Introduction: Cellular senescence is the irreversible growth arrest subsequent to oncogenic mutations, DNA damage, or metabolic insult. Senescence is associated with ageing and chronic age associated diseases such as cardiovascular disease and diabetes. The involvement of cellular senescence in acute kidney injury (AKI) and chronic kidney disease (CKD) is not fully understood. However, recent studies suggest that such patients have a higher-than-normal level of cellular senescence and accelerated ageing. Methods: This study aimed to discover key biomarkers of senescence in AKI and CKD patients compared to other chronic ageing diseases in controls using OLINK proteomics. Results: We show that senescence proteins CKAP4 (p-value < 0.0001) and PTX3 (p-value < 0.0001) are upregulated in AKI and CKD patients compared with controls with chronic diseases, suggesting the proteins may play a role in overall kidney disease development. Conclusions: CKAP4 was found to be differentially expressed in both AKI and CKD when compared to UHCs; hence, this biomarker could be a prognostic senescence biomarker of both AKI and CKD.

Rheumatoid arthritis (RA) is a chronic autoimmune condition, characterised by joint pain, damage and disability, which can be addressed in a high proportion of patients by timely use of targeted biologic treatments. However, the patients, non-responsive to the treatments often suffer from refractoriness of the disease, leading to poor quality of life. Additionally, the biologic treatments are expensive. We obtained plasma samples from N = 144 participants with RA, who were about to commence anti-tumour necrosis factor (anti-TNF) therapy. These samples were sent to Olink Proteomics, Uppsala, Sweden, where proximity extension assays of 4 panels, containing 92 proteins each, were performed. A total of n = 89 samples of patients passed the quality control of anti-TNF treatment response data. The preliminary analysis of plasma protein expression values suggested that the RA population could be divided into two distinct molecular sub-groups (endotypes). However, these broad groups did not predict response to anti-TNF treatment, but were significantly different in terms of gender and their disease activity. We then labelled these patients as responders (n = 60) and non-responders (n = 29) based on the change in disease activity score (DAS) after 6 months of anti-TNF treatment and applied machine learning (ML) with a rigorous 5-fold nested cross-validation scheme to filter 17 proteins that were significantly associated with the treatment response. We have developed a ML based classifier ATRPred (anti-TNF treatment response predictor), which can predict anti-TNF treatment response in RA patients with 81% accuracy, 75% sensitivity and 86% specificity. ATRPred may aid clinicians to direct anti-TNF therapy to patients most likely to receive benefit, thus save cost as well as prevent non-responsive patients from refractory consequences. ATRPred is implemented in R.

Organ function relies on microvascular networks to maintain homeostatic equilibrium, which varies widely in different organs and during different physiological challenges. The endothelium role in this critical process can only be evaluated in physiologically relevant contexts. Comparing the responses to oxygen flux in primary murine microvascular EC (MVEC) obtained from brain and lung tissue reveals that supra-physiological oxygen tensions can compromise MVEC viability. Brain MVEC lose mitochondrial activity and undergo significant alterations in electron transport chain (ETC) composition when cultured under standard, non-physiological atmospheric oxygen levels. While glycolytic capacity of both lung and brain MVEC are unchanged by environmental oxygen, the ability to trigger a metabolic shift when oxygen levels drop is greatly compromised following exposure to hyperoxia. This is particularly striking in MVEC from the brain. This work demonstrates that the unique metabolism and function of organ-specific MVEC (1) can be reprogrammed by external oxygen, (2) that this reprogramming can compromise MVEC survival and, importantly, (3) that ex vivo modelling of endothelial function is significantly affected by culture conditions. It further demonstrates that physiological, metabolic and functional studies performed in non-physiological environments do not represent cell function in situ, and this has serious implications in the interpretation of cell-based pre-clinical models.

Disease activity in rheumatoid arthritis (RA) is influenced by activation of circulating and synovial immune cells. Regulatory T cells (Tregs) and monocytes are key cells that drive inflammation in RA. This study investigated if a relationship exists between disease activity in RA and circulating Treg and monocyte numbers and phenotypes. A potential sialic acid (Sia) mediated link between Tregs and monocytes was also probed in vitro. Peripheral blood mononuclear cells (PBMCs) were isolated from RA patient (n = 62) and healthy control (n = 21) blood using density gradient separation. Flow cytometry was used to count and phenotype Treg and monocyte subsets, and to sort healthy control Tregs for Sia cell culture experiments. The effects of Sia on activated Treg FoxP3 and NFκB expression was assessed by flow cytometry and concentrations of secreted TNFα, IL-10 and IFNγ determined by ELISA. High disease activity RA patients who were unresponsive to disease modifying anti-rheumatic drugs (n = 31), have significantly lower relative numbers (percentages) of CD4+CD25+CD127− Tregs (p < 0.01) and memory CD45RA−FoxP3+ Tregs (p < 0.01), compared to low disease activity responders (n = 24). Relative numbers of non-classical CD169+ monocytes are associated with disease activity in RA (p = 0.012). Sia reduced Treg expression of FoxP3, NFκB and cytokines in vitro. A strong association has been identified between non-classical CD169+ monocytes and post-treatment disease activity in RA. This study also indicates that Sia can reduce Treg activation and cytokine release. We postulate that such a reduction could be mediated by interaction with sialyted proteins captured by CD169+ monocytes.

OBJECTIVES Predicting response to anti-tumour necrosis factor alpha (anti-TNFα) drugs at baseline remains an elusive goal in rheumatoid arthritis (RA) management. The purpose of this study was to determine if baseline genetic variants of PTPRC, AFF3, myD228, CHUK, MTHFR1, MTHFR2, CD226 and a number of KIR and HLA alleles could predict response to anti-TNF-α in rheumatoid arthritis patients. METHODS Peripheral blood samples were collected from 238 RA patients treated with anti-TNFα drugs. Genotyping was performed using biochip array technology by Randox Laboratories Ltd. and sequence specific polymerase chain reaction. Linear regression analysis was performed to investigate the role of these genotypes in predicting response to treatment, as defined by European League Against Rheumatism (EULAR) response classification and absolute change in disease activity score (DAS28). RESULTS Of 238 RA patients analysed, 50.4% received adalimumab, 29.7% received etanercept, 14.8% received infliximab, 3.4% certoluzimab and 1.7% golimumab. The MTHFR1 variant rs1801133 was significantly associated with the EULAR response, p=0.044. Patients with the HLA-DRB1*0404 allele displayed a significantly larger reduction in DAS28 compared to non-carriers (mean -2.22, -1.67 respectively, p=0.033). CD226 rs763361 was the only SNP variant significantly associated with ΔDAS28 (p=0.029). CONCLUSIONS This study has investigated individual allele associations with reductions in DAS28 across a range of anti-TNFα treatments. A combined predictive model indicates that patients with the HLA-DRB1*0404 allele and without the CD226 rs763361 polymorphism exhibit the largest reduction in DAS28 after anti-TNF-α treatment.

Microvascular endothelial cells (MVEC) are plastic, versatile and highly responsive cells, with morphological and functional aspects that uniquely match the tissues they supply. The response of these cells to oxygen oscillations is an essential aspect of tissue homeostasis, and is finely tuned to maintain organ function during physiological and metabolic challenges. Primary MVEC from two continuous capillary networks with distinct organ microenvironments, those of the lung and brain, were pre-conditioned at normal atmospheric (∼ 21 %) and physiological (5 and 10 %) O2 levels, and subsequently used to compare organ-specific MVEC hypoxia response. Brain MVEC preferentially stabilise HIF-2α in response to hypoxia, whereas lung MVEC primarily accumulate HIF-1α; however, this does not result in significant differences at the level of transcriptional activation of hypoxia-induced genes. Glycolytic activity is comparable between brain and lung endothelial cells, and is affected by oxygen pre-conditioning, while glucose uptake is not changed by oxygen pre-conditioning and is observed to be consistently higher in brain MVEC. Conversely, MVEC mitochondrial activity is organ-specific; brain MVEC maintain a higher relative mitochondrial spare capacity at 5% O2, but not following hyperoxic priming. If maintained at supra-physiological O2 levels, both MVEC fail to respond to hypoxia, and have severely compromised and delayed induction of the glycolytic shifts required for survival, an effect which is particularly pronounced in brain MVEC. Oxygen preconditioning also differentially shapes the composition of the mitochondrial electron transport chain (ETC) in the two MVEC populations. Lung MVEC primed at physioxia have lower levels of all ETC complexes compared to hyperoxia, an effect exacerbated by hypoxia. Conversely, brain MVEC expanded in physioxia display increased complex II (SDH) activity, which is further augmented during hypoxia. SDH activity in brain MVEC primed at 21 % O2 is ablated; upon hypoxia, this results in the accumulation of near-toxic levels of succinate in these cells. Our data suggests that, even though MVEC are primarily glycolytic, mitochondrial integrity in brain MVEC is essential for metabolic responses to hypoxia; these responses are compromised when cells are exposed to supra-physiological levels of oxygen. This work demonstrates that the study of MVEC in normal cell culture environments do not adequately represent physiological parameters found in situ , and show that the unique metabolism and function of organ-specific MVEC can be reprogrammed by external oxygen, significantly affecting the timing and degree of downstream responses. ![Figure][1]</img> In brief Hypoxia sensing by microvascular endothelial cells (MVEC) is organ-specific, and efficacy of response is affected by external oxygen. While glycolytic capacity is mostly maintained in MVEC regardless of organ or origin, mitochondrial function is required for adequate sensing and timely metabolic shift to glycolysis. Hyperoxygenation of MVEC compromises mitochondrial function, glycolytic shift and survival to hypoxia. Highlights ### Competing Interest Statement The authors have declared no competing interest. [1]: pending:yes

Anthracycline-based chemotherapy is a common treatment for cancer patients. Because it is delivered intravenously, endothelial cells are exposed first and to the highest concentrations, prior to diffusion to target cells. Not surprisingly, vascular dysfunction is a consequence of anthracycline therapy. While chemotherapy-induced endothelial damage at administration sites has been investigated, the effects of lower doses encountered by distant microvascular networks has not. The aim of this study was to investigate the impact of epirubicin, a widely used anthracycline, on healthy endothelial cells to elucidate its effects on microvascular physiology. Here, endothelial cells were briefly exposed to low doses of epirubicin to recapitulate levels in circulation following dilution in the blood and compound half-life in circulation. Both immediate and prolonged responses to treatment were assessed to determine changes in endothelial function. Epirubicin caused a decrease in proliferation and viability in hUVEC, with lower doses resulting in a senescent phenotype in a large proportion of cells, accompanied by a significant increase in pro-inflammatory cytokines and a significant decrease in metabolic activity. Epirubicin exposure also impaired endothelial function with delayed wound closure, reduced angiogenic potential and increased monolayer permeability downstream of VE-cadherin internalization. Primary lung endothelial cells obtained from epirubicin-treated mice similarly demonstrated reduced viability and functional impairment. In vivo, epirubicin treatment resulted in persistent reduction in lung vascular density and significantly increased infiltration of myeloid cells. Modulation of endothelial status and inflammatory tissue microenvironment observed in response to low doses of epirubicin may predict risk for long-term secondary pathologies associated with chemotherapy.

Hand osteoarthritis (HOA) includes different subsets; a particular and uncommon form is erosive HOA (EHOA). Interleukin- (IL-) 1β plays a crucial role in the pathogenesis of osteoarthritis (OA); it is synthesized as an inactive precursor which requires the intervention of a cytosolic multiprotein complex, named inflammasome, for its activation. The aim of this study was to investigate the involvement of IL-1β and the NOD-like receptor pyrin domain containing 3 (NLRP3) inflammasome in patients with EHOA and nonerosive HOA (NEHOA) compared to healthy controls. In particular, we evaluated the gene expression of IL-1β and NLRP3, the serum levels of IL-1β, IL-6, IL-17, and tumor necrosis factor- (TNF-) α, and the protein levels of IL-1β and NLRP3. We also assessed the relationships between IL-1β and NLRP3 and clinical, laboratory, and radiological findings. Fifty-four patients with HOA (25 EHOA and 29 NEHOA) and 20 healthy subjects were included in the study. Peripheral blood mononuclear cell (PBMC) gene and protein expressions of IL-1β and NLRP3 were quantified by quantitative real-time PCR and western blot. IL-1β, IL-6, IL-17, and TNF-α serum levels were determined by ELISA. IL-1β gene expression was significantly reduced (p = 0.0208) in EHOA compared to healthy controls. NLRP3 protein levels were significantly increased in the NEHOA group versus the control (p = 0.0063) and EHOA groups (p = 0.0038). IL-1β serum levels were not significantly different across the groups; IL-6, IL-17, and TNF-α were not detectable in any sample. IL-1β concentrations were negatively correlated with the Kellgren-Lawrence score in the whole population (r = −0.446; p = 0.0008) and in NEHOA (r = −0.608; p = 0.004), while IL-1β gene expression was positively correlated with the number of joint swellings in the EHOA group (r = 0.512; p = 0.011). Taken together, our results, showing poorly detectable IL-1β concentrations and minimal inflammasome activity in the PBMCs of HOA patients, suggest a low grade of systemic inflammation in HOA. This evidence does not preclude a possible involvement of these factors at the local level.

Osteoarthritis (OA) was previously thought of as 'wear and tear' as humans age, however there is increasing evidence to support an inflammatory theory. The nucleotide-binding and oligomerization domain-like receptor containing protein 3 (NLRP3) inflammasome has been implicated in the pathogenesis of a number of arthritic disorders, producing proinflammatory cytokines and degradative enzymes such as Interleukin-1 beta (IL-1β), Tumour necrosis factor alpha (TNF-α) and Matrix metalloproteinase-3 (MMP-3) which drive cartilage degeneration and synovial inflammation. This review aims to summarise the evidence of NLRP3 involvement in OA. Currently, treatment options focus on management of the disease and to date there is no cure. The development of novel biomarkers for OA could improve diagnosis, treatment and management. Importantly, this review provides detail on the involvement of the NLRP3 inflammasome in OA pathology and how its members could act as potential biomarkers to assist clinical decisions.

Autoimmune diseases (ADs) are currently treated with anti-inflammatory and immunosuppressive drugs, aimed at reducing symptoms of disease in order to improve quality of life for patients. However, for a significant number of patients these therapies are ineffective, leading to an increased risk of irreversible damage and eventual disability in certain cases. Growing evidence has implicated glycosylated proteins and their cognate receptors in modulation of the autoimmune response. This review will summarize these findings with particular focus on sialic acid-binding immunoglobulin-like lectin (Siglec)-1 and Siglec-2 involvement in AD. Fluctuations in these glycosylation-dependent pathways could act as sentinels of disease activity or drug responses. If validated, protein modification and cellular response markers could help clinicians achieve remission earlier.