although smoking is considered as the major cause of the disease, persistent or recurrent infections seem to play a particular role in the disease establishment and progression. COPD is characterized by dysregulated immunity that has been associated with the bacterial colonization and infections. The establishment of culture-independent techniques has shed new light on the relationships between bacterial ecology and health status and expanded our knowledge on the lung microbiome. Interactions between the host and lung microbiome result in inflammation and activation of resident cells. The lung microbiome contains populations of symbionts and pathobionts in balance which lose their equilibrium and disturb the balance of T-helper and regulatory T-cells (Treg) upon infection, or lung disease. In COPD factors such as disease severity, exacerbations, degree of inflammation, and type of treatment used (e.g inhaled or systemic steroids and antibiotics) affect the composition of lung microbiota. Recent data indicate that the presence of specific bacterial taxa in the airways has the potential to influence the host immune response and possibly to interfere with disease phenotype. Although, there is a growing body of evidence for the role of microbiome in COPD several unanswered questions still exist for its clinical relevance., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The oxytocin receptor (OTR) and the 5-hydroxytryptamine 2A receptor (5-HTR 2A ) are expressed in similar brain regions modulating central pathways critical for social and cognition-related behaviors. Signaling crosstalk between their endogenous ligands, oxytocin (OT) and serotonin (5-hydroxytryptamine, 5-HT), highlights the complex interplay between these two neurotransmitter systems and may be indicative of the formation of heteroreceptor complexes with subsequent downstream signaling changes. In this study, we assess the possible formation of OTR-5HTR 2A heteromers in living cells and the functional downstream consequences of this receptor-receptor interaction. First, we demonstrated the existence of a physical interaction between the OTR and 5-HTR 2A in vitro , using a flow cytometry-based FRET approach and confocal microscopy. Furthermore, we investigated the formation of this specific heteroreceptor complex ex vivo in the brain sections using the Proximity Ligation Assay (PLA). The OTR-5HTR 2A heteroreceptor complexes were identified in limbic regions (including hippocampus, cingulate cortex, and nucleus accumbens), key regions associated with cognition and social-related behaviors. Next, functional cellular-based assays to assess the OTR-5HTR 2A downstream signaling crosstalk showed a reduction in potency and efficacy of OT and OTR synthetic agonists, carbetocin and WAY267464, on OTR-mediated Gαq signaling. Similarly, the activation of 5-HTR 2A by the endogenous agonist, 5-HT, also revealed attenuation in Gαq-mediated signaling. Finally, altered receptor trafficking within the cell was demonstrated, indicative of cotrafficking of the OTR/5-HTR 2A pair. Overall, these results constitute a novel mechanism of specific interaction between the OT and 5-HT neurotransmitters via OTR-5HTR 2A heteroreceptor formation and provide potential new therapeutic strategies in the treatment of social and cognition-related diseases.

Oxytocin mediates its behavioural effects via the centrally expressed oxytocin receptor (OTR). Oxytocin signalling has been implicated in multiple disorders involving centrally regulated pathways, including obesity, autism, schizophrenia and depression. The OTR has been described to have a complex downstream signalling pathway and an increased understanding of oxytocinergic signalling is needed for the development of novel and better treatments for centrally regulated disorders. The ghrelin receptor (GHSR), known primarily for its role in centrally regulated energy balance and food intake, has in more recent years also been shown to play a role in mood disorders, including anxiety and depression. Although there have been suggestions of crosstalk between both signalling systems, these have largely been unexplored to date. Here we show, to our knowledge for the first-time, compelling evidence for the formation of an OTR and GHSR heterocomplex, resulting in significant modulation of OTR downstream signalling. Co-localized expression of the OTR and GHSR is shown in a heterologous cellular expression system and in primary cultures of the hypothalamus and hippocampus. A physical interaction between the OTR and GHSR is confirmed using flow-cytometry based fluorescence resonance energy transfer (fcFRET). Interestingly, co-expression of the GHSR results in a significant attenuation of OTR-mediated Gαq signalling and changes in receptor trafficking within the cell. Together, these data demonstrate a potential functional relevance of an OTR/GHSR heterocomplex and its ability to alter OTR signalling, which is poised to have important implications for future therapeutic strategies, involving oxytocinergic signalling. This article is part of the Special Issue entitled 'Receptor heteromers and their allosteric receptor-receptor interactions'., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Malignant pleural effusion (MPE) is the lethal consequence of various human cancers metastatic to the pleural cavity. However, the mechanisms responsible for the development of MPE are still obscure. Here we show that mutant KRAS is important for MPE induction in mice. Pleural disseminated, mutant KRAS bearing tumour cells upregulate and systemically release chemokine ligand 2 (CCL2) into the bloodstream to mobilize myeloid cells from the host bone marrow to the pleural space via the spleen. These cells promote MPE formation, as indicated by splenectomy and splenocyte restoration experiments. In addition, KRAS mutations are frequently detected in human MPE and cell lines isolated thereof, but are often lost during automated analyses, as indicated by manual versus automated examination of Sanger sequencing traces. Finally, the novel KRAS inhibitor deltarasin and a monoclonal antibody directed against CCL2 are equally effective against an experimental mouse model of MPE, a result that holds promise for future efficient therapies against the human condition.

Background: Major barriers in using classical FOXP3+ regulatory T cells (Tregs) in clinical practice are their low numbers in the circulation, the lack of specific cell surface markers for efficient purification and the loss of expression of Treg signature molecules and suppressive function after in vitro expansion or in a pro-inflammatory microenviroment. A surface molecule with potent immunosuppressive function is the human leukocyte antigen-G (HLA-G), which is normally expressed in placenta protecting the "semi-allogeneic" fetus from maternal immune attack. Because HLA-G expression is strongly regulated by methylation, we asked whether hypomethylating agents (HA) may be used in vitro to induce HLA-G expression on conventional T cells and convert them to Tregs., Methods: Human peripheral blood T cells were exposed to azacytidine/decitabine and analyzed for HLA-G expression and their in vitro suppressor properties., Results: HA treatment induces de novo expression of HLA-G on T cells through hypomethylation of the HLA-G proximal promoter. The HA-induced CD4 + HLA-G pos T cells are FOXP3 negative and have potent in vitro suppression function, which is dependent to a large extent, but not exclusively, on the HLA-G molecule. Converted HLA-G pos suppressors retain their suppressor function in the presence of tumor necrosis factor (TNF) and preserve hypomethylated the HLA-G promoter for at least 2 days after azacytidine exposure. Decitabine-treated T cells suppressed ex vivo the proliferation of T cells isolated from patients suffering from graft-versus-host disease (GVHD)., Discussion: We propose, in vitro generation of HLA-G-expressing T cells through pharmacological hypomethylation as a simple, Good Manufacturing Practice (GMP)-compatible and efficient strategy to produce a stable Treg subset of a defined phenotype that can be easily purified for adoptive immunotherapy., (Copyright © 2017 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

HDL has important immunomodulatory properties, including the attenuation of lipopolysaccharide (LPS)-induced inflammatory response. As lecithin-cholesterol acyltransferase (LCAT) is a critical enzyme in the maturation of HDL we investigated whether LCAT-deficient (Lcat(-/-)) mice present an increased LPS-induced inflammatory response. LPS (100μg/kg body weight)-induced cytokine response in Lcat(-/-) mice was markedly enhanced and prolonged compared to wild-type mice. Importantly, reintroducing LCAT expression using adenovirus-mediated gene transfer reverted their phenotype to that of wild-type mice. Ex vivo stimulation of whole blood with LPS (1-100ng/mL) showed a similar enhanced pro-inflammatory phenotype. Further characterization in RAW 264.7 macrophages in vitro showed that serum and HDL, but not chylomicrons, VLDL or the lipid-free protein fraction of Lcat(-/-) mice, had a reduced capacity to attenuate the LPS-induced TNFα response. Analysis of apolipoprotein composition revealed that LCAT-deficient HDL lacks significant amounts of ApoA-I and ApoA-II and is primarily composed of ApoE, while HDL from Apoa1(-/-) mice is highly enriched in ApoE and ApoA-II. ApoA-I-deficiency did not affect the capacity of HDL to neutralize LPS, though Apoa1(-/-) mice showed a pronounced LPS-induced cytokine response. Additional immunophenotyping showed that Lcat(-/-) , but not Apoa1(-/-) mice, have markedly increased circulating monocyte numbers as a result of increased Cd11b(+)Ly6C(med) monocytes, whereas 'pro-inflammatory' Cd11b(+)Ly6C(hi) monocytes were reduced. In line with this observation, peritoneal macrophages of Lcat(-/-) mice showed a markedly dampened LPS-induced TNFα response. We conclude that LCAT-deficiency increases LPS-induced inflammation in mice due to reduced LPS-neutralizing capacity of immature discoidal HDL and increased monocyte number., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Background and Aim: To report on the prevalence of inflammatory bowel disease (IBD) related intestinal dysplasia and cancer in northwestern Greece., Patients and Methods: Single referral center retrospective study. The policy among all gastroenterologists of the area regarding medical treatment, patient follow up and bowel surveillance strategies including risk factors is the same., Results: We analyzed 1494 colonoscopies from 696 consecutive IBD patients (494 UC). The follow up time [median, IQR] was 16 [8-23] years and the age at diagnosis was 28 [21-49] years. The number of patient years at risk was 16.219. Disease location for UC was: pancolitis 761 (59%), left sided colitis 455 (35%), and proctitis 69 (6%). Disease location for CD was: colitis 142 (66%), ileitis 45 (22%) and ileocolitis 21 (10%). Disease activity was in remission in 1240 (83%) of them. In total, 498 (72%) patients were on mesalazine, 169(24%) on immunosuppression and 29 (4%) on biologicals. Biopsies were taken randomly in 1429 (96%) endoscopies and were targeted in 65 (4%) of them. We recorded 69 (9.4%) cases with dysplasia and 10 (1.4%) cases with intestinal cancer (9 in UC). No difference was found for dysplasia and cancer in patients who followed up for 10-20 years or for more than 20 years., Conclusions: The prevalence of dysplasia and cancer is increased in UC compared to CD but the prevalence of high-grade dysplasia is comparatively low. Intestinal cancer prevalence is increasing after the first decade and then practically remains stable., (Copyright © 2010 European Crohn's and Colitis Organisation. Published by Elsevier B.V. All rights reserved.)

The relationship between TNF and immune pathology forced an intense research into the regulation of its biosynthesis that extends to multiple mechanisms controlling the utilization of its mRNA. These posttranscriptional mechanisms gradually and variably impose a series of flexible rate-limiting controls to modify the abundance of the TNF mRNA and the rate of its translation in response to environmental signals. Mechanistically, these controls consist of signaling networks converging to RNA-binding proteins and microRNAs, which in turn target a code of secondary or tertiary ribonucleotide structures located on the TNF mRNA. The outcome of these interactions is the stringent control of this mRNA's maturation, localization, turnover and translation. A wealth of molecular and genetic data highlighted that if these posttranscriptional interactions fail, they perturb cellular responses to provide the impetus for TNF-mediated inflammatory disease. Here, we highlight the parameters guiding the posttranscriptional regulation of TNF mRNA and their relevance to homeostasis and pathology., (Copyright (c) 2010 S. Karger AG, Basel.)

Competitive interactions between coinfecting genotypes of the same pathogen can impose selection on virulence, but the direction of this selection depends on the mechanisms behind the interactions. Here, we investigate how host immune responses contribute to competition between clones in mixed infections of the rodent malaria parasite Plasmodium chabaudi. We studied single and mixed infections of a virulent and an avirulent clone and compared the extent of competition in immunodeficient and immunocompetent mice (nude mice and T cell-reconstituted nude mice, respectively). In immunocompetent mice, the avirulent clone suffered more from competition than did the virulent clone. The competitive suppression of the avirulent clone was alleviated in immunodeficient mice. Moreover, the relative density of the avirulent clone in mixed infections was higher in immunodeficient than in immunocompetent mice. We conclude that immune-mediated interactions contributed to competitive suppression of the avirulent clone, although other mechanisms, presumably competition for resources such as red blood cells, must also be important. Because only the avirulent clone suffered from immune-mediated competition, this mechanism should contribute to selection for increased virulence in mixed infections in this host-parasite system. As far as we are aware, this is the first direct experimental evidence of immune-mediated apparent competition in any host-parasite system.

We studied the contribution of CD4 T cell help to survival and competitive fitness of CD8 memory T cells specific for influenza virus nucleoprotein. In agreement with recent studies, the optimal generation of functional memory CD8 T cells required CD4 help, although long-term maintenance of resting CD8 memory T cells did not absolutely depend on the presence of CD4 T cells. Nonetheless, CD4 T cells were essential during differentiation of CD8 memory T cells to imprint on them the capacity to compete effectively with other memory T cells. CD8 memory cells generated with help survived better in secondary polyclonal hosts, and co-transfer into lymphopenic hosts together with "un-helped" CD8 memory cells showed improved homeostatic expansion of CD8 memory cells that had been generated with CD4 help. Therefore, the requirement for CD4 help in CD8 T cell memory extends to homeostatic parameters that ensure the maintenance and competitive fitness of memory clones.

This study describes a double-transgenic model in which monoclonal CD8 F5 T cells are chronically exposed to self Ag (nucleoprotein) in the periphery, but are not affected during thymic development. Chronic exposure of CD8 T cells to their cognate Ag rendered them unable to proliferate or produce cytokines in response to antigenic stimulation in vitro. However, the cells still retained some killer function in vivo and continuously eliminated APC expressing high levels of Ag. In addition, when crossed with mice expressing Ag in the anterior pituitary gland (triple-transgenic mice), F5 T cells migrated to this site and killed growth hormone producing somatotrophs. The anergic state was reversible upon transfer into Ag-free recipients, resulting in full recovery of in vitro responsiveness to Ag. Anergic CD8 T cells express higher levels of CD5, a negative regulator of T cell signaling, whereas after transfer and residence in Ag-free hosts, CD5 levels returned to normal. This suggests that up-regulation of negative T cell regulators in peripheral T cells exposed to chronic stimulation by Ag may prevent full functionality and thus avoid overt autoreactivity.